ViroSeq 2.8 English 5002961-RevA 3130

DRAFTSeptember 16, 2009 11:45 am,

ViroSeq IFU 3130 Title.fm

Celera Corporation

Alameda, CA 94502USA

ABBOTTMax-Planck-Ring 265205 WiesbadenGermany+ 49-6122-580

*+H3764J94580.*

1401 Harbor Bay Parkway

ViroSeq™

HIV-1 Genotyping System v2.0For Use with the Applied Biosystems® 3130/3130xl Genetic Analyzers and

ViroSeq® HIV-1 Genotyping System Software v2.8

Instructions For Use

0197

4J94-58

5002961 Rev. A CDx

DRAFTSeptember 16, 2009 11:45 am, ViroSeq IFU 3130 Title.fm

ViroSeq™ HIV-1 Genotyping System v2.0 i

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1Intended Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Indications for Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Summary and Explanation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Principles of the Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

To Reorder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Key to Symbols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Sample Preparation and Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2ViroSeq Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Warnings and Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Storage and Handling Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Specimen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Required Materials Not Provided . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Work Area Setup. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Preventing RNA Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Run Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Sequencing Runs on the Applied Biosystems® 3130 and 3130xl Genetic Analyzers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Required Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

ViroSeq® HIV-1 Genotyping System Software v2.8 Analysis. . . . . . . . . 27Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Computer Requirements for Running ViroSeq® Software v2.8 . . . . . . . . . . . . . . . . . . . . 27

ViroSeq™ HIV-1 Genotyping System v2.0 ii

Installing, Uninstalling and Using the Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Editing Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Antiretroviral Drug Resistance Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Mutations Tables. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Software Error Messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

ViroSeq® Software and Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Performance Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Maintenance and Calibration of the Applied Biosystems® 3130 and 3130xl Genetic Analyzers . . . . . . . . . . . . . . . . . . . . . . . . . 54Required Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

Care of the Applied Biosystems® 3130 and 3130xl Genetic Analyzers . . . . . . . . . . . . . . 54

Capillary Array. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

Flushing and Filling the Water Trap. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

Water Wash. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

Polymer Block. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

Spatial and Spectral Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Autosampler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64References Cited . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

Appendix A: Auditing and Software Access Control . . . . . . . . . . . . . . . 66AB Navigator. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

Enabling Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

ViroSeqTM HIV-1 Genotyping System v2.0 Page 1 of 68

Section 1: Introduction

Intended UseThe ViroSeq™ HIV-1 Genotyping System is intended for use in detecting HIV genomic mutations that confer resistance to specific types of antiretroviral drugs, as an aid in monitoring and treating HIV infection. Specifically, the ViroSeq HIV-1 Genotyping System can be used to:• Detect HIV-1 Subtype B viral resistance in plasma samples collected in EDTA

with a viral load ranging from 2,000 to 750,000 copies/mL• Genotype the entire HIV-1 protease gene from codons 1 to 99 and two-thirds of

the reverse transcriptase (RT) gene from codons 1 to 335In order to use the ViroSeq HIV-1 Genotyping System:• The user (operator or technologist) must be trained in its use.• Interpretation and application of the results should be done by a qualified

physician.The kit is not to be used as a screening test for HIV or as a diagnostic test to confirm the presence of HIV infection.

Indications for UseIncludes the following populations:• HIV-1 infected individuals at drug therapy failure (with increased viral load)

before therapy switch• HIV-1 infected individuals at initial presentation, before initial drug therapy

Summary and ExplanationHuman Immunodeficiency Virus type 1 (HIV-1) is the etiologic agent responsible for the Acquired Immunodeficiency Syndrome (AIDS) pandemic.1,2 Treatment of HIV-1 infections with potent antiretroviral therapy can result in suppression of HIV-1 replication.3,4 However, the eradication of HIV-1 is problematic since latent infections provide a mechanism for lifelong persistence and re-emergence of the virus.5,6 Frequently, treatment failures occur as a result of poor adherence to treatment regimens and/or the emergence of virus strains resistant to drugs.7 These HIV-1 mutant strains can be resistant to one or more drugs in each of the six classes of antiretroviral drugs — nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, fusion inhibitors, entry inhibitors, and HIV integrase strand transfer inhibitors.8,9,10 In addition, resistance to a given drug can generate cross-resistance to other drugs of the same class.7,11 However, treatment failure does not always result in resistance to all drugs in a regimen.12 Therefore, determining the best salvage therapy for patients failing multiple regimens is a formidable challenge.A key factor in identifying new treatment strategies is knowledge of the viral resistance genotype as indicated by mutations present in the viral swarm in patient plasma.13 Retrospective and prospective intervention-based studies have provided evidence supporting the clinical utility of resistance testing.14-16 These studies indicate that the presence of drug resistance is an independent risk factor for treatment failure. Currently, resistance testing is recommended for persons with HIV infections when they enter into care, regardless of whether therapy is immediately initiated. Repeat testing at the initiation of antiretroviral therapy should be considered. HIV drug resistance testing should be performed when changing antiretroviral regimens following virologic failure. Resistance testing is also recommended for pregnant women prior to the initiation of antiretroviral therapy and for women who become pregnant while on therapy.17,18

The ViroSeq™ HIV-1 Genotyping System detects mutations in the RT and protease regions of the pol gene and provides a report indicating genetic evidence of viral resistance. It is a complete system that provides reagents for viral RNA isolation from plasma, RT-PCR, and sequencing.19,20 The entire protease gene and two-thirds of the RT gene are amplified to generate a 1.8 kb amplicon. The amplicon is used as a sequencing template for seven primers that generate an approximately 1.3 kb consensus sequence. The ViroSeq® HIV-1 Genotyping System Software assembles, edits, and identifies mutations within this 1.3 kb sequence. The software compares the consensus sequence with a known reference, HXB-2, to determine mutations present in the sample. Finally, the ViroSeq software uses a proprietary algorithm to analyze the mutations and generate a drug resistance report.

Principles of the ProcedureOperator training is critical to performing the ViroSeq HIV-1 Genotyping assay and achieving accurate results.

The ViroSeq™ HIV-1 Genotyping System is based on six major processes:• Sample Preparation• Reverse Transcription (RT)• Polymerase Chain Reaction (PCR)• Cycle Sequencing• Automated Sequence Detection• Software Analysis

To Reorder

Key to Symbols

4J94-58 ViroSeq™ HIV-1 Genotyping System v2.0 For Use with the Applied Biosystems® 3130/3130xl Genetic Analyzers and ViroSeq® HIV-1 Genotyping System Software v2.8Instructions for Use

= Celera Catalog Number = Biological Risks

= Applied Biosystems Catalog Number = Temperature Limitation

= Contains 48 assays = Consult Instructions For Use

= Electrical Shock/Fire Hazard =

Authorized Representative in the European Community

= Harmful = In Vitro Diagnostic Medical Device

= Toxic = Manufacturer

= Laser Hazard = Infection Risk

= Light Sensitive

CDx

ABI

Xn

of 68 ViroSeqTM HIV-1 Genotyping System v2.0

Section 2: Sample Preparation and Processing

ViroSeq Assay Kit

Item

ViroSeq™ HIV-1 Genotyping System v2.0, with:• ViroSeq™ HIV-1 Sample Preparation

Module• ViroSeq™ HIV-1 RT-PCR Module• ViroSeq™ HIV-1 Sequencing Module• ViroSeq™ HIV-1 8E5 Control Module

4J94-93

ViroSeq™ HIV-1 Sample Preparation Module Cap Color Quantity

HIV Viral Lysis Buffer, tube43% Guanidine Thiocyanate<2% Dithiothreitol<1% N-Lauroylsarcosine

R 20/21/22 Harmful by inhalation, in contact with skin and if swallowed.R 32 Contact with acids liberates very toxic gas.R 36/38 Irritating to eyes and skin.S 26 In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.S 35 This material and its container must be disposed of in a safe way.S 36/37/39 Wear suitable protective clothing, gloves and eye/face protection.S 46 If swallowed, seek medical advice immediately and show this container or label.

Clear 2 x 14.4 mL

RNA Diluent, tube Clear 3 x 1.6 mL

ViroSeq™ HIV-1 RT-PCR Module Cap Color Quantity

HIV RT Mix, tube<0.1% dATP, dCTP, dGTP, dTTP<0.1% non-infectious synthetic oligonucleotide HIV-1 primers

Blue 1 x 384 µL

RNase Inhibitor, tube20 U/L RNase Inhibitor

White 1 x 48 µL

MuLV Reverse Transcriptase, tube50 U/L Recombinant Murine Leukemia Virus Reverse Transcriptase

Purple 1 x 48 µL

= 4848

Contact with Acids or Bleach releases a toxic gas.

Contains GuanidineThiocyanate

HIV PCR Mix, tube<0.1% dATP, dCTP, dGTP, dTTP, dUTP<0.1% non-infectious synthetic oligonucleotide HIV-1 primers

Blue 1 x 1.42 mL

AmpliTaq Gold® DNA Polymerase, tube5 U/L AmpliTaq Gold® DNA Polymerase

Gold 1 x 24 µL

AmpErase® UNG, tube1 U/L Uracil N-glycosylase

Green 1 x 48 µL

DTT (100 mM), tube1.4% Dithiothreitol

Yellow 1 x 20 µL

DNA Mass Ladder, tube<0.1% DNA mass ladder

Clear 1 x 36 µL

Agarose Gel Loading Buffer, tube Clear 1 x 240 µL

RNA Diluent, tube Clear 1 x 1.6 mL

ViroSeq™ HIV-1 Sequencing Module Cap Color Quantity

HIV SEQ Mix A, tubeBigDye® Terminator Ready Reaction Mix<0.1% non-infectious synthetic oligonucleotide HIV-1 primers and nucleotides<0.1% AmpliTaq® DNA Polymerase, FS<0.1% Magnesium Chloride

White 1 x 576 µL

HIV SEQ Mix B, tubeBigDye® Terminator Ready Reaction Mix<0.1% non-infectious synthetic oligonucleotide HIV-1 primers and nucleotides<0.1% AmpliTaq® DNA Polymerase, FS<0.1% Magnesium Chloride

White 1 x 576 µL

HIV SEQ Mix C, tubeBigDye® Terminator Ready Reaction Mix<0.1% non-infectious synthetic oligonucleotide HIV-1 primers and nucleotides<0.1% AmpliTaq® DNA Polymerase, FS<0.1% Magnesium Chloride

White 1 x 576 µL

HIV SEQ Mix D, tubeBigDye® Terminator Ready Reaction Mix<0.1% non-infectious synthetic oligonucleotide HIV-1 primers and nucleotides<0.1% AmpliTaq® DNA Polymerase, FS<0.1% Magnesium Chloride

White 1 x 576 µL

HIV SEQ Mix F, tubeBigDye® Terminator Ready Reaction Mix<0.1% non-infectious synthetic oligonucleotide HIV-1 primers and nucleotides<0.1% AmpliTaq® DNA Polymerase, FS<0.1% Magnesium Chloride

Red 1 x 576 µL

ViroSeq™ HIV-1 RT-PCR Module (continued) Cap Color Quantity


Warnings and Precautions1. This test is for use with human plasma collected with EDTA anticoagulant

only. Do not use plasma collected in heparin with this procedure as it has been shown to inhibit PCR.

2. Do not pipette by mouth.

3. Do not eat, drink, or smoke in laboratory work areas. Wear appropriate personal protective equipment when handling specimens and kit reagents (e.g., safety glasses, gloves, and protective clothing). Wash hands thoroughly after handling specimens and kit reagents.

4. Avoid microbial and ribonuclease contamination of reagents when removing aliquots from reagent bottles. We recommend the use of sterile disposable pipettes and pipette tips.

5. Do not pool reagents from different lots or from different bottles of the same lot.

6. Do not use this kit after its expiration date.

7. Minimize exposure to chemicals. Exposure includes contact, ingestion, or inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation.

8. Dispose of unused reagents and waste in accordance with good laboratory practices and local, state/provincial, or national environmental and health regulations.

9. LASER HAZARD. Exposure to direct or reflected laser light at 40 mW for 0.1 seconds can burn the retina and leave permanent blind spots. Never look directly into the laser beam or allow a reflection of the beam to enter your eyes. Follow the manufacturer’s recommendations for appropriate protective eye-wear and clothing when using the Applied Biosystems® 3130 or Applied Biosystems® 3130xl Genetic Analyzers.

10. BIOLOGICAL RISKS. Biological samples such as tissues and blood have the potential to transmit infectious diseases. Handle all specimens as if they are infectious. Use safe laboratory procedures such as those outlined in Biosafety in Microbiological and Biomedical Laboratories22 and in the NCCLS Document M29-T.23

11. CAUTION: The controls contain human sourced and/or potentially infectious components. Components sourced from human blood have been tested and found to be nonreactive for HBsAg, anti-HCV, anti-HIV-1/HIV-2 and HTLV-I by FDA licensed tests. No known test method can offer complete assurance that products derived from human sources will not transmit infection. Therefore, all human sourced materials should be considered potentially infectious. The HIV-1 particles contained in the positive control are defective, however, studies have shown a limited capability to revert to an infectious form. This may need to be taken into consideration in the event of a significant accidental exposure incident.

12. Components containing sodium azide may react with lead and copper plumbing to form highly explosive metal azides. When disposing solutions that contain sodium azide down laboratory sinks, flush the drains with large volumes of water to prevent azide buildup.

13. Thoroughly clean and disinfect all work surfaces with a freshly prepared solution of 0.5% sodium hypochlorite in deionized water. Do NOT use bleach (0.5% sodium hypochlorite) with glassware or surfaces which have been exposed to HIV Viral Lysis Buffer.

14. Information for European customers: for product not classified as dangerous per European Directive 1999/45/EC Safety data sheet available for professional user on request. For those materials not provided by Celera, refer to the manufacturer’s Safety Data Sheet for additional information.

HIV SEQ Mix G, tubeBigDye® Terminator Ready Reaction Mix<0.1% non-infectious synthetic oligonucleotide HIV-1 primers and nucleotides<0.1% AmpliTaq® DNA Polymerase, FS<0.1% Magnesium Chloride

Red 1 x 576 µL

HIV SEQ Mix H, tubeBigDye® Terminator Ready Reaction Mix<0.1% non-infectious synthetic oligonucleotide HIV-1 primers and nucleotides<0.1% AmpliTaq® DNA Polymerase, FS<0.1% Magnesium Chloride

Red 1 x 576 µL

ViroSeq™ HIV-1 8E5 Control Module Cap Color Quantity

HIV-1 8E5 Positive Control, tubeThe Positive Control was prepared by diluting cultured HIV-1 type B virus (8E5) in HIV RNA-negative human plasma, non-reactive by USFDA-licensed tests for antibody to HIV-1/2, HCV, HTLV-I, and HBsAg. The 8E5 virus contains an intact but defective viral genome that contains a single base insertion at codon 219 of the RT gene.21 Viral load is 50,000 to 100,000 copies/mL.

Red 5 x 500 µL

HIV-1 8E5 Negative Control, tubeThe Negative Control contains normal human plasma tested to be free of HIV RNA, non-reactive by US FDA-licensed tests for antibody to HIV-1/2, HCV, HTLV-I, and HBsAg.

Clear 5 x 950 µL

ViroSeq™ HIV-1 Sequencing Module (continued) Cap Color Quantity

POTENTIAL BIOHAZARD. Human source material. Use Universal Precautions.


Storage and Handling Requirements

Specimen

A. Specimen Collection

The ViroSeq HIV-1 Genotyping System is intended for use with plasma samples only. Collect 5 mL of whole blood in a sterile EDTA anticoagulant tube (Vacutainer™ PPT™ Brand tubes, Becton-Dickinson #362788 or equivalent) and immediately invert the tube 8 to 10 times to mix.

IMPORTANT!

• This assay has been validated only for use with patient samples collected in EDTA.

• Specimens collected with heparin are not suitable for this assay.• Plasma specimens containing the following have been shown to not interfere

with test results:– Lipids up to 30 mg/mL– Bilirubin up to 0.6 mg/mL– Hemoglobin up to 5 mg/mL

We recommend that plasma be separated from whole blood within 30 minutes of collection, but no later than 120 minutes after collection, if using PPT tubes or their equivalent.• Centrifuge the tubes at 1,000 to 2,000 x g at room temperature (15 to 25°C) for

15 minutes.• As soon as possible, transfer the plasma from the PPT tubes (or equivalent) to

sterile, 1.5 mL polypropylene tubes and store them at –65 to –80°C until used.

B. Specimen Transport

The transportation of human whole blood products, including plasma, must comply with country and local regulations for the transport of etiological agents. You may ship the tubes with the plasma at 2 to 8°C for delivery within 24 hours, or ship the tubes with the plasma at –70°C or less on dry ice.

C. Specimen Storage

Store plasma samples at –65 to –80°C. Do not store for greater than 6 months. Do not freeze-thaw the plasma more than two times.

Required Materials Not ProvidedNote: When reagent storage recommendations are observed, both unopened and open reagent tubes are stable until the expiration date. Do not use this kit or any of its reagent components after the expiration date.

ViroSeq Assay Kit

Module Storage Conditions Comments

ViroSeq™ HIV-1 Sample Preparation Module

ViroSeq™ HIV-1 RT-PCR Module

Store at • –15 to –25°C • In a manual defrost freezer

that is designated amplicon-free

Once opened, the following components should be transferred to and stored in the amplified DNA area at 2 to 8°C:• DNA Mass Ladder• Agarose Gel Loading

Buffer

Note: All other components should be stored at –15 to –25°C in the amplicon-free area.

ViroSeq™ HIV-1 8E5 Control Module

Store at • –15 to –25°C • In a manual defrost freezer

that is designated amplicon-free

• Five vials each of positive and negative plasma control.

• Thaw one vial each of the positive and negative plasma control.

• Use with each sample run.

• Do not refreeze.

ViroSeq™ HIV-1 Sequencing Module

Store in the Post-Amplification area at:• –15 to –25°C • In a manual defrost freezer

that is designated for amplified DNA only

These materials are light sensitive. Avoid prolonged exposure to light.

Item Storage Conditions

PCR Cleanup Kit 4J94-73 < -15°C

Sequencing Consumables Kit 4J94-92 15°C to 30°C

Item

Applied Biosystems® 3130xl Genetic Analyzer with:• Data Collection Software v.3.0 • Firmware version 6286200-02 or 6286250-02• PC computer with:

– Microsoft® Windows ® XP Professional edition, Service Pack 2

– 2 GHz Intel® Pentium® 4 processor– 1 GB RAM– Dual 36 GB hard drives

3130XLIMPORTANT! No software should be downloaded from the internet for this intended use.

Applied Biosystems® 3130 Genetic Analyzer with:• Data Collection Software v.3.0 • Firmware version 6278250-02• PC computer with:

– Microsoft® Windows® XP Professional edition, Service Pack 2


3130IMPORTANT! No software should be downloaded from the internet for this intended use.

Applied Biosystems® 3130xl Capillary Array, 50 cm 4315930

Applied Biosystems® 3130 Capillary Array, 50 cm 4333466

Lower Polymer Block Cleaning Kit 4359572

Sequencing Analysis Software v5.3.1 with KB™ Basecaller v1.4

4360967To obtain additional licenses, please contact your Applied Biosystems representative.IMPORTANT! Do not download software from the internet for this intended use.

GeneAmp® PCR System 9600 Thermal CyclerorGeneAmp® PCR System 9700 Thermal Cycler

N801-0001or

N805-0001

MicroAmp® 96-Well Tray (to hold Reaction Tube with attached cap)

N801-0541

MicroAmp® 96-Well Support Base N801-0531

Reservoir Septa 4315932

96-Well Plate Base 4317237

96-Well Plate Retainer 4317241

96-Well Plate Septa 4315933

MicroAmp® Optical 96-Well Reaction Plate N801-0560

POP-6™ Polymer for 3130/3130xl Genetic Analyzers 4363783 (3500 µL) or 4352757 (7000 µL)

Running Buffer, 10X 402824

ABI


Materials Not Provided

The user must supply the following equipment, materials, and reagents.(Use according to Manufacturer’s instructions unless otherwise specified.)

a. Aerosol-resistant tips, RNase-free, 10 to 1,000 µLb. Agarose gel, containing ethidium bromidec. Agarose gel electrophoresis system (gel box with power supply capable of

10 V/cm of agarose gel)d. Aluminum foil tapee. BioSafety hood, laminar flow, clean hoodf. Bleach (for cleaning – make a 10% solution prior to use)g. Centrifuge, microtube quick-spin (2,000 x g)h. Centrifuge, refrigerated (21,000 to 25,000 x g), with hermetically-sealed rotor

for 1.5 mL tubesi. Centrifuge with microtiter plate rotorj. Centrifuge with vacuum pump (if using CENTRI•SEP 96™ Well Plates) must

have:

– Capacity to create a vacuum

– Swinging-bucket rotor

k. CENTRI•SEP 96™ Well Platesl. EDTA, 0.5 M, pH 8.0 (Molecular Biology grade)m. Ethanol, 70% (for cleaning)n. Ethanol, 100%, non-denatured (Molecular Biology grade)o. Ethidium bromidep. Gloves, powder-freeq. Isopropanol 100%, anhydrous (ACS grade)r. Lab coat/gownss. Lint-free tissuet. Pipettors, capable of pipetting from 0 to 1,000 Lu. Screwtop tubes, 1.5 mL (sterile, RNase/DNase-free), must withstand 21,000

to 25,000 x gv. Sodium Acetate, 3.0 M, pH 5.2w. TBE buffer, 10Xx. Transfer pipets, fine-tipy. Ultraviolet light box (300 nM UV lamp)z. Vortex mixer, platform head (2500 rpm)aa. Water, sterile, deionized, RNase/DNase-freeab. Freezer (manual defrost), capable of achieving storage conditions cited

throughout the product labelingac. Refrigerator, capable of achieving storage conditions cited throughout the

product labeling

Work Area SetupDifferent parts of the ViroSeq HIV-1 Genotyping System procedure should be performed in different work areas. Each work area must have dedicated pipettors, equipment, and supplies in order for the assay to be routinely successful. There are three suggested work areas:• Area 1: Sample Preparation (Pre-Amplification) — used for performing Sample

Preparation and working with HIV-1 samples.• Area 2: Pre-Amplification — used for performing the Reverse Transcription and

PCR setup steps.• Area 3: Post-Amplification (Amplified DNA) — dedicated to PCR

amplification, Cycle Sequencing and Automated Sequence Detection, and other activities that require the handling of amplified DNA.

Ideally, Areas 1 and 2 should be separate from each other to prevent the potential transfer of exogenous RNA and DNA into the RT and PCR setup work area. However, if Areas 1 and 2 are located in the same room, they must be clearly delineated. Benchtop biological safety cabinets may serve to isolate the areas in this room.The pipettors and other equipment used in Area 1 are routinely exposed to bloodborne human pathogens and should not be used in any other area outside of Area 1.When possible, use a dedicated space such as a biological safety cabinet with a UV source for the reagent setup of the RT and PCR steps. The UV germicidal lamps in most biological safety cabinets damage DNA left on exposed surfaces, making it unsuitable for subsequent amplification.Maintain strict physical isolation between the amplified DNA workspace (Area 3) and the other two areas to avoid the transfer of amplified DNA out of Area 3.Because of the type of equipment used in the amplified DNA workspace (Area 3), a relatively large space is required. This space requirement generally exceeds the space requirement for Areas 1 and 2.

Preventing RNA DegradationFor genotyping success, it is critical that you prevent RNA degradation in your test samples.RNA is degraded by RNases. These enzymes occur naturally in cells and are liberated during cell lysis. RNases can remain in samples derived from cells if purification is not complete. Also, because RNases are secreted by your skin, RNases can be introduced into samples by contact with surfaces that you have touched. You should wear gloves at all times when handling specimens, reagents, equipment, and disposables.RNases are very stable and do not need any cofactors, so they linger on surfaces and remain functional under a wide range of environmental conditions. They have a high activity, so only a small amount of RNase contamination can cause significant loss in a sample of RNA. Sources of RNase contamination include:• General laboratory glassware and plasticware• Skin and hair• Contaminated solutions

WorkflowYou may complete the procedure over a period of two or more days. When stopping at the specified points, you must store the prepared samples as specified in the table below until you are ready to continue.

IMPORTANT! Keep all samples, controls, and reagents cold (2 to 8°C) prior to making the mastermix. Once the mastermix is made, store it at room temperature, but keep the original reagents on ice..

Hi-Di™ Formamide, 25 mL

Contains Formamide.

R 61 May cause harm to the unborn child.R 36/38 Irritating to eyes and skin.S 53 Avoid exposure - Obtain special instructions before use.S 24/25 Avoid contact with skin and eyes.S 35 This material and its container must be disposed of in a safe way.S 36/37/39 Wear suitable protective clothing, gloves and eye/face protection.S 45 In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).

4311320

BigDye® Terminator v1.1 Sequencing Standard 4336791

Item ABI

At the completion of. . . Store the samples at. . . For no longer than. . .

A. Sample Preparation –65 to –80°C 2 weeks

B. Reverse Transcription –15 to –25°C 2 weeks

C. PCR –15 to –25°C 2 weeks

D. Cycle Sequencing –15 to –25°C 3 days

E. Purified Sequences –15 to –25°C 1 week


Run SizeThe ViroSeq HIV-1 Genotyping System Kit contains enough reagents to perform 48 tests. We recommend that individual runs be performed in batches of 12 samples, which allows four runs per kit. Runs with fewer samples increase the number of freeze-thaw cycles and reagent manipulations, and may result in decreased performance or contamination.

IMPORTANT! Do not freeze-thaw the reagents more than five (5) times.

ControlsNote: A positive and a negative control must be included in every run.

The positive (8E5) control is a non-infectious24 HIV-1 virus at a concentration of 50,000 to 100,000 copies per mL. A 1:10 dilution of the positive control (5,000 to 10,000 copies/mL) must be used with every run to ensure the appropriate performance of the reagents. It is designed to mimic patient samples throughout all aspects of the ViroSeq System testing procedure, from Section A, Sample Preparation, through Section 4, ViroSeq® HIV-1 Genotyping System Software v2.8 Analysis.The negative control is normal human plasma that has been tested to be free of HIV-1 RNA. It must be used from Section A, Sample Preparation, through Section D1, To Quantify and Purify the PCR Products, to ensure that no contamination is present. Use of a negative control is not necessary when performing the cycle sequencing procedure.

Protocol

A. Sample Preparation

Note: Time to complete 12 samples is a total of 2.5 to 3.0 hours; hands-on time is 1.5 hours.

IMPORTANT! Perform this procedure in Pre-Amplification Area 1.

A1. To Set Up

A2. To Isolate the HIV-1 Viral RNA

1. Precool a refrigerated centrifuge and rotor to 2 to 8°C, according to the manufacturer’s instructions.

2. Thaw plasma samples and one tube each of the positive and negative controls at room temperature (15 to 25°C).

3. Keep only one reagent or sample tube open at a time.

1. Vortex the plasma samples 3 to 5 seconds to mix.

2. Centrifuge 1 to 2 seconds at 2,000 x g to collect the contents at the bottom of the tube.

3. Label tubes according to your laboratory protocol, then aliquot 0.5 mL of the plasma into a fresh, RNase- and DNase-free screw-top tube.

4. Prepare a low viral RNA control.Combine in a microfuge tube:

– 50 L HIV-1 8E5 Positive Control– 450 L HIV-1 8E5 Negative Control (negative plasma)

and label appropriately.

5. Place an orientation mark on the side of the tube and cap.

6. Insert the samples in the rotor with the orientation mark facing the outside rim of the rotor.

Note: Use a hermetically sealed rotor with an O-ring to prevent splashing and aerosols.

7. Centrifuge at 21,000 to 25,000 x g for 60 min. at 2 to 8°C.

8. While the samples are centrifuging:• Thaw the Viral Lysis Buffer at room temperature.

R 20/21/22 Harmful by inhalation, in contact with skin and if swallowed.R 32 Contact with acids liberates very toxic gas.R 36/38 Irritating to eyes and skin.S 26 In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.S 35 This material and its container must be disposed of in a safe way.S 36/37/39 Wear suitable protective clothing, gloves and eye/face protection.S 46 If swallowed, seek medical advice immediately and show this container or label.

Note: If precipitate is observed, heat the Viral Lysis Buffer to 37°C to dissolve the precipitate, then cool to room temperature before use.

• Thaw the RNA Diluent and store it at 2 to 8°C.• Prepare the 70% ethanol, and store it at 2 to 8°C for use in step 20.Note: Use Molecular Biology grade, 100% ethanol and RNase/DNase-free water only.

CHEMICAL HAZARD. Ethanol is a flammable liquid and vapor. Exposure may cause eye, skin, and upper respiratory tract irritation. Prolonged or repeated contact may dry the skin. Exposure may cause central nervous system depression and liver damage. Keep away from heat, sparks, and flame. Wear appropriate protective eyewear, clothing, and gloves. Refer to the manufacturer’s Safety Data Sheet for additional information.

9. Remove the tubes immediately after the rotor stops moving.

10. Carefully remove the supernatant from the tubes, using a fine-tip transfer pipet. Do not disturb the pellet (which may not be visible).

TIP: When aspirating, start with the tip of the transfer pipet just below the meniscus line and move the tip down, along the wall opposite the orientation mark, as you aspirate. Remove as much supernatant as possible without disturbing the pellet.

11. Add 600 L of Viral Lysis Buffer to each pellet.

12. Vortex 3 to 5 seconds to mix.


14. Let the samples sit at room temperature (15 to 25°C) for 10 minutes to ensure complete lysis of the virus.

Contact with Acids or Bleach releases a toxic gas.

Contains GuanidineThiocyanate


B. Reverse Transcription

IMPORTANT! Perform this procedure in Pre-Amplification Area 2.

B1. To Set Up

B2. To Run the RT ReactionsNote: Use only the GeneAmp® 9600 or 9700 thermal cycler located in Work Area 2.Note: Program the thermal cycler prior to the reaction setup (refer to step 5). Please refer to the thermal cycler operation manual for detailed programming instructions.

15. Add 600 L of room temperature (15 to 25°C) isopropanol to each sample.

CHEMICAL HAZARD. Isopropanol is a flammable liquid and vapor. Exposure may cause eye, skin, and upper respiratory tract irritation. Prolonged or repeated contact may dry skin and cause irritation. Exposure may cause central nervous system effects such as drowsiness, dizziness, and headache. Wear appropriate protective eyewear, clothing, and gloves. Refer to the manufacturer’s Safety Data Sheet for additional information.

16. Vortex each tube for 3 to 5 seconds to mix.

17. Insert the samples in the rotor with the orientation mark facing the outside rim of the rotor.

18. Centrifuge the samples 12,500 to 15,000 x g at room temperature for 15 min.

19. Carefully remove the supernatant with a fine-tip transfer pipet. Do not disturb the pellet (which may not be visible).

IMPORTANT! Do not discard the supernatant with bleach waste because toxic gas will form.

20. Add 1 mL of cold (2 to 8°C) 70% ethanol (prepared at step 8) to each tube.

21. Vortex each tube for 3 to 5 seconds to mix.

22. Insert the tubes in the rotor with the orientation mark facing the outside rim of the rotor.

23. Centrifuge 12,500 to 15,000 x g at room temperature for 5 min.

24. Carefully remove the supernatant with a fine-tip transfer pipet. Do not disturb the pellet (which should now be visible).Remove as much ethanol as possible.

25. Centrifuge 1 to 2 seconds at 2,000 x g to collect the residual fluid at the bottom of the tube.

26. Carefully remove any residual ethanol with another fine-tip transfer pipet. Air dry the tubes, with the caps off, for 1 to 5 min, or until no ethanol is visible.

IMPORTANT! It is critical to remove all visible ethanol. Residual ethanol inhibits the RT-PCR reaction.

27. Resuspend each pellet in cold (2 to 8°C) RNA Diluent according to the guidelines below.

28. Vortex vigorously for 10 seconds to resuspend the pellet. Some insoluble material may remain.

29. Centrifuge 1 to 2 seconds at 2,000 x g to collect contents at the bottom of the tube.

If the viral load is. . . Then add this quantity of RNA Diluent. . .

>15,000 copies per mL 100 L

2,000 to 15,000 copies per mL 50 L

unknown 50 L

Controls

Undiluted positive 100 L

Low positive 50 L

Negative 50 L

30. When the procedure is complete:• Proceed to the Reverse Transcription steps, or• Stop and store the samples in a –65 to –80°C freezer.

IMPORTANT! Do not freeze-thaw the RNA more than two (2) times. Do not store the RNA for more than two weeks.

1. If necessary, thaw the RNA samples. Be sure to also thaw the positive and negative control RNAs.

2. Vortex for 3 to 5 seconds to mix.

3. Label one 0.2 mL MicroAmp® Reaction Tube for each RNA sample.

4. Thaw the HIV RT Mix and DTT.Vortex for 3 to 5 seconds to mix.

5. Remove the RNase Inhibitor and MuLV Reverse Transcriptase from the kit.

6. Centrifuge all the reagent tubes and samples for 1 to 2 seconds at 2,000 x g to collect the contents at the bottom.

7. Store all reagents and samples at 2 to 8°C or on ice until use.

1. Prepare the RT Mastermix according to the following table. When finished, return the stock solutions to –15 to –25°C.

Note: Prepare sufficient volume for 1 to 2 extra reactions to compensate for pipetting loss.

2. Vortex the RT Mastermix for 2 to 3 seconds to mix.


IMPORTANT! The RT Mastermix must be at room temperature (15 to 25°C) when you add it to the reaction tubes. Do not leave it at room temperature for more than 30 min.

4. Add 10 L of the viral RNA to 0.2 mL MicroAmp® Reaction Tubes and return the stock viral RNA to ice.

IMPORTANT! Use a clean tip for each addition.

Reagent Volume for 1 Reaction (µL)

Volume for 5 Reactions (µL)


HIV RT Mix 8 40 120

RNase Inhibitor 1 5 15

MuLV Reverse Transcriptase

1 5 15

DTT, 100 mM 0.4 2.0 6.0

Final volume 10.4 52.0 156.0


C. PCR

C1. To Set UpIMPORTANT! Perform this procedure in Pre-Amplification Area 2.

C2. To Perform the PCR Reaction5. Immediately cap and place the MicroAmp® Reaction Tubes into a thermal

cycler that has been set to the conditions below, and begin the program. Allow the samples to ramp, with the instrument, to 65°C. While the RNA is heating, leave the RT Mastermix at room temperature.

Note: Pause the run after the 42°C, 5 min. step and proceed to step 6 immediately.

Note: Set the volume on the thermal cycler to 20 L when prompted.

IMPORTANT! The GeneAmp® 9600 and 9700 instruments stay paused for only 10 minutes. Step 6 must be performed within 10 minutes.

6. While the instrument is paused, perform the following:a. Immediately remove the RNA samples from the thermal cycler.b. Add 10 L of the room temperature RT Mastermix to each reaction tube,

and cap the tubes.c. Vortex the tray with samples for 3 to 5 seconds to mix.d. Centrifuge the sample tray for 1 to 2 seconds at 2,000 x g to collect the

contents at the bottom.

7. Return the samples to the thermal cycler and press Resume.

8. After completion of the RT program, hold the samples in the thermal cycler for at least 10 minutes at 4°C, but do not exceed 18 hours.

9. When the program is complete:• Proceed to the PCR steps, or• Stop and store the samples at –15 to –25°C until you are ready to perform

the PCR, but do not store for more than two weeks.

1. If the samples are frozen, thaw them at room temperature (15 to 25°C).

2. Centrifuge the sample tray or tubes for 1 to 2 seconds at 2,000 x g to collect the contents at the bottom.

3. Thaw the HIV PCR Mix.

4. Vortex for 3 to 5 seconds to mix.

5. Remove the AmpliTaq Gold® DNA Polymerase and AmpErase® UNG reagents from the kit. Thaw and vortex for 1 to 2 seconds.

6. Centrifuge all reagents for 1 to 2 seconds at 2,000 x g to collect the contents at the bottom.

Temperature (°C) Time Process

65 30 seconds Relaxes the RNA secondary structure

42 5 minutes Cools to the optimal enzyme activity temperature

Manually Pause and perform step 6, then press Resume

42 60 minutes Reverse transcription

99 5 minutes Inactivates MuLV Reverse Transcriptase

4 Hold (>10 minutes)

Holds until you are ready to proceed

1. Prepare the PCR Mastermix.Combine in a sterile 1.5 mL microcentrifuge tube:

Note: Prepare sufficient volume for 1 extra reaction to compensate for pipetting loss.

2. Vortex the PCR Mastermix for 3 to 5 seconds to mix.


4. Add 30 L of PCR Mastermix to each RT reaction tube containing HIV-1 cDNA.The final volume is now 50 L.

5. Proceed with the samples to the thermal cycler in Area 3.Do not return to Area 2 for the remainder of the day.

6. Note: Use only the GeneAmp® 9600 or 9700 thermal cycler located in Work Area 3.Note: Please refer to the thermal cycler operation manual for detailed programming instructions.Set the thermal cycler program as follows:

Note: Set the volume on the thermal cycler to 50 L when prompted.

7. Transfer the tubes to the thermal cycler, and start the program.

8. When the program is complete:• Proceed to the purification steps, or• Stop and store the samples at –15 to –25°C.

IMPORTANT! Do not leave tubes on hold for more than 24 hours. The residual UNG activity may destroy your amplified DNA.

Reagent Volume for 1 Reaction (µL)



HIV PCR Mix 29.5 147.5 442.5

AmpliTaq Gold DNA Polymerase

0.5 2.5 7.5

AmpErase UNG 1 5 15

Final volume 31 155 465

Temperature(°C) Time Cycles

50 10 min 1

93 12 min 1

93 20 sec4064 45 sec

66 3 min

72 10 min 1

4 Hold —


D. Cycle Sequencing

IMPORTANT! Perform this procedure in Post-Amplification Area 3.

D1. To Quantify and Purify the PCR Products

To run the agarose gel of unpurified PCR product:

Enzymatic Method for PCR Product Clean-up

To purify the PCR products:

Note: Use only the GeneAmp® 9700 thermal cycler.Note: Program the thermal cycler prior to the reaction setup (refer to step 3). Please refer to the thermal cycler operation manual for detailed programming instructions.

Dilution of purified PCR product for Cycle Sequencing:

1. Thaw the DNA Mass Ladder at room temperature (15 to 25°C). This will take at least 30 minutes.Vortex 10 to 15 seconds to mix before using.

2. Thaw the Agarose Gel Loading Buffer at room temperature. This will take at least 30 minutes.

Note: If crystals form, heat the solution to 65°C for 10 minutes.Vortex 10 to 15 seconds to mix before using.

3. Prepare a 1% agarose gel using TBE and containing 0.5 µg/mL of ethidium bromide (or use a purchased 1% agarose gel).

CHEMICAL HAZARD. Ethidium bromide causes eye, skin, and respiratory tract irritation and is a known mutagen (that is, it can change genetic material in a living cell and has the potential to cause cancer). Wear appropriate protective eyewear, clothing, and gloves. Refer to the manufacturer’s Safety Data Sheet for additional information.

4. Prepare a 1X TBE gel buffer solution containing 0.5 µg/mL of ethidium bromide.

5. Place the prepared gel in the gel box.

6. Add enough 1X TBE gel buffer to cover the gel.

7. For each sample, mix:• 5 µL of Agarose Gel Loading Buffer• 5 µL of unpurified PCR product

8. Load the DNA Mass Ladder solution.a. 6 µL in Lane 1b. 3 µL in Lane 2

This gives the following results:

9. Load the remaining lanes with 10 µL each of the samples

IMPORTANT! Be sure to record which sample is loaded in which lane.

10. Electrophorese at 10 V/cm until the bromphenol blue has migrated at least 5 cm into the gel.

11. Examine the gel with UV light.

12. Photograph your gel using an exposure time that does not saturate the film and shows the differences in intensity of the mass ladder fragments.

Bands 6 µL Lane(ng)

3 µL Lane(ng)Location Size (kb)

Top 2.0 100 50

Second 1.2 60 30

Third 0.8 40 20

13. Evaluate the quantity of PCR products in each sample by comparing the intensity of each band to the intensities of the DNA Mass Ladder bands.

Note: Do NOT dilute at this step.

14. Proceed to purification of PCR products with PCR Cleanup Reagent for samples determined to have an intensity equal to or greater than the 20 ng mass ladder band.

Note: The RT-PCR product band must have an intensity equal to or greater than the 20 ng mass ladder band to ensure a high quality sequence.

1. Carefully pipette 3 µL of PCR Cleanup Reagent into each tube that now contains 45 µL of PCR product with a band intensity greater than or equal to 20 ng/5 µL.

2. a. Cap the tubes.b. Vortex the tubes or tray with sample for 3 to 5 seconds to mix.c. Centrifuge the tubes or sample tray for 1 to 2 seconds at 2,000 x g to collect the contents at the bottom.

The final volume is now 48 µL.

3. Program the thermal cycler using the following program:

Note: Set the reaction volume on the thermal cycler to 48 L when prompted.

4. Transfer tubes or tray with samples to the thermal cycler and start the program.

5. When the program is complete, proceed to the dilution steps.

1. Dilute the purified PCR product for each sample with deionized, distilled water (ddH2O) according to the following table.


37 15 min 1

80 15 min 1

4 Hold —

If the band intensity was. . . Then. . .

20 to 40 ng adjust the sample volume to 60 L.

40 to 60 ng make a 1:2 dilution of your sample with ddH2O(1 part sample and 1 part water).

60 to 100 ng make a 1:4 dilution of your sample with ddH2O(1 part sample and 3 parts water).

>100 ng make a 1:10 dilution of your sample with ddH2O(1 part sample and 9 parts water).


D2. To Set Up Cycle SequencingNote: Use only the GeneAmp® 9600 or 9700 thermal cycler located in Work Area 3.Note: Program the thermal cycler prior to the reaction setup (refer to step 7). Please refer to the thermal cycler operation manual for detailed programming instructions.Note: Do not set up sequencing reactions for the negative control.

IMPORTANT! The sequencing mixes are light sensitive. Do not expose the mixes to light for extended periods.

D3. To Purify the Sequences

There are three methods recommended for the purification of BigDye® Terminator sequence reactions. Use one of the following methods.

D3. a To Purify the Sequences With Ethanol/EDTA

Illustration:

2. Cap and vortex each diluted sample for 3 to 5 seconds to mix.


4. When the procedure is complete, proceed to Cycle Sequencing.

1. Thaw the HIV-1 sequencing mixes at room temperature (15 to 25°C).

2. Set up the reaction format. You can use one of the following options.• One MicroAmp® Reaction Tube for each HIV SEQ Mix• MicroAmp® 8-Tube Strip(s) in a MicroAmp® tray• A MicroAmp® Optical 96-Well Reaction Plate

IMPORTANT! If you are using a MicroAmp® Optical 96-Well Reaction Plate, you must consider the layout of the samples as well as the instrument platform you use.

A 96-well Reaction Plate loaded with sample 1 (S1) beginning in column 1 of the 96-well plate, S2 in column 2, and so forth. Row H is not used unless you want to run 13 samples on one plate.

If using a 3130 Genetic Analyzer for the automated sequence detection step, only 7 columns of samples may be analyzed at one time. The remaining columns of samples and 10 µL from each well containing the positive control must be transferred to a second plate. Refer to To Prepare Purified Sequencing Reactions for Analysis on the 3130 Genetic Analyzer.

3. In each tube or well, combine as follows:

4. Close the tube caps or place a MicroAmp® Optical 96-Well Reaction Plate cover over the plate.

5. Centrifuge at room temperature for 5 to 10 seconds.

6. Transfer the samples to the thermal cycler.

7. Set the thermal cycler program as follows:

Note: Set the reaction volume on the thermal cycler to 20 L when prompted.

IMPORTANT! Do not leave your samples on hold for more than 24 hours. Store your samples at –15 to –25°C.

8. Start the thermal cycler.

9. When the program is complete:• Proceed to the purification steps, or• Stop and store the samples at –15 to –25°C.Do not store the samples for more than 3 days at –15 to –25°C.

1. Remove the MicroAmp® tray from the thermal cycler, and remove the caps from each tube or the cover from the plate.

2. To each sequencing reaction, add 5 µL of 125 mM EDTA (prepared from the 0.5 M EDTA).

Component Volume for One Reaction (L)

Add one of the following HIV-1 SEQ mixes to each tube or plate well:HIV SEQ Mix AHIV SEQ Mix BHIV SEQ Mix CHIV SEQ Mix DHIV SEQ Mix FHIV SEQ Mix GHIV SEQ Mix H

12

Diluted, purified PCR product 8

Final Volume 20


96 10 sec2550 5 sec

60 4 min

4 Hold —


D3.b To Purify With Ethanol/Sodium Acetate

D3.c To Purify With CENTRI•SEP 96™ Well Plates

IMPORTANT! CENTRI•SEP 96 plates are temperature sensitive. Do not store plates below 4°C. Plates must be at room temperature prior to use.

3. To each sequencing reaction, add 60 µL of 100% ethanol.

CHEMICAL HAZARD. Ethanol is a flammable liquid and vapor. Exposure may cause eye, skin, and upper respiratory tract irritation. Prolonged or repeated contact may dry the skin. Exposure may cause central nervous system depression and liver damage. Keep away from heat, sparks, and flame. Wear appropriate protective eyewear, clothing, and gloves.Prepare enough for one reaction more than you need. Refer to the manufacturer’s Safety Data Sheet for additional information.

4. Seal the tubes with strip caps or the plate with adhesive aluminum foil tape.

5. Mix thoroughly by vortexing.

6. Centrifuge the tray/plate at 2,000 x g for 20 minutes at room temperature.

7. As soon as the centrifuge stops, carefully remove the caps or foil tape without disturbing the pellets.

8. Immediately place an absorbent paper towel or lint-free tissue on top of the tray/plate and invert.

9. Place the tray or plate in the centrifuge in the inverted position, on top of a lint-free tissue or paper towel, and centrifuge at 150 x g for 30 seconds.

10. Add 150 µL of 70% ethanol to each well, then seal with caps or foil tape.

11. Centrifuge at 2,000 x g for 5 minutes at room temperature.

12. As soon as the centrifuge stops, carefully remove the caps or foil tape without disturbing the pellets.


14. Place the tray or plate in the centrifuge in the inverted position, on top of a lint-free tissue or paper towel, and centrifuge at 150 x g for 30 seconds.

15. When the centrifuge stops and drying is complete, remove the tray/plate and seal with strip caps or with adhesive aluminum foil tape, and proceed to:• Section 3, Sequencing Runs on the Applied Biosystems® 3130 and

3130xl Genetic Analyzers, or• Store at –15 to –25°C in the dark. Analyze the samples within one week.

IMPORTANT! Store in the dark. The products of sequencing reactions are light-sensitive.

1. Prepare the sodium acetate/ethanol solution by combining the following per reaction:2 L of 3.0 M sodium acetate, pH 5.250 µL of 100% ethanol

CHEMICAL HAZARD. Ethanol is a flammable liquid and vapor. Exposure may cause eye, skin, and upper respiratory tract irritation. Prolonged or repeated contact may dry the skin. Exposure may cause central nervous system depression and liver damage. Keep away from heat, sparks, and flame. Wear appropriate protective eyewear, clothing, and gloves.Prepare enough for one reaction more than you need. Refer to the manufacturer’s Safety Data Sheet for additional information.

IMPORTANT! Prepare a fresh solution for each set of precipitations.

2. Add 52 L of the sodium acetate/ethanol solution to each sequencing reaction.

3. Seal the tubes with strip caps or the plate with adhesive aluminum foil tape.

4. Mix by either inverting three times or vortexing.

IMPORTANT! Mix thoroughly at this step. This is essential for efficient precipitation.

5. Centrifuge at 2,000 x g for 20 minutes.

6. As soon as the centrifuge stops, remove the caps or foil tape without disturbing the pellets.


8. Centrifuge 150 x g for 1 minute.

9. Add 150 L of 70% ethanol to each well.

10. Centrifuge at 2,000 x g for 5 minutes.


12. Centrifuge 150 x g for 1 minute.

13. When the centrifuge stops and drying is complete, remove the tray/plate and seal with strip caps or with adhesive aluminum tape, and proceed to:• Section 3, Sequencing Runs on the Applied Biosystems® 3130 and



1. Make sure the CENTRI•SEP 96 plates are at room temperature.

2. Remove the adhesive-foil sealing film from the bottom of the CENTRI•SEP 96 plate, and then from the top.

3. Prepare the CENTRI•SEP 96 plate.a. Stack the CENTRI•SEP 96 plate on top of a MicroAmp® Optical 96-Well

Reaction Plate, and tape the two plates together with a base.b. Centrifuge the plate at 700 x g for 2 minutes to pack the column.c. Remove the tape and separate the plates.d. Discard the remaining liquid in the wash plate by shaking it vigorously.e. Wash and save the MicroAmp® Optical 96-Well Reaction Plate for

repeated use as a wash plate.

4. Place a 96-well collection plate on a 96-well base, then stack the CENTRI•SEP 96 plate on top.

5. Tape the plates to the 96-well base, making sure that you align the alphanumeric indices on all the plates.


Quality ControlThe ViroSeq™(8E5) Control Module, containing both positive and negative controls, is provided with the ViroSeq HIV-1 Genotyping System. A positive and negative control must be used with every run. The HIV-1 8E5 Positive Control is required to monitor both RT-PCR success and Genotyping success. The HIV-1 8E5 Negative Control is required to monitor RT-PCR success only.

RT-PCR Success

RT-PCR success is determined in section “D1. To Purify and Quantify the PCR Products.” To ensure a high quality sequence, the intensity of the RT-PCR band must be equal to or greater than the intensity of the 20 ng mass ladder band in the agarose gel. Plasma samples with viral loads above 2,000 copies per mL should provide at least 20 ng of RT-PCR product at this step. As the viral load decreases below 1,000 copies per mL, the chance for a successful PCR decreases. However, any sample with a DNA quantitation of 20 ng or greater can be sequenced to provide a genotype, regardless of the starting concentration in plasma. The HIV-1 8E5 Positive Control has a starting viral load of 50,000 to 100,000 copies/mL; the low positive control (1:10 dilution) has a viral load of 5,000 to 10,000 copies/mL. The negative control has no detectable HIV-1 RNA.

Limitations

Reagents and Chemistry

• Only trained users are allowed to perform the ViroSeq assay.• Accurate and reliable results are dependent on proper sample collection and

storage prior to testing.• Blood collected using heparin tubes is unsuitable for use with PCR and this

assay.• Testing for antiretroviral drug resistance has only been validated on patient

samples with viral loads between 2,000 and 750,000 copies per mL. In a typical HIV positive patient population in the U.S. that is on HIV highly active antiretroviral therapy (HAART), about 25% of the patients can be expected to have suppressed viral loads. These samples may not always generate interpretable results with the ViroSeq HIV-1 Genotyping System if the viral load is below 1,000 copies/mL. Users should test samples with a viral load of 2,000 copies/mL or greater.

• Regardless of viral load, the agarose band intensity of the RT-PCR product must be equal to or greater than the intensity of the 20 ng mass ladder band to produce a high-quality sequence.

• The presence of AmpErase® UNG in the ViroSeq HIV-1 Genotyping System reduces the risk of contamination with previously amplified product only. Sample to sample contamination, or contamination from the positive control, can still occur. Careful adherence to work area setup and the protocol reduces the possibility of contamination.

• Only Applied Biosystems thermal cyclers and automated DNA sequencers can be used with this assay.

6. Load the sequencing reactions. Using a multichannel pipettor, transfer the 20-µL sequencing reactions to the individual wells in the CENTRI•SEP 96 plate.• Carefully place samples on the centers of the gel beds. Do not place the

samples on the side of the wells.• Do not touch the gel bed with the pipette tips.

7. Centrifuge the plate at 700 x g for 2 minutes.

8. Discard only the CENTRI•SEP plate (you should have approximately 20 µL in each well of the collection plate).

9. Dry the samples in a centrifuge with vacuum pump equipped with the appropriate rotor. Do not overdry the samples.

10. When the centrifuge stops and drying is complete, remove the tray/plate and seal with strip caps or with adhesive aluminum tape, and proceed to:• Section 3, Sequencing Runs on the Applied Biosystems® 3130 and



The expected RT/PCR results for the ViroSeq controls are:

Positive/Low Positive Control Negative Control

Expected Value >40 ng/5 L (undiluted positive)>20 ng/5 L (low positive)

Expected Value 0 ng

If. . . Then. . . If. . . Then. . .

the band intensity is:• Positive,

undiluted:<40 ng

• Low positive:<20 ng

run is invalid. Repeat testing.

any band is present on the agarose gel

sample to sample contamination has occurred.• All sample results

are invalid• Repeat from “A.

Sample Preparation” through “D1. To Purify and Quantify the PCR Products”

all of the other sample reactions failed (less than 20 ng/5 µL).

a system failure has occurred.• Repeat all samples

and controls

repeated failure occurs contact Abbott Molecular support


Section 3: Sequencing Runs on the Applied Biosystems® 3130 and 3130xl Genetic Analyzers

IntroductionThe ViroSeq™ HIV-1 Genotyping System v2.0 has been approved for in vitro diagnostic use with the ViroSeq® HIV-1 Genotyping System Software v2.8 on the Applied Biosystems® 3130 Genetic Analyzer and the Applied Biosystems® 3130xl Genetic Analyzer. Both instruments are fully automated, fluorescence-based capillary electrophoresis platforms that simultaneously analyze samples within a single run. As summarized below, key differences between the two platforms include the number of 96-well plates that can be loaded on the instrument and the number of capillaries in the capillary array.Note: The number of capillaries in the instrument's capillary array determines run size.

Together, these parameters define the throughput capacity of the instrument and allow sites to determine the system configuration that is most compatible with current and future sequencing volume.

96-Well Plate MapFor each 3130xl Genetic Analyzer run, injections are made from each well of 16 wells of two consecutive plate columns, starting with the column that contains well A1 (e.g., wells A1 though H2, A3 through H4, etc.). A full plate requires six runs to inject all 96 wells.For each 3130 Genetic Analyzer run, injections are made from each well of 4 consecutive wells within a plate column starting with the column that contains well A1 (e.g., wells A1 through D1, E1 through H1, A2 through D2, etc.). It takes four runs to inject 16 wells, and 24 runs to inject a full plate of 96 wells.

• Ensure that the instrument is calibrated before proceeding with the run. Refer to Maintenance and Calibration of the Applied Biosystems® 3130 and 3130xl Genetic Analyzers.

• For illustrations and diagrams that explain how to conduct a specific task on the Applied Biosystems® 3130/3130xl Genetic Analyzer, refer to the Applied Biosystems® 3130/3130xl Genetic Analyzer Getting Started Guide (PN 4352715) and the Maintenance, Troubleshooting, Reference Guide (PN 4352716).

Required Materials

Parameter 3130 Genetic Analyzer 3130xl Genetic Analyzer

Number of 96-Well Plates 1 2

Number of Capillaries in Array 4 16

Number of Runs/One 96-Well Plate 24 6

ROWS

1 2 3 4 5 6 7 8 9 10 11 12

A

B

C

D

E

F

G

H

COLUMNS

: Single Run on 3130 Genetic Analyzer (4-capillary array)

: Single Run on 3130xl Genetic Analyzer (16-capillary array)

Item

ABI PRISM® 3130xl Genetic Analyzer with:• Data Collection Software v.3.0• Firmware version 6286200-02 or 6286250-02 • PC computer with:


– 2 GHz Intel® Pentium® IV processor– 1 GB RAM– Dual 36 GB hard drives

3130XLIMPORTANT! No software should be downloaded from the internet for this intended use.

Applied Biosystems® 3130 Genetic Analyzer with:• Data Collection Software v.3.0 • Firmware version 6278250-02• PC computer with:

– Microsoft® Windows XP Professional edition, Service Pack 2



Applied Biosystems® 3130xl Capillary Array, 50 cm 4315930

Applied Biosystems® 3130 Capillary Array, 50 cm 4333466


Sequencing Analysis Software v5.3.1 with the KB™ Basecaller v1.4

4360967To obtain additional licenses, please contact your Applied Biosystems representative.IMPORTANT! Do not download software from the internet for this intended use.

GeneAmp® PCR System 9600 Thermal CyclerorGeneAmp® PCR System 9700 Thermal Cycler

N801-0001or

N805-0001










Contains Formamide. R 61 May cause harm to the unborn child.R 36/38 Irritating to eyes and skin.S 53 Avoid exposure - Obtain special instructions before use.S 24/25 Avoid contact with skin and eyes.S 35 This material and its container must be disposed of in a safe way.S 36/37/39 Wear suitable protective clothing, gloves and eye/face protection.S 45 In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).

4311320

ABI


Protocol

To Prepare for Automated Sequence Detection

To Start the Computer WorkstationIMPORTANT! You must start the computer workstation before starting the instrument.

To Start the Instrument

To Set Up the Instrument for the RunRefer to the Applied Biosystems® 3130/3130xl Genetic Analyzer Getting Started Guide and the Maintenance, Troubleshooting, Reference Guide for detailed installation, maintenance, and instructions for general use of the instruments.

Note: After opening the instrument door, you must allow the autosampler homing sequence to complete before issuing further commands. The instrument is not available until all commands are completed, including steps within wizards and manual control commands, and the autosampler is in the home position.

Data Collection Software

IMPORTANT! Do not rename the computer. The instrument computer was assigned a unique name before the 3130/3130xl Genetic Analyzers Data Collection software was installed. Doing so may cause the Data Collection software to malfunction.

To determine the 3130 or 3130xl firmware and Data Collection software versions installed on your system, click Help > About from the menu or select GA Instruments from the Data Collection window.

IMPORTANT! Use only the following versions of software:

ABI 3130 Data Collection software v.3.0 with Firmware v. 6278250-02or

ABI 3130xl Data Collection software v.3.0 with Firmware v. 6286200-02 or 6286250-02

Never move or delete any system file or folder unless specifically directed to do so by an Applied Biosystems representative or by the Applied Biosystems®

3130/3130xl Genetic Analyzer Getting Started Guide. Doing this could render the software inoperable.

To Start the Data Collection Software

1. Power on the computer and monitor.

2. In the Log-in to Windows dialog box, enter the user name and, if applicable, enter a password, then click OK.

1. On the instrument, ensure that the:• Oven door is closed and locked• Instrument doors are closedNote: If the doors are open during power on, a yellow warning light will continue to blink until the doors are closed.

2. Press the on/off button on the front of the instrument.

Note: While the instrument is booting up and performing self-checks, the yellow status light blinks.

3. Ensure the green status light is on and not blinking before proceeding.

Note: If the green status light does not come on, start the Data Collection software and view the event log at:

E:\AppliedBiosystems\UDC\DataCollection\Log\Instrument Name

1. Prepare and have the following reagents and consumables on hand:• POP-6™ Polymer for 3130/3130xl Genetic Analyzers • A 50 cm, 16-capillary array or a 50 cm, 4-capillary arrayNote: Replace the capillary array after 100 runs and check that the capillary array information is correct in the Data Collection database.• Running Buffer, 10X Note: To prepare 1X Running Buffer, dilute 5 mL of 10X Running Buffer, with 45 mL of deionized water.

CHEMICAL HAZARD. POP-6 polymer causes eye, skin, and respiratory tract irritation. Wear appropriate protective eyewear, clothing, and gloves

2. Ensure that there is sufficient POP-6 polymer for the run, and add polymer if needed.

Note: If adding more polymer, ensure that the polymer is from the same lot.Note: Change the polymer if it has been on the instrument for 7 days. Do not use expired polymer or polymer containing precipitated material.

3. Fill and/or change the buffer and water reservoirs before each run.• Anode reservoir. Fill to the fill line with fresh 1X Running Buffer

(16 mL).• Cathode reservoir. Fill to the fill line with fresh 1X Running Buffer

(16 mL).• Water reservoirs. Fill to the fill line with fresh deionized water (16 mL

each).

4. Check for bubbles in the pump block, lower polymer block, interconnect tube, polymer supply tube, and channels.Remove all bubbles with the Bubble Remove Wizard.

5. Check:• That the polymer block fits securely on the instrument.• For dried polymer around the polymer block, and clean as necessary.• That the capillary tips are not crushed or damaged.

6. Perform a spatial calibration if you have just installed a new or used capillary array on the instrument.

7. Perform a spectral calibration if needed. Refer to To Perform A Spectral Calibration.

1. Select Start > All Programs > Applied Biosystems > Data Collection> Run 3130 Data Collection v3.0 or Run 3130xl Data Collection v3.0.

The Service Console is displayed.

As each application activates, the red circles (off) change to yellow triangles (activating), and then to green squares (on) when they are fully functional.

When all the applications are running (all green squares—this could take several minutes), the Foundation Data Collection Viewer window is displayed.

Enter the user login and password. The login ID should be a user with Instrument Protocols access (refer to Appendix A: Auditing and Software Access Control).


To Create Required Files for the ViroSeq HIV-1 Genotyping System v2.0The following three files, Instrument Protocol, Analysis Protocol and Results Group, must be created one time by the user. These files are used for all subsequent plate records. Refer to To Create a Plate Record.

To View the Default Run Module for a Sequencing Run

The Run Module specifies information about how the sample is run (e.g., the duration of the run, the run temperature, and the injection time).

To Create a ViroSeq Instrument Protocol

An instrument protocol contains all the settings needed to run the instrument. An instrument protocol contains the protocol name, type of run, run module, and dye set.

To View an Instrument Protocol

2. Click the + to expand subfolders in the left window pane. All application folders are now visible and ready to access.

1. Select GA Instruments > ga3130 or ga3130xl > Module Manager.

2. To view the default sequencing run parameters, double-click StdSeq50_POP6_1 from the Run Module list. All the parameters for the default module are displayed.The default module installed with 3130/3130xl Data Collection software cannot be edited.

3. Click Cancel to close the default Run Module.

1. Select GA Instruments > ga3130 or ga3130xl > Protocol Manager.

2. In the Instruments Protocol section, click New. The Protocol Editor opens.

3. Complete the Protocol Editora. Name:

ViroSeq_Instrument_Protocolb. Description: Optionalc. Type: Regulard. Run Module:

StdSeq50_POP6_1e. Dye Set: E-BigDyeV1

4. Click OK.


2. In the Instrument Protocols section, double-click the instrument protocol you want to view. The protocol editor dialog box displays all the parameters for the instrument protocol.

Default Run Module 3130Default Run Module 3130xl


To Edit an Instrument Protocol

To Create a ViroSeq Analysis Protocol


2. In the Instrument Protocols section, click on the instrument protocol to highlight and click Edit.

3. In the Protocol Editor dialog box, edit the parameters that you want to change.

4. Click OK.

5. In the Warning dialog box, click Yes to overwrite the previous settings.


2. In the Analysis Protocol section, click New. The Sequencing Analysis Protocol Editor opens. Complete the Sequencing Analysis Protocol Editor as described in the following steps.

3. General tab:• Name: ViroSeq_Analysis_Protocol• Description: optional• Sequence File Formats: uncheck all check boxes.

4. Basecalling tab:• Basecaller: KB.bcp• DyeSet / Primer: KB_3130_POP6_BDTv1.mob• Processed Data: True Profile• Ending Base: At PCR Stop• Quality Threshold: Do not assign N’s to Basecalls.

5. Mixed Bases tab:• Uncheck the Use Mixed Base Identification check box.


To View an Analysis Protocol

To Edit an Analysis Protocol

To Create a ViroSeq Results Group

A Results Group is a component of Data Collection software that is used to name, analyze, and organize sample files.

6. Clear Range tab:• Uncheck all check boxes.


2. In the Analysis Protocols section, double-click the analysis protocol you want to view. The protocol editor dialog box displays all the parameters for the analysis protocol.


2. In the Analysis Protocols section, click on the analysis protocol to highlight and click Edit.

3. In the Sequencing Analysis Protocol Editor dialog box, edit the parameters that you want to change.

4. Click OK.

1. Select GA Instruments > Results Group.

2. Click New. The Results Group Editor window displays.

3. Complete the General tab:a. Results Group Name: enter ViroSeq_Results_Group.b. Results Group Owner: the owner name can be used in naming and

sorting sample files.c. Results Group Comment: enter a comment (optional).

4. Complete the Analysis tab:

a. Analysis Type: SequencingAnalysis.b. Analysis Actions: select the check box Do Autoanalysis.

5. Select the Destination tab to define a data storage location. Use the default Root Destination path for data storage:

E:\AppliedBiosystems\udc\datacollection\Data


To Duplicate the ViroSeq Results Group (Optional)

To Create a Plate Record6. Select the Naming tab to customize sample file and run folder names.

a. Complete the Sample File Name Format pane:– Click the Name Delimiter list and select the underscore symbol (_)

to separate the Format elements in the file name.– Click the Format list and then select the following format elements

for the file name in this order: Sample Name, Well Position. As you select the elements for the file name, they are reflected in the Example line. The names of the Format elements eventually truncate, but the Example field remains visible (up to 72 characters).

b. Complete the Run Folder Name Format pane: – Click the Format list and then select the format element Plate Name.

This will group all samples within a plate (across runs) into a single folder.

IMPORTANT! Sample name, run folder name, and path name, combined, cannot exceed 250 characters.

7. Click OK to save the Results Group.

1. Select GA Instruments > Results Group.

2. Click the ViroSeq_Results_Group to highlight.

3. Click Duplicate.

4. When the Duplicate Results Group dialog box opens, rename the ViroSeq Results Group.

Note: A useful way to name a Results Group is to use the current date as the initial identifier, followed by an underscore character and additional information, (e.g., Date [DDMMYY]_TEXT).

IMPORTANT! The duplicate Results Group name must be unique.

5. Click OK to save the duplicate ViroSeq Results Group.

1. Select GA Instruments > ga3130 or ga3130xl > Plate Manager.

2. Click New to open the New Plate dialog box.

3. Complete the information in the New Plate dialog box:a. Name: enter a name for the plate.

IMPORTANT! The new Plate Name must be unique.

Note: A useful way to name a Plate is to use the current date as the initial identifier, followed by an underscore character and additional information, (e.g., Date [DDMMYY]_TEXT).b. Description: enter a description for the plate (optional).c. Application: SequencingAnalysis.d. Plate Type: 96-Welle. Owner Name: enter an owner name. f. Operator Name: enter an operator name.g. Click OK. The SequencingAnalysis Plate Editor opens.


To Edit a Plate Record

To Prepare Purified Sequencing Reactions for Analysis on the 3130xl Genetic AnalyzerNote: For Instructions on preparing purified sequencing reactions for analysis on the 3130 Genetic Analyzer, refer to the next section.

To Prepare Purified Sequencing Reactions for Analysis on the 3130 Genetic Analyzer

4. Complete the information in the SequencingAnalysis Plate Editora. In the Sample Name column of a row, enter the appropriate sample

name, then click the next cell. Sample names are limited to a maximum of 32 characters.– When naming the samples, use letters, numbers, and the following

punctuation only:-_(){} # +. DO NOT USE SPACES.– The sample name must be identical for all seven primer sequences of

a sample.– The sample name and primer identification must be separated by two

consecutive underlines (Shift plus_).– The primer identification contains the primer name and any other

information unique to the specific sequence. For example: Patient196__A, Patient196__B, Patient196__C, Patient196__D, Patient196__F, Patient196__G, Patient196__H.

b. Priority: the value 100 automatically displays in the column.c. In the Results Group 1 column, select the ViroSeq Results Group

created specifically for the plate from the drop-down list. d. In the Instrument Protocol 1 column, select the

ViroSeq_Instrument_Protocol.e. In the Analysis Protocol 1 column, select the

ViroSeq_Analysis_Protocol.Note: Left-click in the column header and press Ctrl+D to fill down whenever a field is the same for all samples. f. Click OK.


2. Select the plate record to edit and click Edit.

3. Edit the parameters in the Plate Editor.

4. Click OK.

1. If your samples are frozen, bring them to room temperature.

IMPORTANT! This must be done immediately before loading the samples.

2. Resuspend your samples by adding 20 L of Hi-Di formamide to each sample well; use a new aliquot of Hi-Di formamide for each experiment.


Note: Samples resuspended in Hi-Di formamide are stable at room temperature for up to 35 hours. Do not leave these samples at room temperature for a longer period of time.

3. Vortex for 10 to 15 seconds.

4. Centrifuge the plate for 30 to 40 seconds at 2,000 g to collect the liquid at the bottom of the plate.

Note: Heat denaturation of the samples is not required.

5. Prepare and install the plate assembly.

1. If your samples are frozen, bring them to room temperature.


2. Resuspend your samples by adding 20 L of Hi-Di formamide to each sample well; use a new aliquot of Hi-Di formamide for each experiment.


Note: Samples resuspended in Hi-Di formamide are stable at room temperature for up to 35 hours. Do not leave these samples at room temperature for a longer period of time.





To Prepare a Plate Assembly

To Place the Plate onto the Autosampler

To Load Samples and Link a Plate to the Plate Record

To Unlink a Plate Record

5. IMPORTANT! Signal degradation may occur for samples left on the 3130 Genetic Analyzer for more than 14 runs (1 run = approximately 2.5 hours).

If the plate contains 7 or fewer columns of samples (including control), prepare and install the plate on the plate assembly as described in the section below. ORIf the plate contains more than 7 columns of samples, continue with step 6 below.

6. Transfer the remaining columns of samples from plate 1 to a new MicroAmp® Optical 96-Well Reaction Plate (plate 2).

Note: Additional plates may be purchased from Applied Biosystems. Refer to Required Materials Not Provided on page 4.

7. Transfer 10 µL of positive control (one-half of the volume from the sequencing setup) from plate 1 to plate 2. Perform this step for all 7 wells containing the positive control.

8. Prepare and install plate 1 on the plate assembly as described in the section below.

9. Seal plate 2 with aluminum foil tape and store at –15 to –25°C in thedark. Analyze the samples within one week.

Note: Load plate 2 after plate 1 is completed. Follow steps 10 to 13 below.

10. Bring plate 2 to room temperature.





13. Prepare and install plate 2 on the plate assembly as described in the section below.

1. Secure a clean and dry plate septa on the sample plate.

IMPORTANT! Never use warped plates.

IMPORTANT! Make sure the plate septa lies flat on the plate. The holes in the plate retainer must align with the holes in the septa or the capillary tips will be damaged.

2. Place the sample plate into the plate base.

3. Snap the plate retainer onto the plate and plate base.

4. Ensure that the plate retainer holes are aligned with the holes in the septa strip.

IMPORTANT! Damage to the array tips will occur if the plate retainer and septa strip holes do not align correctly.

1. Press the Tray button.

2. Place the plate assembly on the autosampler. Use position A or B for the 3130xl Genetic Analyzer or position B for the 3130 Genetic Analyzer.Position the plate with well A1 in the upper right corner, with the notched-end of the plate base away from you.

Note: The plate will only fit into the autosampler when properly positioned.

IMPORTANT! Ensure the plate assembly fits flat in the autosampler. Failure to do so may allow the capillary tips to lift the plate assembly off of the autosampler.

3. When the plate is correctly positioned, the plate position indicator on the Plate View page changes from gray to yellow.Verify that this has happened before proceeding.

4. Close the instrument doors.

Note: Closing the doors returns the autosampler to the home position, placing the tips of the capillaries in buffer.

1. Search for the plate record:Select GA Instruments > ga3130 or ga3130xl > instrument name > Run Scheduler > Plate View.There are two search options:• Find All • AdvancedUnder Find All:• Select Barcode in the Type of Search drop-down list. • If you have a limited number of plates in the database, click Find All. All

plates in the database will display in the plate record section.Under Advanced:a. Select Advanced in the Type of Search drop-down list.b. Use the drop-down list to define search conditions for a category or

multiple categories (Run Name, Results Group Name, Plate Name, etc.)

c. Use the Plate Name for the Plate ID category.d. For each category with a condition selected, type a value (primary search

string) in the Value 1 column.• Click Search. All plates in the database that match the search criteria

display in the plate record section.

2. Select the plate record, then click the plate position indicator that corresponds to the plate being linked. The plate position indicator changes from yellow to green when linked and the green Run Instrument button is active.

3. After a plate is linked, use the Run View window to verify that the runs are scheduled correctly. a. Select GA Instruments > ga3130 or ga3130xl > instrument name > Run

Scheduler > Run View.b. Select a row for any run. The corresponding wells to be injected for that

run are highlighted in the plate diagram.

IMPORTANT! If the well is not highlighted, it will not be injected.

1. Select GA Instruments > ga3130 or ga3130xl > instrument name > Run Scheduler > Plate View.

2. Select the plate record to be unlinked.

3. Click Unlink. The plate position indicator changes from green to yellow when unlinked.


To Start the Run

Do not use data in the process of collection if electrical power fails in the middle of a run.

To Monitor a Run

Monitor a Run in Progress

Monitor the System Messages from a Run in Progress

1. Verify that the active spectral calibration matches your dye set, capillary array length, and polymer type for all scheduled runs.a. Select GA Instruments > ga3130 or ga3130xl > instrument name >

Spectral Viewer.b. In the Dye Set drop-down list, select E-BigDyeV1.c. In the list of Calibrations for the Dye Set drop-down list, select a

spectral calibration.d. Click Set.

2. Click the green Run Instrument button in the toolbar.

IMPORTANT! Do not open the door of the genetic analyzer once the run has begun.

Do not simultaneously perform data analysis or use other software during the run.

3. The Processing Plates dialog box opens, then click OK.

4. The software automatically performs a run validation:• If the validation passes, the run will start.• If any of the validation test fails, the run will not start. Check the event

log for information.

1. Select GA Instruments > ga3130 or ga3130xl > instrument name > Instrument Status. Instrument Status window displays the real time instrument status, including the EP Voltage, EP Current, Laser Power, Laser Current and Oven Temperature.

2. The Event Messages lists:a. The instrument's recent actionsb. The status of each capillary as passed or failed at the end of a spectral

calibrationc. The calibration data at the end of a spatial calibrationd. Information for service engineers.

3. The Error Messages lists:a. Errors that have occurred during the current runb. Information for service engineers. A “fatal” error usually requires that

you restart the Data Collection software.

4. The EPT Chart displays the real time chart of EPT status.

5. System Status changes from green to flashing red when errors occur.

1. Select GA Instruments > ga3130 or ga3130xl > instrument name > Instrument Status > Event Log.

2. Clear error messages by clicking Clear Errors. The System Status light flashes red until all errors are cleared. Take corrective action based upon the error message.

3. The Event Log itemizes events such as errors and general information for all data collection steps. Clear Errors changes the System Status from red to green (ready state).


Use the Capillary Viewer to View Data During a Run

IMPORTANT! Always exit from the Cap/Array Viewer and the Capillaries Viewer windows. During a run, do not leave these pages open for extended periods. This may cause unrecoverable screen update problems. Leave the Instrument Status window open.

Use the Cap/Array Viewer to View Data During a Run

IMPORTANT! Always exit from the Cap/Array Viewer and the Capillaries Viewer windows. During a run, do not leave these pages open for extended periods. This may cause unrecoverable screen update problems. Leave the Instrument Status window open.

To View Raw Data from a Completed RunThere are two formats for viewing data within the Data Collection software under the Run History icon:• the Cap/Array Viewer window, and• the Capillary Viewer window (capillary-by-capillary).

1. Select GA Instruments > ga3130 or ga3130xl > instrument name > Capillaries Viewer. Use the Capillaries Viewer to examine the quality of the raw data during a run for several capillaries at once

2. Select the check boxes of the desired capillaries to be viewed. The capillaries are displayed in the order in which the boxes are checked.

3. Zoom in and out:a. Select a rectangular area over the area of interest by holding down the

mouse button. Release the mouse button to zoom-in.b. Click the Zoom-out button in the toolbar to return to full view.

1. Select GA Instruments > ga3130 or ga3130xl > instrument name > Cap/Array Viewer. View this window during a run in progress to examine the data quality, which is depicted as color data for the entire capillary array.

2. Zoom in and out:a. Select a rectangular area over the area of interest by holding down the

mouse button. Release the mouse button to zoom-in.b. Click the Zoom-out icon button in the toolbar to return to full view

1. Select GA Instruments > ga3130 or ga3130xl > Run History to select a run to view.

2. Search for a run using Barcode or Advanced search. After choosing a run, click the Capillary Viewer or the Array Viewer (under the Run History icon) from the left Viewer window.


To View Analyzed DataWhen a run is finished, the sample files are automatically extracted into a run folder, along with a run log, to a location defined in your preferences or the following default location:

E:\AppliedBiosystems\UDC\Data Collection\Data\ga3130xl\<run folder name>orE:\AppliedBiosystems\UDC\Data Collection\Data\ga3130\<run folder name>

If the data has been analyzed, the data is in the location defined in the following default location:

E:\AppliedBiosystems\UDC\Data Collection\Data\plate folder\ab1 files

Sequencing Analysis Software v5.3.1 with KB Basecaller v1.4

To Use the Sequencing Analysis Software v5.3.1 and Analyze the DataThe Sequencing Analysis Software v5.3.1 will be installed on the hard drive of the instrument computer workstation. This software is used to:• Review basecalled sequences• Reanalyze data to trim to 580

Launch the Sequencing Analysis Software

Set the Analysis Defaults

Create a ViroSeq Analysis Protocol

3. Note: EPT Viewer - The electrophoresis data of the run, such as voltage, current, and oven temperature profiles can be seen in the EPT viewer under Run History.

1. Start the Sequencing Analysis Software v5.3.1 by double-clicking the Sequencing Analysis v5.3.1 desktop shortcut or select Start > Programs > Applied Biosystems > Sequencing Analysis > Sequencing Analysis 5.3.1.

2. In the Login window, enter a valid User Name and password. Click OK.

Note: To check the software name and version number, from the main menu select Help > About Sequencing Analysis. Click OK to close the About dialog.

1. From the Main menu, select Analysis > Analysis Defaults. The Analysis Defaults window opens.

2. In the Analysis Defaults window, under Add Samples Settings, select Analysis Protocol as None. Check the Base Calling (BC) check box. Uncheck the Post Processing (PP) and Print (P) check boxes.

3. In the Analysis Defaults window, under Sequence File Formats, select “Use settings in the sample’s Analysis Protocol”. Click OK.

4. Click OK on the Warning Analysis Defaults dialog.

1. From the Main menu, select Analysis > Analysis Protocol Manager. The Analysis Protocol Manager - DataStore window opens.

2. Click New in the Analysis Protocol Manager - DataStore window and create the ViroSeq Analysis Protocol by clicking on the appropriate tabs as described in the following steps.

3. General tab:• Name: ViroSeqAnalysisProtocol_580Trim• Description: optional• Sequence File Formats: uncheck all check boxes.


Add Sample Files to the Sample Manager

Sample files may be added or removed from the Sample Manager window at any time, except for files that are currently being processed by the program. Refer to Additional Sample Manager Functions.

4. Basecalling tab:• Basecaller: KB.bcp• DyeSet / Primer: KB_3130_POP6_BDTv1.mob• Processed Data: True Profile• Ending Base: check the box and enter 580 in the Bases field.

• Quality Threshold: Do not assign N’s to Basecalls.

5. Mixed Bases tab:• Use Mixed Base Identification: uncheck the check box.

6. Clear Range tab:• Uncheck all check boxes,

7. Click OK to return to the Analysis Protocol Manager - Data Store window.

8. Click Done.

1. Click the Add Samples icon in the toolbar or select File > Add Sample(s).

2. Navigate to the location of the sample folders. The individual sample files are located within the sample folders.

3. To add files to be analyzed, choose one of the steps below:

4. When the desired sample files have been added, click OK in the Add Samples dialog box to close the dialog box.

To add … Then …

All samples within a single folder Select the folder, then click Add Selected Samples.

A single file Open the desired sample folder, select the file, then click Add Selected Samples.

Continuous multiple files Open the desired sample folder, use the Shift key to select sample files, then click Add Selected Samples.

Discontinuous multiple files Open the desired sample folder, use the Ctrl key to select samples, then click Add Selected Samples.

Continuous multiple sample folders Use the SHIFT key to select contiguous sample folders, then click Add Selected Samples.

Discontinuous multiple sample folders Use the Ctrl key to select sample folders, then click Add Selected Samples.


You can remove a sample file from the Sample Manager window at any timeexcept when the program is currently processing that file. You can also rearrange the order that the sample files appear in the Sample Manager window.

Analyze Sample Files Using the Sequencing Analysis Software with 580 Trim

Show the Sample Data

Review Sequencing Analysis DataIMPORTANT! Review the Sequencing Analysis data using the checklist below before proceeding. to the ViroSeq® HIV-1 Genotyping System Software analysis:

1. From the Main menu, select Analysis > Analysis Protocol Manager.

2. Select the ViroSeqAnalysisProtocol_580Trim. Click Apply to All Samples.

3. Click Done.

4. Verify the following settings for each sample:

5. Click the Start Analysis icon or select Analysis > Start Analysis.

6. After analysis, visually check:• that the Spacing, Peak 1 Location, Start Point and Stop Point have been

defined by the software, and• the color of the BC check box. The color indicates the analysis status.

The color status is cleared at the start of each new sample processing.

BC Check Box Box is checked

PP Check Box Box is not checked

P Check Box Box is not checked

Basecaller KB.bcp

DyeSet/Primer KB_3130_POP6_BDTv1.mob

Matrix File None

Spacing 0

Peak 1 Location 0

Start Point 0

Stop Point 0

Check Box Color Indication

Green The basecaller analyzed the sample file correctly.

Blue The basecaller analyzed the sample file successfully, but detected some anomalies that may or may not be serious. Review the error message, the sample score, and the data.

Yellow The basecaller analyzed the sample file successfully, but with poor quality data. Some anomalies that may or may not be serious. Review the error message, the sample score, and the data.

Red A software failure occurred. Check the software error messages.

1. Use the Show check box to show data for one or more samples files or double-click on the sample file name.

2. To show the data in the Sample Navigator view, click the Toggle Show Sample Navigator/Sample Manager icon in the toolbar.

There are seven views available in the Sample Manager or Sample Navigator panes:a. Annotationb. Sequencec. Featuresd. Electropherograme. Raw (Data)f. EPTg. Audit

To change the view, select the tab for the desired view.

Item Check for / Action

Review or Correct Ending Base

Verify that the Ending Base for a subset of sample electropherograms are at 580 bases. If not, be sure to select the ViroSeqAnalysisProtocol_580Trim and re-analyze data.

Review or Correct Start Point

Inspect a subset of samples to verify that the Start Point begins after the initial dye blob.If not, reset the Start Point value to begin after the initial dye blob in a sample or determine the average Start Point for a subset of samples. a. Enter the new start point value in the Peak 1 column.

Verify that the value in the Start Point column matches Peak 1.

Note: For a subset of samples, use Fill Down to enter values for all samples. b. Check the BC check box(es) for the sample(s).c. Click the Start Analysis icon or select Analysis >

Start Analysis.

View Description

Annotation Summary of the sample information written by the data collection and analysis software

Sequence The nucleotide (base) sequence text called for the data. Gray- colored sequence text represents the trimmed bases.The view is available only after basecalling is done.

Feature The features that were found in the sequence by the post processing (clear range).

Electropherogram A four-color picture of analyzed data with peaks representing the bases. The original bases, edited bases or complementary bases can be displayed.This is the default view that is displayed when a sample files are shown and is available only after basecalling is done.

Raw The raw data collected by the instrument.

EPT A plot of run voltage, current, power and temperature values.

Audit Information about modifications to the data (base change, deletion, inserting, change in analysis setting, sample name change). This window contains data only if the Audit Trail feature is activated in the Authentication and Audit tab of the Options dialog box.


Save the Analyzed DataSelect File > Save All Samples or click the Save All Samples icon from the toolbar to save the Analyzed sample files.

Additional Sample Manager Functions

To Remove a Single File From Sample Manager

To Remove Multiple Files From Sample Manager

To Print Sample Data

Users and Authentication Set-up

To Set-up New UsersIMPORTANT! Only Administrators can set up and change information in the Users, Authentication, and Audit tabs.

Sample names Verify sample file names are correct. Use the same sample name for all samples of a project. Sample file names:• Must be less than 59 characters• Should include any information needed to identify a

sample.• Must have two consecutive underscores to separate

the sample name and the sequence primer. The ViroSeq software requires the double underscore to assemble and create projects.

• IMPORTANT! The information to the left of the double underscore must be identical for all six or seven sequences of a sample. The information to the right of the double underscore is unique to the individual sequence and should include the primer letter.

DyeSet/Primer file KB_3130_POP6_BDTv1.mob

BaseCaller KB.bcp

Sufficient signal strength

Review the signal strength numbers for each base.If the total A, C, G, and T signal strength value is below 400, inspect the raw and analyzed data and discard any noisy data.

Sequence Segments IMPORTANT! Six of the seven segments must be successfully sequenced in order to proceed with the analysis in the ViroSeq software. One of the missing segments can be A or D.

1. Click on the sample row number.The entire row will be highlighted.

2. Click Remove Samples icon or select File > Remove Samples or use the Delete key. The Sequencing Analysis software removes the selected file from the list.

1. To remove all files, select File > Remove All Samples from the Main menu.

2. To remove several adjacent files:a. Click on the sample row number of the first file in the group.b. Hold down the Shift key and click on the sample row number of the last

file in the group.c. Click the Remove Samples icon or select File > Remove Samples or use

the Delete key.

3. To remove multiple files that are not next to each other:a. Hold down the Ctrl key while clicking on the sample row number of

each file to be removed.b. Click the Remove Samples icon or select File > Remove Samples or use

the Delete key.

Item Check for / Action

1. To temporarily change the page orientation, paper type, panels/page, and so forth, select File > Page Setup to open a special Page Setup dialog box.

2. Adjust the settings as needed. Then click OK to close the dialog box.

3. In the row number column, select one or more samples to print.

4. Select File > Print to open the Print dialog box.

5. Select the appropriate options for printing. Click Print to close the Print dialog box and open the standard Print dialog box for the printer.

6. Make any required changes in the Print dialog box, and then click Print to start printing.

1. Select Tools >Options to open the Options dialog box.

2. Select the Users tab, then click New.

3. Fill-in the appropriate user name, password, first and last names, then select the level of user from the User Group drop-down list.

4. Admin - has complete access to manage User accounts and Audit set up, create/edit Analysis Protocol and Settings, Reports, Sample Manager, and Sequencing Analysis.Scientist - has access to create/edit Analysis Protocol and Settings, Reports, Sample Manager, and Sequencing Analysis.Analyst - has access to Reports, Sample Manager, and Sequencing Analysis.


Section 4: ViroSeq® HIV-1 Genotyping System Software v2.8 Analysis

IntroductionViroSeq® software processes the six or seven primer sequence files that correspond to a single plasma sample to generate a project. A project is an assembly of the sample files containing all the sequencing information required to produce a genotype. The project format supports manual review and editing of the electropherogram data to generate a final consensus sequence for the HIV-1 protease and reverse transcriptase (RT) genes.

IMPORTANT! For information about known limitations and anomalies in the ViroSeq® HIV-1 Genotyping System Software v2.8, refer to the Release Notes included with the ViroSeq Software v2.8 CD.

Terminology Used in This Section

Materials Required

Computer Requirements for Running ViroSeq® Software v2.8The following requirements must be met to run ViroSeq software on an offline, stand-alone computer system. The software cannot be installed on Windows NT.• Java™ Runtime Environment (JRE)

Microsoft® Windows® 2000 - JRE version 1.4.2_10, or

Microsoft® Windows® XP - JRE version 1.4.2_10.

Note: JRE is included on the ViroSeq software installation CD and is automatically installed with the ViroSeq software.

Installing, Uninstalling and Using the SoftwareBefore installing or uninstalling ViroSeq® software v2.8, you must log on to the Windows system using an account with Administrator privileges. Do not attempt to install or uninstall ViroSeq software using a Standard User or Restricted User account.

To Install the Software On a Windows 2000 or XP Computer

Administrative privileges are required for the installation to run properly.

Term Definition

ASCII American Standard Code for Information Interchange. A simple text format that is recognizable across many platforms and computers.

Basecall The nucleotide identity assigned to a signal peak in an electropherogram.

Consensus Sequence A sequence developed from several other aligned sequences.

Edit Cursor A graphical element that highlights a single nucleotide in the consensus sequence and that is the focus for an edit operation.

Electropherogram The processed signals of the fluorescence of sequencing dyes computed by the collection and analysis software provided with sequencing instrumentation.

FASTA An ASCII-formatted text file that contains file name information and a sequence.

IUB International Union of Biochemists

Java A programming language designed to generate applications that can run on different computer operating systems without modification.

Navigation Bar A graphical element showing POIs and providing a means to navigate to the corresponding data.

POI Position of Interest. A nucleotide position that warrants inspection by the user.

Primer Identification The matching of a sequence to a known primer.

Project The software entity that comprises the data, editing history, trim positions, assembled sequences, and so forth, associated with processing the sequence segments from a single HIV-1 sample.

Project File The primary file created and used by the ViroSeq software. It contains all the data necessary to produce a genotype analysis.

Reference Sequence The fixed sequence against which all comparisons are made. The ViroSeq reference sequence is based on HXB-2 (GenBank K03455).

Sample File A file containing the sample sequence data, generated in the Sequencing Analysis Software.

Sequence A string of nucleotide bases.

Sequence Segment The sequence data resulting from one primer.

Trimming Marking data for exclusion from use in deriving the consensus sequence.

VM Virtual Machine. The Windows Java™ interpreter that allows the computer to understand the Java language.

XML Extensible Markup Language. A flexible text format used in exchange of data on the Web and elsewhere.

Description

ViroSeq® HIV-1 Genotyping System Software v2.8 4J94-22

Configuration 1 Configuration 2 Configuration 3

Operating system requirements: Operating system requirements: Operating system requirements:

• Microsoft® Windows® 2000, service pack 4

• Microsoft® Windows® XP, service pack 2

• Microsoft® Windows® XP, service pack 2

Computer Platform: Computer Platform: Computer Platform:

• Intel® Pentium® IV (Single Core)

• Intel® Pentium® M (Single Core)

• Intel® Core™ 2 Duo (Dual Core)

• 2.0 GHz or above • 1.86 GHz or above • 2.4 GHz or above

• 1 GB RAM or above • 512 MB RAM or above • 2 GB or above

• 40 GB hard drive or above • 40 GB hard drive or above • 40 GB hard drive or above

• Keyboard, mouse • Keyboard, mouse • Keyboard, mouse

• Monitor supporting screen resolution of 1280 x 1024



1. Close all other desktop applications.

2. Insert the ViroSeq software v2.8 CD into the CD drive.

3. The installer should start automatically. If it does not, perform the following actions:a. Double-click My Computer on the desktop.b. Using the right-mouse button, select the drive that contains the CD.c. Select Explore.d. In Windows Explorer, open the ViroSeq software folder.

Double-click Setup.exe.

4. Accept the terms of the License Agreement.


To Uninstall the Software On a Windows 2000 or XP Computer

The software can be easily removed from the computer using the Uninstall program. The uninstaller will remove all program files and the desktop icon, but it will not remove any user-modified files from the system.

To Start the Software

There are three ways to start the software:

To Log Into the Software

For security purposes, the software requires each user to log on to the software. The User Login dialog displays when the software is launched. Enter a valid user name and password, then click OK. The user name as well as the system date and time are recorded in the History Window. Refer to Software Error Messages.If logging in for the first time, the default user name is Admin and the password is hiv1.

To Change the Administrator Password

To Add New Users

Only trained operators are allowed to use the ViroSeq software.

To Remove a User

To Change User Passwords

The Administrator may change user passwords in the Administrator Access dialog window after selecting Edit > Change Passwords. Non-administrators may change user passwords in the User Access dialog after selecting Edit > Change Passwords. When the User Access dialog appears, enter the Current Password, New Password, and Re-Enter Password fields, then click OK.The following rules apply to user names and passwords:• User names must be unique. They are not case sensitive.• Spaces are allowed in the user name and password.• Passwords are case sensitive.• Passwords cannot be blank.• Passwords must be at least 6 characters long, but not longer than 14 characters.• Passwords are encrypted as asterisks when displayed on screen.• Passwords can be made up of letters, numbers, and special (printable) characters.• The default password hiv1 cannot be used when adding a new user.

5. Follow the instructions on the installer screen.

Note: The software is installed in the C: directory. A different directory may be selected, if desired. The ViroSeq v2.8 icon is automatically installed on the desktop.

1. From the Windows Start menu, select Start > Programs >ViroSeq v2.8 > Uninstall.

2. The InstallShield Wizard dialog is displayed.

3. Click Remove.

4. Click Next.

5. A confirmation dialog is displayed.

6. Click Yes to remove the application.

7. The uninstall process will complete automatically, and then display the Maintenance Complete dialog window.

1. Double-click the ViroSeq v2.8 desktop icon.

WARNING! Do not open more than one instance of the ViroSeq software v2.8 application concurrently.

2. Launch ViroSeq software from the Start menu.a. Click the Start menu.b. Select Programs > ViroSeq v2.8 > ViroSeq v2.8.

3. Launch the software from C:\ViroSeq v2.8.a. Click the C:\ViroSeq v2.8 directory.b. Double-click the ViroSeq icon.

1. Type your location name into the Installation Site box.

Note: Abbreviations and numerical identifications are acceptable.

2. For Default Password, type hiv1.

3. Type your password in the New Password box and in the Re-Enter Password box.

Note: Your password is case sensitive and must be at least six but not more than 14 characters long.

4. Click OK.

1. In the Navigation Window, select Edit > Change Passwords.

Note: A ViroSeq project must be opened in order for the Navigation Window to appear.

2. In the User Information panel, type the new user name you want to add into the User Name box.

3. In the Employee ID box, type an employee identification name or number.

4. Type a password in the New Password box and in the Re-Enter Password box.

5. Click Add User.The new user is entered in the Registered Users panel.

6. You can continue adding users as needed, then click OK when you have finished.

1. In the Navigation Window, select Edit > Change Passwords.The Administrator Access dialog box opens.

2. In the Registered Users panel, select the name you want to remove.The Remove button becomes enabled.

3. Click Remove to delete the selected user, then click OK.


To Verify the Software Installation

Verify that the ViroSeq HIV-1 Genotyping System Software v2.8 was installed correctly by analyzing the QA10 demonstration sample files (.ab1) located in the C:\ViroSeq v2.8 directory.

Verify that the resulting drug resistance mutations match the information in the table below:

Project Overview

Main Steps in a Project

To Create a New Project

ViroSeq software displays existing, assembled projects in the Project Status window. View the Project Status window by going to the Navigation Window and selecting File > Open Project. The Status column indicates whether or not the software found all the input segments.

To Open an Existing Project

IMPORTANT! Do not open and edit the same project file in more than one instance of the ViroSeq software. Editing the same data segments in multiple instances of ViroSeq software at the same time will result in unexpected software behavior.

The Navigation Window

After assembling a project, ViroSeq software graphically presents the consensus sequence in the Navigation window. The Navigation window has two parts:• Navigation bar• Segment alignment section

1. Click New in the Project Status window. The Open dialog box opens.

2. Navigate to C:\ViroSeq v2.8 > Projects > Demo > QA10 folder.

3. Select one sequence data file and click Open.The software assembles and aligns the data to generate a consensus sequence using .ab1 files found in the QA10 folder. The Navigation and View*Edit windows open, displaying the completed assembled project for QA10.

4. Select File > Generate Resistance Report to preview the Drug Resistance Mutations listed on page 1 of the Antiretroviral Drug Resistance report.

QA10 RT Protease

ResistanceMutations

M41LA62VT69insV118IM184VT215Y

L10IL24IM46IF53LI54VA71VV82A

DNA Sequencing Data Files Check for:• Correct sequence quality• Defined boundaries• Correct sample names• Appropriate DyeSet/Primer file

Create a New Project • Find and select sequencing files• Assemble the project

Review Project Data Verify that:• A sufficient number of sequencing files are assembled• Sample names are correct• Sequencing files are aligned in the correct order and

orientation

Edit Data • Enter patient, lab, and sample-specific information• Distinguish peaks and noise• Trim segment ends• Verify and reconcile mixed basecalls and mismatched

bases• Compare with reference sequence

Prepare the Resistance Report • Print or save it in XML format• Save the FASTA file

1. Launch ViroSeq software and click New in the Project Status window.The Open dialog box is displayed.If ViroSeq software is already open, select File > New Project.

2. Navigate to the folder containing your Sequencing Analysis software files and open it.

3. Select one sequence data file and click Open. The software assembles and aligns the primer sequence files from a sample to generate a consensus sequence and creates a project.A Progress window opens while the software is assembling the project.

Note: The software creates separate projects for each unique sample name given to the data in the Sequencing Analysis software. There is a limit to the number of projects that can be assembled at one time. A conservative limit is 14 projects.When complete, the Progress window closes and the Navigation and View*Edit windows open, displaying one of your assembled projects.

If the Status is . . . Then . . .

OK All of the expected segments were found and the project assembled successfully.

? ViroSeq software encountered assembly issues.Possible causes include:• One or more segments could not be identified• Fewer than 6 segments were assembled

Note: Do not proceed if fewer than six segments are assembled into the project. One of the missing segments can be A or D.

1. In the Project Status window, select the project you want.If the project you want is not in the Project Status window:a. Click Find to open the Open dialog box.b. Navigate to the folder where the project is stored.c. Select the project you want.

2. Click Open. The Navigation and View*Edit windows for the project open.


About the Navigation BarThe Navigation bar is in the top portion of the Navigation window. It provides an overview of the number and locations of positions of interest (POIs). These appear as vertical lines in a gray bar.

Numbers on the horizontal scale represent codon positions for each gene. POIs are color coded as shown in the following table.

About the Segment Alignment SectionThe segment alignment section is displayed in the lower part of the Navigation window. This portion graphically shows how the sequence segments overlap.

The label on each segment consists of the project name and the primer that was used. The segments point in the direction of polymerization.Verify that the sequences are positioned as expected.• Forward direction - primers A, D, B, C• Reverse direction - primers F, G, H

In rare cases, segment D may be aligned at the wrong location. After assembling the project, check the position of segment D. It should completely cover the protease gene and the beginning of the RT gene. If segment D is not positioned as specified, please assemble the project again without segment D.

About the View*Edit Window

The View*Edit window displays the consensus sequence generated by the software. It also contains navigation and editing tools.

Color Coding for Positions of Interest (POIs)

Color Meaning

Gray = background color No difference relative to the reference

Red Selected POIs

Black Mismatches between segments

Green Known or unknown difference between reference and consensus sequence

Blue Multibase position

Yellow Shows areas covered by only one segment during assembly

White User-confirmed basecall

Note: You must save your project first.

Parts of the View*Edit Window and Their Descriptions

Item Name Description

1 Jump left button Jumps to the leftmost POI.

2 Reverse jump button Jumps to the POI to the left of the active POI.

3 Trim button Trims and untrims the selected end of a segment in the electropherogram.

4 Revert button Reverts the current POI to its original base assignment.

5 Editing palette Edits the active POI.Buttons represent letter codes for bases and mixes of bases.

6 Auto jump on edits check box

When checked, causes an automatic jump to the next POI after each edit.

7 Reverse jump check box

Used after editing the current position, jumps to the next POI to the left.This box is only active when the Auto jump on edits check box is selected.

8 Navigation Options button

Opens the Navigation Options dialog box. In this dialog box you select the active POIs.Active POIs are those POIs visited by the jump buttons.

9 Forward jump button Jumps to the POI to the right of the active POI.

10 Jump right button Jumps to the rightmost POI.

11 Labels panel Labels each line in the sequence panel. Also gives the name of primer and direction of polymerization for each sequence.

12 Codon number Gives the codon number for the reference sequence.

13 Reference Translation Gives the one-letter code for the amino acid translation of the code for the reference sequence.

14 Consensus Translation Gives the one-letter code for the amino acid translation of the code for the consensus sequence.

15 Reference Sequence Bases of the reference sequence.


Editing Palette

File Menu

16 Consensus Sequence Bases of the consensus sequence.

17 Box around base Current POI.

18 Sequence peaks Electropherogram for a sequence segment. ViroSeq software shows all sequence segments that overlap at a position.

19 Scroll bar Horizontal scroll bar for the sequence panel.

20 Position information box

Explains why a position has been identified as a POI.

21 Close View Edit Closes the View Edit window.

IUB Codes

IUB Codes

A = adenosine S = G or C (Strong—3 H bonds)

C = cytidine W = A or T (Weak—2 H bonds)

G = guanosine Y = C or T (pYrimidine)

T = thymidine B = C, G, or T

U = uracil D = A, G, or T

K = G or T (Keto) H = A, C, or T

M = A or C (aMino) V = A, C, or G

R = A or G (puRine) N = aNy base

Amino Acid Codes

Amino Acid Three-Letter Code One-Letter Code

Alanine Ala A

Arginine Arg R

Asparagine Asn N

Aspartate or Aspartic acid Asp D

Cysteine Cys C

Glutamine Gln Q

Glutamate or Glutamic acid Glu E

Glycine Gly G

Histidine His H

Isoleucine Ile I

Leucine Leu L

Parts of the View*Edit Window and Their Descriptions (continued)

Item Name Description

Lysine Lys K

Methionine Met M

Phenylalanine Phe F

Proline Pro P

Serine Ser S

Threonine Thr T

Tryptophan Trp W

Tyrosine Tyr Y

Valine Val V

File Menu Options

Menu Item Keyboard Shortcut Description

New Project Ctrl+N The Open dialog is displayed.

By clicking the New button you can select and open a Sequencing Analysis file in a folder and the software assembles all sequences in that folder into new projects.

Open Project Ctrl+O The Project Status window is displayed.

Select a project from the list, or by clicking the Find button, you can select and open a ViroSeq project that has already been assembled.

Save Ctrl+S Saves the open project with any edits that you have made.

Save consensus [FASTA] Ctrl+F Stores the consensus sequence in an ASCII text file with the extension .fasta.

Generate Resistance Report Ctrl+G Allows you to view and print the Resistance Report.

Export Resistance Report Ctrl+X Allows you to export the Resistance Report.

Quit Ctrl+Q Exits the ViroSeq software.

Amino Acid Codes

Amino Acid Three-Letter Code One-Letter Code


Edit Menu

Window Menu

Help Menu

Parts of an Assembled Project

The assembled project consists of: • Electropherograms for the sequence segments

ViroSeq software represents bases as peaks with a different color for each of the four bases.

• Consensus sequence of the sequence segmentsWith seven different primers, sequence segments overlap and cover each section of the targeted gene at least twice and in both directions. (The exceptions are RT codons at approximately 315-335.)

• Reference sequenceViroSeq software compares the consensus sequence to a reference sequence, HXB-2.

• Consensus and reference translationsViroSeq software shows the amino acid sequence in the reference virus strain and all possible amino acid translations of the consensus sequence.

• Codon numberViroSeq software numbers codons based on the reference sequence.

Review the Assembled Project

Once ViroSeq software has assembled a project, you must review and verify basecalls at POIs in the consensus sequence. You can accept or change the basecalls where the software detects differences between the consensus sequence and the reference sequence. You decide whether the suggested basecall is justified by looking at the electropherograms. For example, dye blobs at the beginning of a sequence or several repeats of the same base can lead the software to misjudge the sequence and to suggest a variation where there is none.

POIs

A POI is a base in the consensus sequence that is one or more of the following:• Different from the HIV-1 reference strain• Found in the internal table of known resistance positions• Identified as mixed base in at least one segment at that position• An insertion relative to the reference sequence• Different between segmentsNote: One or more POI criteria may apply to a given consensus position.

Base Color Codes

To Set Navigation Options

The Navigation Options window opens when you click the Navigation Options button in the View*Edit window.

Edit Menu Options


Toggle Position Cursor Ctrl+T Turns the positioning line of the cursor on and off.

Edit Report Information Ctrl+E Opens the Edit Report Information dialog box, where you enter the patient, laboratory, and technician information for your final report header.

Change Passwords — When logged in as administrator, opens the Administrator Access dialog box, where you can change passwords and perform other administrative functions.

When logged in as a user, opens the User Access dialog, in which you can change your password.

Login As New User — Opens the User Login window.

Window Menu Options


History — Opens the History log.

Help Menu Options


About ViroSeq — Displays product information.

Base Color

Adenine Green

Cytosine Blue

Guanine Black

Thymine Red

Mixed bases Pink


Navigation Options Explained

To Confirm or Edit the POIFor each position of interest (POI) you select in the Navigation Options, you must manually confirm or edit the position.The cursor in the View*Edit window is a box around the letter code of a base or POI in the consensus sequence. You can either confirm or edit any mismatches between overlapping segments or mutations that have been called by the software.

To Distinguish Peaks from Noise

A peak is an electrophoretic signal that has a definable maximum and clearly resolved slopes on either side. Peaks represent the signal strength of the fluorescent dye bound to each DNA sequence fragment. Peaks are read and analyzed by the Data Collection and Sequencing Analysis programs.

Note: Shoulders on peaks are defined as a peak only if they also have a definable maximum.Noise is the background signal present in an electropherogram. It can be caused by the instrument or by the quality of the capillary array, polymer, sample, buffer, or Hi-Di formamide.Mixture peaks should be distinct from noise peaks. However, when high levels of continuous noise are present in a sequence, real data can be obscured and smaller peaks cannot be distinguished from noise.

Editing Sequence of Events

• Trim segment ends to remove poor quality sequence segments.• Verify and reconcile mixed basecalls and mismatched bases.• Compare areas with mobility problems with other sequence segments and with

the reference sequence to determine the correct peak position.

To Edit a Project

Reasons for Using Editing GuidelinesIMPORTANT! Operator to operator consistency in the manner of basecalling is essential. The sequence editing guidelines provide you with a method for achieving that consistency.

In most cases, the ViroSeq software accurately identifies mixtures. In those cases where you need to make a determination, use these guidelines to distinguish real mixtures.

IMPORTANT! The quality of the sequence data determines the accuracy of basecalls. The lower the background or noise, the fewer the number of edits you will have to make.

Summary of the Editing Guidelines for Identifying MixturesYou can call a mixture if:

1. Two opposite sense sequence segments contain secondary peaks clearly above the local noise (approximately double the noise level).

2. One sequence segment contains a secondary peak at least 30% of the primary peak and three times the local noise level.Take note of resistance mutations in regions of single coverage – only editing guideline #2 can be used to identify a resistance mutation.

3. One sequence segment contains a secondary peak at least 30% of the primary peak and the segment in the opposite direction confirms the mixture.

Option Description

Global Settings

Additional variants from reference Locates mutations that are not used in the ViroSeq software algorithm.

Multibase positions Allows you to go to the consensus sequence positions that are derived from segments that have mixtures, or more than one nucleotide, at a position.

Mismatches between segments Locates differences in a consensus sequence position derived from forward or reverse primer segments that do not agree at that position.

Show saved edits Locates bases that were manually edited and saved in the project.

Insertions Locates extra bases that are not part of the reference sequence.

Note: For information on how the software handles insertions, please refer to Insertion Mutations.

Features from Gene Profile

Show Resistance positions Makes every mutation used in the drug resistance algorithm a POI, whether or not the consensus sequence differs from the reference sequence.

Note: This can only happen if you uncheck the Only show if variant box.

Only show if variant Checking this box, in addition to the Show Resistance positions box, selects only those drug resistance codons in the consensus sequence for which mutations are detected that have a role in determining drug resistance.This feature may be used when creating new projects. Use with saved or edited projects is not recommended.

Poorly-Resolved PeaksWell-Resolved Peaks

1. Click Navigation Options to open the Navigation Options window.

2. Make sure all the check boxes for the options in the Navigation Options window are selected.

3. Select and trim areas of poor sequence quality to improve basecalling.On the Navigation bar, dense groupings of vertical bars represent areas where poor sequence quality of one or more primers may exist.

4. Confirm or edit the basecalls.a. Begin at the leftmost POI in the protease gene by clicking on the

button to edit or confirm the basecall.b. Press the spacebar to confirm your selection.c. Proceed to the next POI by pressing to edit or confirm the basecall.

Repeat until you have edited or confirmed all the POIs in the consensus sequence.

Note: If overlapping sequences do not agree in the majority of positions, investigate for possible sample mix-up or naming errors.

5. In the Navigation Options window, clear all the check boxes except Show Resistance positions and click OK.

6. Repeat the edit/confirm procedure starting at the far left and moving to the right.

Note: You can use editing guideline 1 in the following summary to identify mixtures that the software may have missed.


Hierarchy for Project Editing

1. For a first pass of the data, select all navigation features. This allows you to stop at all positions where a mixture has been called. This occurs because at least one of the sequences shows a 30% mixture.

2. Evaluate if the 30% mixture is real (that is, above the noise level).

3. If the 30% criteria are met, check if the opposite sequence shows a confirming peak (Guideline 3). If the confirming peak is present, call the mixture.

4. If the opposite strand does not show a confirming peak, determine if the 30% peak (in the original strand) is 3 times the background. If the 3 times the background criterion is met, call the mixture (Guideline 2). This is the rule for a mixture in one direction only.

5. After you have made one pass through the data, the navigation options should be changed to stop only at the resistance positions. Remember — you must deselect the Only show if variant feature.

6. Make a second pass through all the data, looking for mixtures which meet Guideline 1 (2 background in both directions). At this time, also review the edits and automatic calls made in the other resistance positions.

Examples

Example of Good Sequence DataThe following example shows very low background and secondary peaks.

Example of Poor Sequence DataThe following example shows very high background, and secondary peaks cannot be confidently called. False mixtures can be called due to background noise. This sample should be repeated.

Example of Diminished PeaksDiminished peaks, as seen in the example below, may appear across the entire sequence segment or may appear in limited regions within a sequence segment. These peaks are primarily caused by the presence of dye blobs at the beginning, middle or end of a sequence segment. Less frequently, the diminished peaks are caused by low signal intensity. If diminished peaks are evident, refresh the View*Edit window or trim the dye blobs that appear at the beginning of the sequence segment using Sequencing Analysis (refer to the Review Sequencing Analysis Data section). If the trimming does not resolve this observation, then re-inject or repeat Cycle Sequencing.

Examples of Guideline 1

Pass

Guideline 1: Two opposite sense sequence segments contain secondary peaks clearly above the local noise (approximately double the noise level).

The ViroSeq® software automatically identifies mixtures at 30% of the primary (larger) peak. In some situations, you see mixtures that are not called by the software because they are below 30%.

To edit a basecall in this situation (second position of codon 20), follow Guideline 1 to accurately and consistently make the correct basecall. The correct basecall in this example is W.


Fail

Below is an example of a mixture peak that is present in both directions, but cannot be called because it is the same size as the surrounding background noise peaks. The peaks themselves are noise.


Pass

Guideline 2: One sequence segment contains a secondary peak at least 30% of the primary peak and three times the local noise level.In rare situations a mixture sequence is seen in only one direction. To call this mixture, the single segment mixture peak should be well defined and unambiguous. The ViroSeq software identifies this type of mixture as “. . .Mismatch,. . .Mixture”.

The example above shows a mixture where one sequence segment contains a secondary peak above 30%, and the other sequence segment shows no mixture (it does not have a resolvable maximum). The mixture present in the top sequence is at least 30% of the primary peak, and significantly greater than background.

Fail

Below is an example of a mixture (first position of codon 32) that cannot be called. This is a false mixture that is caused by a dye blob at the start of primer B.


Pass

Guideline 3: One sequence segment contains a secondary peak at least 30% of the primary peak and the segment in the opposite direction confirms the mixture.

The ViroSeq™ HIV-1 Genotyping System sequence primers provide double coverage for the full length of the Protease and up to approximately codon 315 of the RT gene, and single coverage from approximately codon 315 to 335. The mixture is seen in both the forward and reverse directions, making mixture calls more reliable. In many cases, the mixture is greater than 30% of the primary peak in one direction and less than 30% in the other. The lower sequence (the segment in the opposite direction) is used to confirm the presence of the mixture.


Fail

Below is an example of a false mixture (third position of codon 254) that is caused by the high continuous background noise. The correct basecall is C.

Exceptions to the GuidelinesThe examples below show anomalous positions at RT151 and RT215 that do not follow the editing guidelines. When there are mixtures at these positions, they frequently show a pattern like that illustrated below.Compare these examples with your sample. If the patterns match then a mixture can be called for these positions.

Editing Data

About Trimming with ViroSeq® Software

Sequences in the beginning or the end of a segment are often of poor quality. ViroSeq software automatically trims these sequences. The software also allows for manual trimming.The trimmed part of a sequence is still visible as part of an electropherogram but it appears on a gray background on the screen. ViroSeq software ignores differences between trimmed sequences and the reference sequence when determining the consensus sequence. However, you can still use this information (in the grayed area) when making your basecall.

Example of Trimming

Before Trimming

Trimmedsequence


After Trimming

To Trim Segment Ends Manually

Carefully review areas with insertions. These interpretations affect the results in the Antiretroviral Drug Resistance Report.

To Find POIs There are four methods for moving the edit cursor to a new position:• Click the Forward ( ) or Reverse ( ) jump button to move to the next

POI as specified in the Navigation Options.• Use the scroll bar at the bottom of the View*Edit window to move through the

sequence. The edit cursor stays in the center of the window.• Use the left or right arrow keys on the keyboard.• Click once on the left or right side of the consensus sequence to move the Edit

window frame. The base where you click becomes the center POI.

To Edit a Basecall

To Edit a Mutation

To Confirm a Basecall

To Revert to the Original Basecall

To Save Your EditsTo save your edits go to the File menu and select Save.The Progress window opens and indicates when the file has been saved. An entry is made in the History window, and the file is saved in the Projects\Completed folder within the ViroSeq software directory.• You can save your consensus sequence in an ASCII text format (FASTA format)

by selecting File > Save consensus [FASTA]The file is saved in the directory C:\ViroSeq v2.8\Projects\Completed\Reports.

• The Antiretroviral Drug Resistance Report can be saved in XML format by selecting File > Export Resistance Report.The file is saved in the directory C:\ViroSeq v2.8\Projects\Completed\Export.

Note: Do not edit or modify the results after an XML version of the resistance report is saved.

1. In the View*Edit window, select the region of poor quality by double-clicking this portion in the electropherogram. The region from the point where you double-click to the closest end becomes highlighted in black.

2. Click Trim.The selected sequence is trimmed and now appears on a gray background.

Note: Trimming only excludes the use of trimmed data when making consensus basecalls. The software does not exclude trimmed data from use when it is assembling data. Therefore, if noisy data is causing poor assembly or alignment results, it must be trimmed using the Sequence Analysis software.

3. To untrim a segment:a. Select a smaller region of data and double-click to highlight it in black.b. Click Trim.

1. Make the edit.a. Click the button in the editing palette that corresponds to the type of

mutation you are editing or enter the appropriate base call letter (IUB code) for the POI using the keyboard stroke.

b. Press the spacebar to confirm the edit.

2. Follow the editing guidelines for mixtures.

1. Two opposite sense sequence segments contain secondary peaks clearly above the local noise (approximately double the noise level).

2. One sequence segment contains a secondary peak at 30% of the primary peak and three times the local noise level.Take note of resistance mutations in regions of single coverage – only editing guideline #2 can be used to identify a resistance mutation.

3. One sequence segment contains a secondary peak at 30% of the primary peak and the segment in the opposite direction confirms the mixture.

IMPORTANT! Do not use sequences with high levels of continuous noise.

1. Confirm a basecall by pressing the spacebar on the keyboard.

2. To remove the confirmation, press the spacebar again.

1. Select the basecall you want to change.

2. Click Revert.The edit reverts to the original basecall made by the software.


To Review the Project in the History WindowThe History window shows a permanent record of added, rejected, and trimmed segments, as well as edited basecalls.

To View the History Window

Note: If you make edits to one base and save multiple times, the final edit is recorded correctly in History.

Insertion Mutations

In rare cases, insertions can result in an inaccurate Drug Resistance Report.

1. ViroSeq software is not designed to resolve cases for which there are two dominant species of virus and one has no insertions while the other does. This situation is easy to detect using ViroSeq and is covered below.

2. Using gene sequencing to genotype viruses with insertions may yield non-unique solutions for the location of insertions and the existence of mutations in the immediate vicinity of an insertion. This limitation is illustrated and discussed below.

3. In samples where an insertion is present, at least two primer sequences are required for correct base calling of the insertion. Ensure that the area with the insertion has at least two primer sequences that are untrimmed. If a single primer sequence is available in the area of the insertion, inaccurate results will be generated.

The ViroSeq software can detect the insertion mutations when the mutation is present in a homogeneous population. However, the ViroSeq software cannot interpret a sample with a mixture of two or more virus populations where one population contains an insertion and the other does not. This limitation exists with all sequencing methodologies. Insertion mutations in a mixture result in sequence shifts that are different for each virus population, causing the basecalls downstream of the insertion to be seen as mixtures. This phenomenon is plainly visible in the ViroSeq software as a series of strong mixtures beginning at the end of the insertion and continuing until the end of the sample sequence for that primer.In the example below, Virus 2 has a three-base insertion that offsets the alignment of the two mixture sequences.

The high incidence of mutations in the HIV-1 genome creates ambiguity with determining the locations of insertions. This ambiguity can make it difficult to determine whether or not a certain resistance mutation exists. A prime example is the insertion that occurs in the vicinity of codon 69. There are three important resistance mutations in this vicinity:• D67N• T69D• K70RConsider the following examples that show two possible interpretations of the insertion.

Example 1

Example 1 shows a case where the placement of the insertion results in K70T.

Example 2

Example 2 shows a case where the placement of the insertion results in K70R.Both placements are equally probable and valid because both have the same number of differences between the reference and consensus sequences.The insertion at codon 69 occurs at a very low frequency, less than 1%, in patientson prolonged therapy.25 A recent report indicates that the investigators were unable to detect mixtures (wild type and mutant) in patients with virus containingthe codon 69 insertion.26 This result suggests that the insertion mutation replaced the wild type rapidly, and mixtures, if any, would be present at extremely low frequencies.

Antiretroviral Drug Resistance Report

To Create the Antiretroviral Drug Resistance Report

You can include laboratory and sample-specific information into a project by using the Edit Report Information menu item. The laboratory-specific information can be saved as a default and printed in every Antiretroviral Drug Resistance Report. It is consistent with every user in your laboratory.Technician, patient and sample information that is unique to each sample can be entered for every project using the Edit Report Information menu item.• The special character, double quotation marks (“), is not supported for entry into

the resistance report header. Avoid using this in any of the Edit Report Information fields.

• In the Comments field, you can enter a maximum of five lines and up to 256 characters, including spaces. If you enter text that exceeds this limitation, it may not be displayed on the report.

• In the Comments field, do not use the Enter key, as this will result in loss of information.

1. From the Window menu, select History.The History window opens.

2. Scroll through the log using the vertical scroll bar on the right to view your activities.

Consensus: GAC AGT rsk Amw wrr wGr ArA

Virus 1:(no insertion)

GAC AGT ACT AAA TGG AGA AAA

Virus 2:(with insertion)

GAC AGT GGG ACT AAA TGG AGA

codon # 67 68 69 70 — — 71

Reference Translation

D S T K W

Consensus Translation

N S S T S R W

Reference Sequence

GAC AGT ACT AAA – – – – – – TGG

Consensus Sequence

AAC AGT TCT ACA TCT AGA TGG

codon # 67 68 69 — — 70 71

Reference Translation

D S T K W

Consensus Translation

N S S T S R W

Reference Sequence

GAC AGT ACT – – – – – – AAA TGG

Consensus Sequence

AAC AGT TCT ACA TCT AGA TGG


• If you edit report fields in an existing project, it is recommended that you complete generating and saving the resistance report before opening another project in the ViroSeq® software.

To Create a Default Header

To Enter Sample-Specific Information

Note: When editing report information, the changes must be saved in order for edits to take effect.

To View the Report.

To Print

To Interpret the Antiretroviral Drug Resistance Report

Laboratory and Sample InformationThis portion of the report is user defined. In the Edit menu, the Edit Report Information option opens a dialog box for information entry.

About Mutations and ResistanceIt is well recognized that development of resistance to a drug is a continuing process with some mutations conferring low-level resistance, others conferring higher-level resistance, and combinations having additive or synergistic effects.27,29

Interaction between mutations conferring resistance to a drug is well defined for some drugs, but not others. Additional studies are required to decipher the exact contributions of each of these combinations of mutations to the development of high-level resistance. In instances where the data do not clearly indicate evidence of a high level of resistance, the mutations detected may indicate impending resistance. These are reported as markers of Possible Resistance.In some instances, mutations may cause hypersusceptibility to a particular drug.

HIV-1 Antiretroviral ResistanceAntiretroviral resistance is reported for three classes of drugs:• Nucleoside Reverse Transcriptase Inhibitors• Non-Nucleoside Reverse Transcriptase Inhibitors• Protease InhibitorsEvidence of Resistance is reported in the far right column on page 1 of the report. It is given for each drug within a class and is based on the mutations found in the sample. The presence of a specific mutation or combination of mutations results in one of the following calls:• Resistance indicates a high level of evidence of resistance.• Possible Resistance indicates possible evidence of resistance. Mutations or

combinations of mutations that do not meet the criteria required for Resistance, but are suggestive of resistance.

• None indicates insufficient evidence for resistance.

Disclaimer LevelsThe Evidence of Resistance levels are labeled with either none or up to three asterisks. The meanings of the asterisks are described in the following table:

1. Select Edit > Edit Report Information.

2. In the Edit Report Information window, type in the information that you want to appear as a default on all resistance reports.

3. Click Set As Default, then click OK to close the dialog box.

Note: To edit your default information, type in your changes, then click Set As Default and OK.

1. From the Edit menu, select Edit Report Information.

2. In the Edit Report Information window, type in the assay operator, patient, and sample information and/or comments.

Note: The default entries in the Edit Report Information window are a series of five dashes. If the user does not customize the Edit Report Information window, then the series of dashes is automatically included in the drug resistance report, the FASTA file, and the XML file.

3. Click OK to close the dialog box.

4. Click Save to save changes.

1. Select File > Generate Resistance Report.

Use the buttons at the top of the window to reduce or enlarge your view, or to page through a multipage report.

2. Select the Save icon to save the generated report as a Portable Document Format (PDF) file.

3. The Save Report dialog is displayed. Select the directory where the resulting PDF report file will reside and click Save.

1. Select File > Generate Resistance Report if the report is not already open. At the top of the ViroSeq Report window, click the Print icon.

2. When the Print dialog box opens, select your printer and click OK.

Disclaimer Label DisclaimerMeaning in Terms of

Analytical and Clinical Studies

(no label) none All mutations present have been verified in clinical and analytical studies.

* NOTE: At least one mutation used to determine Evidence of Resistance for this drug has not been fully validated.

All mutations present have been validated in clinical studies but may not have been verified in analytical studies.

** NOTE: At least one mutation used to determine Evidence of Resistance for this drug has not been clinically verified.

All mutations present have been verified in analytical studies but may not have been verified in clinical studies.

*** NOTE: For at least one mutation used to evaluate Evidence of Resistance for this drug, both notes above apply.

At least one mutation was not verified in both clinical and analytical studies.


The Protease Inhibitor drug class is labeled with a + indicating a disclaimer label.

Mutation Notation at Resistance PositionsMutations are represented using style, color, and punctuation conventions to indicate their relevance to the development of drug resistance.• {Red Bold Curly Bracket} – mutations which by themselves confer viral

resistance and result in a Resistance call.• [Blue Bold-Italics Square Bracket] – mutations which by themselves confer the

possibility of viral resistance and result in a Possible Resistance call. • (Black Parenthesis) – mutations which contribute the least to the development

of resistance. More than one of these mutations is required to produce a Possible Resistance call. This mutation must appear with at least one other mutation to confer the possibility of viral resistance.

• <Green Angle Bracket> – This mutation counters resistance.

Additional MutationsThe Additional Mutations section of the report identifies amino acids that differ from the reference sequence (HXB-2, accession number K03455) at the indicated codon positions and may be useful as a baseline determination of virus genotype. The performance characteristics of the additional mutations have not been established. Additional mutations are specified either for the Protease gene or for the Reverse Transcriptase gene.

Notation KeyPages three and four of the report contain the color key and the HIV-1 Resistance Mutation List that is used in the algorithm for determining drug resistance.

AlgorithmThe interpretation is based on a proprietary algorithm that determines the relative impact of mutations in the reverse transcriptase and protease open reading frames on the development of resistance to antiretroviral drugs. Mutations included in the algorithm were based on:• Mutations defined by the FDA as having established clinical utility.28

• Mutations listed as primary and secondary in the recommendations by the International AIDS Society-USA.13,30

– This list is periodically updated and can be found at the IAS-USA web site ( http://www.iasusa.org/).

• Mutations listed in GART14 and VIRADAPT15 studies as being predictive of drug resistance.

• Recommendations by the Resistance Collaborative Group based on their analysis of several prospective and retrospective studies.31

• Additional mutations were factored into the schema based on more recent reports and consultations with experts in the field to provide an interpretation consistent with current literature.8,16,32-50 The report has separate sections for Nucleoside Reverse Transcriptase Inhibitors (NRTIs), Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) and Protease Inhibitors (PIs). The mutations used in determining resistance are listed schematically along with the drug name in the report.

An algorithm was developed to consider all possible combinations of mutations that can confer resistance to a given drug. We used a numerical scoring system structured so that the combinations of mutations known to generate resistance would be assigned a level of evidence for resistance if detected in the assay. To determine the level of evidence for resistance to a given drug, the scores of the mutations that are present and pertinent to that drug are summed, giving a Total Score for that drug. For each drug, a particular codon position is only counted once using the highest mutation score among the mutations present at that codon position.

A low Total Score (below 0.5) is interpreted as insufficient for resistance. A medium Total Score (between 0.5 and 1) is interpreted as possible resistance. A high Total Score (1.0 or more) is interpreted as resistant. The table below shows how the Total Score maps to the Level of Evidence for Resistance rating.

We used the following criteria when assigning scores to specific mutations:• Mutations with the greatest contribution towards development of resistance,

usually those that confer resistance when present as a single mutation, were assigned the highest score (1.0). Correspondingly, these mutations were also associated with the greatest increases in IC50 (50% Inhibitory Concentration) when introduced into wildtype (WT) strains of HIV-1 and were clearly associated with resistance in multiple clinical trials.

• Mutations with less pronounced effects and requiring accumulation of two or more mutations for conferring resistance were assigned intermediate scores. For example, when considering resistance to AZT, we assigned scores of 0.625 for the T215F/Y mutation, 0.375 for the M41L mutation, and 0.25 for the K70R mutation. The T215F/Y substitution results in a 12-16 fold increase in IC50 for AZT whereas the M41L or K70R mutations cause a 3-5 fold increase. The combination of M41L and K70R results in a 9-fold increase in IC50.

• Mutations with the least impact on the development of resistance but observed in patients treated with the drug and demonstrating small effects on the IC50 values in culture, were assigned low scores. Some of these mutations can enhance the effects of the major mutations. In some instances multiple mutations in this category, in combination with mutations with intermediate scores, could result in resistance.

• Mutations causing an increase in susceptibility to drugs were assigned a negative score based on the level of susceptibility.

For each drug, the tables on the following pages list the mutations that are relevant to resistance to the drug and the assigned score for each mutation.

Disclaimer Label Interpretation

+

The protease inhibitor (PI) evidence of resistance interpretations were developed to estimate the expected virological response to standard doses of protease inhibitors with pharmacokinetic boosting by ritonavir. This has become the most common method of administering each of the protease inhibitors, except nelfinavir, to ensure adequate drug levels in all patients. Boosted PIs are more active in the presence of resistance than non-boosted PIs.

Relationship of Total Score to Level of Evidence for Resistance

Total Score Level of Evidence for Resistance

1.0 Resistance

0.5 to <1.0 Possible Resistance

<0.5 None


Mutations TablesA. Resistance to NRTIs

A1. Zidovudine, AZT, RETROVIR®

AZT

Mutation Score

T69ins 1.2

Q151M 1.2

Q151L 0.7

T215F/Y 0.625

T215C/D/E/I/S/V 0.5

M41L 0.375

D67E/G/N 0.25

K70R 0.25

L210W 0.25

K219E/N/Q/R 0.25

V75I 0.125

F77L 0.125

F116Y 0.125

E44D 0.0625

A62V 0.0625

V118I 0.0625

K65N 0.125

M184I/V 0.2

K65R 0.25

A2. Stavudine, d4T, ZERIT®

d4T

Mutation Score

T69ins 1.2

Q151M 1.2

V75A/M/S/T 1.0

Q151L 0.7

T215F/Y 0.625

T215C/D/E/I/S/V 0.5

M41L 0.375

K65R 0.25

D67E/G/N 0.25

K70R 0.25

L210W 0.25

K219E/N/Q/R 0.25

V75I 0.125

F77L 0.125

F116Y 0.125

E44D 0.0625

A62V 0.0625

V118I 0.0625

M184I/V 0.2

A3. Didanosine, ddI, VIDEX®

ddl

Mutation Score

T69ins 1.0

L74I/V 1.0

Q151M 1.0

K65R 0.75

Q151L 0.5

M41L 0.25

K65N 0.25

T69D/N 0.25

V75A/M/T 0.25

V75S 0.25

L210W 0.25

T215C/D/E/F/I/S/Y/V 0.25

D67E/G/N 0.125

K70E 0.125

V75I 0.125

F77L 0.125

F116Y 0.125

K219E/N/Q/R 0.125

E44D 0.0625

A62V 0.0625

V118I 0.0625


A4. Lamivudine, 3TC, EPIVIR®, Emtricitabine, FTC, EMTRIVA®

3TC & FTC

Mutation Score

M184I/V 1.0

K65R 0.5

T69ins 0.5

K65N 0.25

Q151M 0.25

T215F/Y 0.25

E44D 0.125

K70E 0.125

V75I 0.125

F77L 0.125

F116Y 0.125

V118I 0.125

Q151L 0.125

A62V 0.0625

M41L 0.05

D67E/G/N 0.05

K70R 0.05

L210W 0.05

K219E/N/Q/R 0.05

A5. Abacavir, ABC, ZIAGEN®

ABC

Mutation Score

T69ins 1.0

Q151M 1.0

K65R 0.75

L74I/V 0.75

Y115F 0.5

Q151L 0.5

T215F/Y 0.3

M41L 0.25

K65N 0.25

M184I/V 0.25

T215C/D/E/I/S/V 0.25

L210W 0.2

K70E 0.125

V75I 0.125

F77L 0.125

F116Y 0.125

D67E/G/N 0.1

E44D 0.0625

A62V 0.0625

V118I 0.0625

A6. Tenofovir, TDF, VIREAD®

TDF

Mutation Score

T69ins 1.2

K65R 1.0

Q151M 0.375

M41L 0.35

T215Y 0.35

L210W 0.3

K65N 0.25

K70E 0.25

Y115F 0.2

T215C/D/E/I/S/V 0.15

D67E/G/N 0.125

K70R 0.125

V75I 0.125

F77L 0.125

F116Y 0.125

Q151L 0.125

T215F 0.125

K219E/N/Q/R 0.125

E44D 0.0625

A62V 0.0625

V118I 0.0625

M184I/V 0.2


B. Resistance to NNRTIs

B1. Nevirapine, NVP, VIRAMUNE®

NVP

Mutation Score

K103H/N/S/T 1.0

V106A/M 1.0

Y181C/I/V 1.0

Y188C/H/L 1.0

G190A/C/E/Q/S/T/V 1.0

A98G 0.5

L100I 0.5

K101E/P 0.5

V108I 0.5

E138K 0.5

V179D/E/F 0.5

P225H 0.5

F227C/L 0.5

M230L 0.5

K238T 0.5

Y318F 0.5

K101Q 0.25

K103R 0.25

B2. Delavirdine, DLV, RESCRIPTOR®

DLV

Mutation Score

K103H/N/S/T 1.0

V106M 1.0

Y181C/I/V 1.0

Y188L 1.0

P236L 1.0

Y318F 1.0

A98G 0.5

L100I 0.5

K101E/P 0.5

V106A 0.5

V108I 0.5

E138K 0.5

V179D/E/F 0.5

Y188C/H 0.5

G190E/Q 0.5

P225H 0.5

F227C 0.5

M230L 0.5

K238T 0.5

K101Q 0.25

K103R 0.25

B3. Efavirenz, EFV, SUSTIVA®

EFV

Mutation Score

K103H/N/S/T 1.0

V106M 1.0

Y188L 1.0

G190C/E/Q/S/T/V 1.0

L100I 0.5

K101P/E 0.5

V106A 0.5

Y181C/I/V 0.5

Y188C/H 0.5

G190A 0.5

P225H 0.5

M230L 0.5

A98G 0.25

K101Q 0.25

K103R 0.25

V108I 0.25

E138K 0.25

V179D/E/F 0.25

F227C 0.25

K238T 0.25

Y318F 0.25


C. Resistance to Protease InhibitorsB4. Etravirine, ETR, INTELENCE™

ETR

Mutation Score

V179F 0.5

Y181C/I/V 0.5

L100I 0.25

K101E/P 0.25

E138K 0.25

Y188L 0.25

G190C/E/Q/S/T/V 0.25

M230L 0.25

A98G 0.125

K103H/N/S/T 0.125

V106A/M 0.125

V179D/E 0.125

Y188C/H 0.125

G190A 0.125

P225H 0.125

F227C/L 0.125

C1. Indinavir, IDV, CRIXIVAN®

IDV

Mutation Score

V82A/F/M/S/T 0.5

I84A/C/V 0.5

L90M 0.5

V32I 0.25

M46I/L 0.25

I47A 0.25

G48V 0.25

I54A/L/M/S/T/V 0.25

N88S 0.25

L24I 0.125

M46V 0.125

I47V 0.125

F53L 0.125

G73A/C/S/T 0.125

L76V 0.125

N88D/T 0.125

L10F/I/R/V 0.0625

A71I/T/V 0.0625

I50L 0.125

C2. Saquinavir, SQV, FORTOVASE®, INVIRASE®

SQV

Mutation Score

G48V 1.0

I84A/C/V 0.5

L90M 0.5

I54A/L/M/S/T/V 0.25

G73A/C/S/T 0.25

V82A/F/S/T 0.25

L10F/I/R/V 0.125

L24I 0.125

M46I/L/V 0.125

F53L 0.125

A71I/T/V 0.125

I50L 0.125

L76V 0.125


C3. Nelfinavir, NFV, VIRACEPT®

NFV

Mutation Score

D30N 1.0

N88D/S/T 1.0

L90M 1.0

L23I 0.5

M46I/L/V 0.5

G48V 0.5

I54A/L/M/S/T/V 0.5

V82A/F/S/T 0.5

I84A/C/V 0.5

I47A 0.25

L10F/I/R/V 0.125

L24I 0.125

A71I/T/V 0.125

G73A/C/S/T 0.125

I50L 0.125

C4. Amprenavir, APV, AGENERASE®, Fosamprenavir, FOS, LEXIVA®

APV/FOS

Mutation Score

I50V 1.0

I47A 0.5

I54L/M 0.5

I84A/C/V 0.5

V32I 0.25

L33F 0.25

M46I/L 0.25

I47V 0.25

L76V 0.25

V82A/F/S/T 0.25

L90M 0.25

L10F/I/R/V 0.125

M46V 0.125

I54A/S/T/V 0.125

A71I/T/V 0.125

G73A/C/S/T 0.125

I50L 0.125

N88S 0.125

C5. Lopinavir/Ritonavir, LPV/r, KALETRA®

LPV/r

Mutation Score

I47A 1.0

I50V 0.5

V32I 0.25

M46I/L 0.25

I54A/L/M/S/T/V 0.25

V82A/F/S/T 0.25

I84A/C/V 0.25

L24I 0.125

L33F 0.125

M46V 0.125

I47V 0.125

G48V 0.125

F53L 0.125

G73A/C/S/T 0.125

L76V 0.125

L90M 0.125

L10F/I/R/V 0.0625

A71I/T/V 0.0625

I50L 0.125


C6. Atazanavir, ATV, REYATAZ®

ATV

Mutation Score

I50L 1.0

N88S 1.0

I84A/C/V 0.5

V32I 0.25

M46I/L 0.25

I47A 0.25

I54A/L/M/S/T/V 0.25

V82A/F/S/T 0.25

N88D/T 0.25

L90M 0.25

L10F/I/R/V 0.125

M46V 0.125

G48V 0.125

A71I/T/V 0.125

G73A/C/S/T 0.125

L24I 0.0625

L33F 0.0625

F53L 0.0625

L76V 0.125

C7. Tipranavir, TPV, APTIVUS®

TPV

Mutation Score

V82L 0.5

V82T 0.375

I84A/C/V 0.375

L33F 0.25

M46I/L 0.25

I47A/V 0.25

I54A/V 0.25

V82F/S 0.25

V32I 0.125

E35G 0.125

K43T 0.125

M46V 0.125

I54L/M/S/T 0.125

Q58E 0.125

G73A/C/S/T 0.125

T74P 0.125

N83D 0.125

L90M 0.125

L10F/I/R/V 0.0625

A71I/T/V 0.0625

V82A 0.0625

I50L/V 0.125

C8. Darunavir, DRV, PREZISTA®

DRV

Mutation Score

I50V 0.375

V32I 0.25

I47A/V 0.25

I54L/M 0.25

L76V 0.25

I84A/V 0.25

V11I 0.125

L33F 0.125

G73A/C/S/T 0.125

I84C 0.125

L89V 0.125

M46I/L/V 0.0625

I54A/S/T/V 0.0625

V82A/F/L/M/S/T 0.0625

L90M 0.0625

I50L -0.125


Software Error Messages

ViroSeq® Software and Results• Only trained users are allowed to use the ViroSeq software.• Accurate and reliable results are dependent on good sequence quality. High

background and noisy data can interfere with precise basecalling.• Understanding and using the editing guidelines found in this document will

assist in making more accurate basecalls, and may help identify mutations that otherwise may be missed.

• Multiple nucleotide changes within the wild type codon are necessary to generate some mutations such as: V82T, V82S, T215Y, etc. In these cases, when mixtures of the wild type and mutant are present, the software report indicates the presence of two amino acids, but also reports amino acids corresponding to other possible codon combinations. For example, a mixture of Val (GTC) and Ser (TCC) at codon 82 results in a report indicating the presence of Val, Ser, Ala (GCC) and Phe (TTC) – V, S, A, and F. This is a limitation of all population sequencing methodologies in that they do not define linkage of the nucleotides from a single template.

In accordance with the FDA Guidance document for HIV-1 genotyping, analytical studies using the ViroSeq HIV-1 Genotyping System have not been completed at the limit of detection for the following mutations:

Message Explanation

A unique non-blank user name must be used. Please select a different user name and try again.

Add User button is clicked in the Administrator Access window when trying to create the user that already exists.

Select OK and type a unique User Name.

Current Password incorrect. Check ‘Caps Lock’ and try again.

An incorrect or blank Current Password was entered in the User Access window.

Select OK and type correct Current Password.

Default Password incorrect. Check ‘Caps Lock’ and try again.

There is an incorrect Default Password in Change Administrator Password dialog, while launching the software for the first time.

Select “OK” and type correct Default Password (hiv1).

New passwords must be the same. Try entering the passwords again.

A valid new password was given in the Change Administrator Password dialog or User Access dialog, but the confirmatory password does not match.

Select OK and type same password in confirmatory password as given in New password field.

Password length should be 6 to 14 characters. Please choose a longer password and try again.

The new password given in the Change Administrator Password dialog, Administrator Access dialog or User Access dialog is of insufficient length.

Select OK and type a password with length of 6 to 14 characters.

The passwords entered were not the same. Please enter the exact same password twice.

A valid new password was given in the Administrator Access dialog, but the confirmatory password does not match.

Or

Add User button is clicked in the Administrator Access dialog with a valid new User Name and mismatched confirmatory password.Select OK and type same password in confirmatory password as given in New password field.

User Name or password incorrect. Check ‘Caps Lock’ and try again.

Incorrect User Name or Password is entered in the User Login dialog.

Select OK and type correct user name and password.

Launch failed. A file is missing or damaged. Please re-install the software. If the problem persists, please contact technical support.

A ViroSeq system file or folder is missing or corrupt.

Select OK and install the ViroSeq software again.

One or more “q” or “?”s were detected in the consensus sequence. ViroSeq will not generate output.

An attempt was made to generate a drug report, FASTA file, and/or XML report after editing base with “?” (with Keyboard) or “q” (by clicking on the Edit Palette).

The drug resistance report, FASTA file, and/or XML report cannot be generated if one or more “q” or “?” is detected in the sequence.

Selected file is invalid. A sample file (ab1) is selected when the software was expecting a project file (vpr). [File > Open Project + Find]

Or

A project file (vpr) is selected when the software was expecting a sample file (ab1). [File > New Project/File > Open Project + New]

Or

Selected file type is wrong in the Open dialog box.Click OK and select correct file as expected.

Sequence data does not cover critical portions of the reference sequence. ViroSeq will not generate output.

An attempt was made to generate a drug report, FASTA file, and/or XML report when reference sequence does not cover between protease codon 9 through RT codon 320. Report cannot be generated if there is no coverage between protease codon 9 through RT codon 320.

The file is in use by another application. You must close the file in that application before ViroSeq can output into that file.

An attempt was made to save new report with the existing file name which is opened with MS word (or any other program which locks file).

Select OK, close the file and then save it again.

Unable to assemble files in: <folder>

An attempt was made to create a project with fewer than two segment files with the same sample ID.

Or

Corrupted sample files were selected for assembly in the Open dialog box.

Select OK and check that two or more, non-corrupted ab1 files with same sample ID’s are presented in the working folder.

Unable to load project: <file name>

The platform Operating System fails in opening Project file (vpr), due to low memory or corrupted disk.

Or

An attempt was made to open a corrupt or incomplete project file.

Select OK and check that the vpr file is not corrupted or read-only.

ViroSeq detected a deletion, frame shifting insertion, and/or multiple insertions. The software has not been validated for such mutations. Output will not be created.

An attempt was made to generate a drug report, FASTA file, and/or XML report when deletion/frame shifting insertion/multiple insertions are present in consensus sequence.

The drug resistance report, FASTA file, and/or XML report cannot be generated if deletion/frame shifting insertion/multiple insertions are detected in the sequence.

ViroSeq detected that an incorrect version of Java Runtime Environment [JRE] is being used. Please refer to installation instructions for installing the appropriate JRE.

Wrong version of JRE is installedSelect OK and copy the correct JRE folder from the CD. Launch the ViroSeq software.

Windows Platform /JRE versionWindows XP & 2000/ JRE 1.4.2_10

ViroSeq software is not supported on Microsoft NT operating system.

An attempt was made to install ViroSeq v2.8 on a Windows NT operating system.

Gene Mutation

RT E44D, K65N, D67E/G, D67N, T69N, K70E, L74I, V75A/I/M/S/T, A98G, L100I, K101E/P/Q, K103H/S/T/R, V106M, V108I, Y115F, E138K, Q151L, V179D/E/F, Y181I/V, Y188H, G190A/C/E/Q/S/T/V, T215C/D/E/I/S/V, K219N/R, P225H, F227L/C, M230L, P236L, K238T, Y318F

Protease L10F/V, V11I, L23I, L24I, V32I, L33F, E35G, K43T, M46L/V, I47A/V, I50L, F53L, I54A/L/M/S/T, Q58E, A71I/T, G73A/C/S/T, T74P, L76V, V82L, N83D, I84A/C, N88D/S/T, L89V

Message Explanation


Genotyping Success

Genotyping success is determined qualitatively during ViroSeq software analysis. In order to successfully genotype a sample, six of the seven ViroSeq system primers must assemble into a project to generate the consensus sequence (A or D, B, C, F, G, H). In addition, a qualitative assessment of the sequence quality should be made to ensure the reliability and accuracy of results. High background noise can interfere with the determination of viral mixtures. The ViroSeq Software v2.8 recognizes a viral mixture when a secondary peak, which is at least 30% of the primary peak, is present.

The HIV-1 8E5 Positive Control is a non-infectious HIV-1 virus and should contain little or no background. Any mutations reported, other than those given above, may signify an editing mistake or problems with sequence quality. A comparison with the other sample results is necessary to determine the cause of failure. For example:• If background peaks are responsible for the failed control, and if these peaks are

seen in every sample, and if all samples have the same color noise for a given base, then perform a new spectral calibration (refer to To Perform A Spectral Calibration); all samples and controls should be repeated from “Reverse Transcription” through “ViroSeq Software Analysis”.

• If the background peak colors are random, then all samples and controls should be repeated from “Reverse Transcription” through “ViroSeq Software Analysis”.

If repeated failure occurs, please contact Abbott Molecular customer support.Note: The HIV-1 8E5 Positive Control has a single-base insertion at codon 219 of the RT gene that renders the virus non-infectious. The 219 mutation does not appear during editing or on the antiretroviral drug resistance report because it is a frame-shifting mutation that is not found in clinical viral samples.

The expected genotyping results for the ViroSeq controls are:

Positive Control Negative Control

Expected Value • All seven primers assemble

• No drug resistance mutations

• Two additional mutations– Protease V3I– RT L214F

• No mixed bases

Do not use a negative control to monitor genotyping success or failure. Negative controls, which contain no DNA, can actually contribute to sample failure due to the adverse electrical effects that empty capillaries have on capillary-based sequencing instruments.

Perform a qualitative visual inspection of the electropherograms to assess the sequence quality and degree of background in the control.Examine the run data for accurate instrument calibration. If the run shows consistent and proportional pull-up peaks (that is, smaller peaks of another color) then perform a new spectral calibration (refer to To Perform A Spectral Calibration.).


Section 5: Performance Characteristics

PerformanceThe performance characteristics of the ViroSeq HIV-1 Genotyping System were evaluated using viral and clinical samples containing defined mutations that confer viral resistance to antiretroviral therapy. The clinical samples used in the analysis were obtained both prospectively and retrospectively from adults and infants and pediatric patients (from 4 months to 17 years of age) on antiretroviral therapy. The presented results define assay sensitivity, specificity, reproducibility, and precision with reference to accurate detection of specific mutations in the RT and protease genes of HIV-1 that confer resistance to approved medications. Clinical specimens were also used in studies to determine sequencing success rates and the ability to detect mutations in mixtures, and to define clinical utility. The ViroSeq HIV-1 Genotyping System’s ability to detect specific mutations that confer viral resistance in a patient sample, to evaluate drug resistance assessments, and to generate a final Antiretroviral Drug Resistance Report that accurately follows a rules-based interpretation algorithm have been evaluated. The use of the ViroSeq HIV-1 Genotyping System will enhance physicians’ capability to assess drug efficacy and design individualized therapeutic strategies in conjunction with other clinical results such as clinical presentation, laboratory markers, and antiretroviral history.Table 1 is a list of mutations that have been fully verified at 2,000 RNA copies/mL in accordance with the FDA “Guidance for Industry: Class II Special Controls Guidance Document: In Vitro HIV Drug Resistance Genotype Assay.”Table 2 is a list of mutations that have been fully verified at 1,000 RNA copies/mL in accordance with the FDA “Guidance for Industry: Class II Special Controls Guidance Document: In Vitro HIV Drug Resistance Genotype Assay.”Table 3 is a list of mutations that are used in the ViroSeq System rules-based algorithm.Table 4 is a list of drugs for which drug resistance is evaluated in the ViroSeq System Antiretroviral Drug Resistance Report.

Fully Verified Performance List

As required by the FDA in the “Guidance for Industry: Class II Special Controls Guidance Document: In Vitro HIV Drug Resistance Genotype Assay”, mutations are listed below that met the greater than 90% sensitivity specification in both:• Analytical Performance Studies at the limit of detection (2,000 copies/mL) with

40% mutant samples• Clinical Validity studies with a panel of 50 clinical isolates (VL = 1,800 to

10,500)Mutations M184I, V82F, and V82S were detected in clinical samples that were part of the high viral load (VL greater than 10,500) Clinical Validity Study. They are not included in the Verified List given in Table 1.

Table 1. Fully Verified Performance List at a Limit of Detection (LOD) of 2,000 copies/mL

In Reverse Transcriptase

M41L A62V K65R T69D K70R L74V F77L K103N

F116Y V118I Q151M Y181C M184V L210W T215Y T215F

K219E K219Q

In Protease

L10I K20R D30N M36I M46I G48V I54V L63P

A71V V82T V82A I84V L90M

Table 2. Fully Verified Performance List at an LOD of 1,000 copies/mL


M41L A62V K65R L74V F77L K103N F116Y V118I

Y181C M184V L210W T215F K219Q

In Protease

L10I D30N M36I M46I G48V I54V L63P A71V

V82T I84V L90M

Table 3. Mutations used in the rules-based interpretation algorithm to evaluate viral resistance include:


M41L* E44D A62V* K65N K65R* D67E D67G D67N

T69D* T69N K70E K70R* L74I L74V* V75A V75I

V75M V75S V75T F77L* A98G L100I K101E K101P

K101Q K103N* K103H K103R K103S K103T V106A V106M

V108I Y115F F116Y* V118I* E138K Q151L Q151M* V179D

V179E V179F Y181C* Y181I Y181V M184I M184V* Y188C

Y188H Y188L G190A G190C G190E G190Q G190S G190T

G190V L210W* T215C T215D T215E T215F* T215I T215S

T215V T215Y* K219E* K219N K219R K219Q* P225H F227C

F227L M230L P236L K238T Y318F Insertion mutation at codon 69 (69 insertion)

In Protease

L10F L10I* L10R L10V V11I L23I L24I D30N*

V32I L33F E35G K43T M46I* M46L M46V I47A

I47V G48V* I50L I50V F53L I54A I54L I54M

I54S I54T I54V* Q58E A71I A71T A71V* G73A

G73C G73S G73T T74P L76V V82A* V82F V82L

V82M V82S V82T* N83D I84A I84C 184V* N88D

N88S N88T L89V L90M*

* Fully verified mutations


Analytical Sensitivity

Two parameters were used to define analytical sensitivity. • Determination of the minimum HIV-1 viral load for reliable mutation detection.• Determination of the lowest amount of mutant virus in a mixture that allowed

reliable mutation detection.

Limit of Detection

Detection of Mutations

The ViroSeq System was able to accurately detect the specific drug resistance mutations listed below at 1,000 copies/mL in 100% mutant samples and at 2,000 copies/mL in 40% mutant samples.Description of the Study. For these studies, the Limit of Detection is defined as the lowest viral load with a sensitivity of 100%. This study utilized a single lot of reagents. Twelve recombinant viruses were formulated in negative plasma as 100% pure and 40/60 mutant/wild type.

Results

• When a mutation was present in 100% of the viral population, the limit of detection for all mutations was 1,000 copies/mL – that is, a 100% sensitivity and 100% specificity was obtained at 1,000 copies/mL for all of the following mutations.

• In subsequent studies at 2,000 copies/mL in a 40/60 mixture, the sensitivity for D67N and T69D was 83% and 50% respectively for one of three reagent lots.

• When a mutation was present in a 40/60 mutant/wild type mixture, the aggregate sensitivity was greater than 97% and the aggregate specificity of detection was greater than 99.9% for all mutations at all viral loads greater than 1,000 copies/mL.

• At a viral load of 2,000 copies/mL only, and at a 40/60 mutant/wild type mixture, the aggregate sensitivity was 98.1% (4 false negatives [FN]/206) and the aggregate specificity was 99.9% (1 false positive [FP]/1422).

• At a viral load of 1,000 copies/mL only, and at a 40/60 mutant/wild type mixture, the aggregate sensitivity was 92.7% (15 FN/206) and the aggregate specificity was 99.9% (1 FP/1422).

• Table 6 (reverse transcriptase) and Table 7 (protease) list the sensitivities of the individual mutations in a 40% mutant mixture at viral loads of 1,000 copies/mL and greater.In addition, each mutation has been assigned a Limit of Detection value.

Table 4. The ViroSeq Antiretroviral Drug Resistance Report reports viral resistance in the following drugs:

Nucleoside RT Inhibitors a Non-Nucleoside RT Inhibitors Protease Inhibitors

EPIVIR®/lamivudine/3TC INTELENCE™/etravirine/ETR AGENERASE®/amprenavir/APV

EMTRIVA®/emtricitabine/FTC RESCRIPTOR®/delavirdine/DLV

APTIVUS®/tipranavir/TPV

RETROVIR®/zidovudine/AZT SUSTIVA®/efavirenz/EFV CRIXIVAN®/indinavir/IDV

VIDEX®/didanosine/ddI VIRAMUNE®/nevirapine/NVP FORTOVASE®/INVIRASE®/saquinavir/SQV

VIREAD®/tenofovir/TDF KALETRA®/lopinavir/ritonavir/LPV/r

ZERIT®/stavudine/d4T LEXIVA®/fosamprenavir/FOS

ZIAGEN®/abacavir/ABC PREZISTA®/darunavir/DRV

REYATAZ®/atazanavir/ATV

VIRACEPT®/nelfinavir/NFV

a The report also evaluates multinucleoside resistance due to the presence of the Q151M complexor the 69 insertion.13 Resistance to COMBIVIR® and TRIZIVIR® can be deduced from thereport.

Table 5. Mutations Detected With 100% Sensitivity at 100% Mutant Samples at 1,000 copies/mL


M41L A62V K65R D67N T69D K70R

L74V V75I F77L K103N V106A Y115F

F116Y V118I Q151M Y181C M184I/V Y188C/L

L210W T215F/Y K219E/Q T69S + SS [Insertion mutation at codon 69 (69 insertion)]

In Protease

L10I/R K20R D30N M36I M46I G48V

I50V I54V L63P A71V V82A/F/S/T I84V

L90M

Table 6. Sensitivity of Detection of Reverse Transcriptase Mutations at 40% Mixture Level

Reverse Transcriptase Mutation

Sensitivity at >1,000 copies/mL

(No. of True Positives)



LOD Claim(comments)

M41L 98.8(82)

100(71)

2,000

A62V 100(41)

100(35)

1,000

K65R 100(13)

100(11)

1,000

D67N 91.6(76)

93(66)

In a subsequent three-lot study, a different lot gave 83% sensitivity at 2,000 copies/mL; the other two lots gave 100% sensitivity.

Not determined (1)

T69D 92.9(13)

100(12)

In a subsequent three-lot study, a different lot gave 50% sensitivity at 2,000 copies/mL; the other two lots gave 100% sensitivity.

2,000

K70R 98.2(54)

100(47)

2,000

L74V 100(14)

100(12)

1,000

V75I 97.6(40)

97.1(34)

6,000 (2)

F77L 100(41)

100(35)

1,000

K103N 98.6(68)

100(59)

2,000


Comments:

1. RT mutation D67N is a mutation that the assay detects with a lower sensitivity. There were seven FN results out of 83 detections at viral loads >1,000 copies/mL. D67N was detected with 100% sensitivity in five of the viral panels (MC1, MC2, MC3, MC4, and MC5). However, with one of the panel members (GT1a) there were seven FN results.

2. The claimed LOD is 6,000 copies/mL for RT mutation V75I (6 true positive determinations). At 2,000 copies/mL and above, there was one FN out of 42 expected detections. At 1,000 copies/mL only, the sensitivity was 100%.

3. The claimed LOD is 6,000 copies/mL for RT mutation Y115F (4 true positive determinations). There was 100% sensitivity at viral loads of 6,000 copies/mL and higher; at 2,000 copies/mL, there was one FN out of four expected detections.

4. RT mutation M184V was detected with 100% sensitivity (28 detections) at 1,000 and 2,000 copies/mL. However, there was one FN at 6,000 copies/mL (out of 84 detections). Therefore, the LOD for this mutation is claimed at 1,000 copies/mL.

5. RT mutation K219E has a total of three FN results, with one FN at 1,000 copies/mL and the other two FNs at 60,000 copies/mL. This was an anomalous result since the mutation was detected with 100% sensitivity at other viral loads (6,000, 20,000, 200,000, and 750,000 copies/mL). Therefore the LOD is claimed at 2,000 copies/mL.

6. RT mutation K219Q is associated with one FN result at 6,000 copies/mL (out of a total of 40 detections) and is detected with 100% sensitivity at 1,000 and 2,000 copies/mL viral load (12/12). Therefore, the LOD for this mutation is claimed at 1,000 copies/mL.

7. Protease mutation M46I is associated with one FN result at 2,000 copies/mL (out of 40 detections) and is detected with 100% sensitivity at 1,000 copies/mL and all viral loads greater than 2,000 copies/mL. Therefore, the LOD for this mutation in a 40% mixture is 1,000 copies/mL.

8. Protease mutation A71V is associated with one FN result at 6,000 copies/mL (out of a total of 12 detections) and is detected with 100% sensitivity at 1,000 and 2,000 copies/mL viral load. Therefore, the LOD for this mutation is claimed at 1,000 copies/mL.

9. Protease mutation V82S is associated with two FN results at 6,000 copies/mL (out of a total of 12 detections) and is detected with 100% sensitivity at 1,000 and 2,000 copies/mL viral load. Therefore, the LOD for this mutation is claimed at 1,000 copies/mL.

V106A 100(25)

100(21)

1,000

Y115F 85.2(23)

95.7(22)

6,000 (3)

F116Y 100(41)

100(35)

1,000

V118I 100(28)

100(24)

1,000

Q151M 97.6(40)

100(35)

2,000

Y181C 100(54)

100(46)

1,000

M184I 100(14)

100(12)

1,000

M184V 98.9(92)

98.7(78)

1,000 (4)

Y188C 100(14)

100(12)

1,000

Y188L 100(13)

100(11)

1,000

L210W 100(53)

100(45)

1,000

T215F 98.8(82)

100(71)

2,000

T215Y 95.8(23)

100(20)

2,000

K219E 78.6(11)

83.3(10)

2,000 (5)

K219Q 97.6(40)

97.1(34)

1,000 (6)

Table 7. Sensitivity of Detection of Protease Mutations at 40% Mixture Level

Protease Mutation





LOD Claim(comments)

L10I 100(12)

100(10)

1,000

L10R 100(14)

100(12)

1,000

K20R 91.7(11)

100(10)

2,000

D30N 100(14)

100(12)

1,000

M36I 100(12)

100(10)

1,000

M46I 97.6(40)

97.1(34)

1,000 (7)

G48V 100(14)

100(12)

1,000

I50V 100(14)

100(12)

1,000

I54V 100(12)

100(10)

1,000

Table 6. Sensitivity of Detection of Reverse Transcriptase Mutations at 40% Mixture Level (continued)

Reverse Transcriptase Mutation





LOD Claim(comments)

L63P 100(26)

100(22)

1,000

A71V 91.7(11)

90.0(9)

1,000 (8)

V82A 96.4(27)

100(24)

2,000

V82F 100(13)

100(11)

1,000

V82S 85.7(12)

83.3(10)

1,000 (9)

V82T 100(27)

100(23)

1,000

I84V 100(26)

100(22)

1,000

L90M 98.8(79)

98.5(67)

1,000 (10)

Table 7. Sensitivity of Detection of Protease Mutations at 40% Mixture Level (continued)

Protease Mutation





LOD Claim(comments)


10. Protease mutation L90M is associated with one FP result at 6,000 copies/mL (out of a total of 80 detections) and is detected with 100% sensitivity at 1,000 and 2,000 copies/mL viral load. Therefore, the LOD for this mutation is claimed at 1,000 copies/mL.

Detection of Mixtures

The ViroSeq System can accurately detect:• 24 mutations when present as a 40% mixture level at 2,000 copies/mL• 11 mutations when present at a 30% mixture level at 2,000 copies/mL• 3 mutations when present at a 20% mixture level at 2,000 copies/mL

Description of the Study. In this study, eleven recombinant clones were formulated at three viral loads (2,000, 50,000, and 750,000 copies/mL) at six viral mixtures(100, 80, 60, 40, 30, and 20% mutant).Results.• At the viral load of 2,000 copies/mL, mutations were detected with >95%

sensitivity at the mixture levels given below.

Analytical Specificity

The ViroSeq System is able to accurately genotype mixtures of viral samples in the presence of infectious agents found in HIV-1 infected patients. This Analytical Specificity Study demonstrates that the presence of HIV-1 related or non-related viruses, at approximately five times the HIV-1 viral load, did not affect the performance of the assay. The presence of these infectious agents did not result in a concordance value below 90% for the 72 samples tested. This proves that none of the infectious viruses decrease the specificity of the ViroSeq System to accurately detect specific mutations (previously listed) in HIV-1 that confer viral resistance to drugs.Results indicate that none of the organisms listed below interfered with or were detected by the ViroSeq assay.

Interfering Substances

Commonly occurring biological substances and anti-reverse transcriptase (anti-RT) drugs or inhibitors that are present in patient plasma were tested for their potential to interfere with the detection of mutations that confer viral resistance by the ViroSeq HIV-1 Genotyping System. In this study any substance that caused less than 90% concordance between the genotype of the test sample and the expected genotype was considered to be interfering with assay performance.

Results indicate that commonly occurring biological substances and anti-reverse transcriptase drugs or inhibitors found in HIV-1 infected individuals did not interfere with the assay. These include:• Antiretroviral drugs AZT, ddI, 3TC, d4T, abacavir, nevirapine, efavirenz.

Note: Reverse transcriptase mutations V118I and Q151M were detected in 14 of 16 samples (88% sensitivity). All other mutations had greater than 94% sensitivity.Note: The anti-retroviral drugs listed above have not been fully validated with regards to actual plasma drug levels. Instead, the plasma samples used were from patients currently taking approved reverse transcriptase inhibitors and had viral loads below the limit of detection of the Roche Ultrasensitive AMPLICOR HIV-1 MONITOR® test (50 copies/mL), thus suggesting that these drugs were fully active.

• Lipid (30 mg/mL), bilirubin (0.6 mg/mL), and hemoglobin (5 mg/mL).

Precision and Reproducibility

The ViroSeq System is able to reproducibly detect specific drug resistance mutations between different sites and/or operators, different manufactured lots, different replicates within the assay, and different runs of the assay.

This claim is supported by both the Analytical Imprecision Study and the Clinical Reproducibility Study. The Imprecision Study shows that the range of run-to-run variability (%CV) was 0.1 to 1.4% and that the range for the intra-assay variability (%CV) was 0.1 to 5.3%. The Clinical Reproducibility Study shows that the ViroSeq System can reproducibly detect specific mutations that confer drug resistance in HIV-1 infected patient samples with low viral loads (1,800 to 10,500 copies/mL) when the mutation comprises 40% or greater of the quasispecies with a %CV of <0.1%, well below the level of significance (>5%).

Imprecision StudyThe Imprecision Study tested six mutant/WT viral mixtures at a viral load of 2,000 copies/mL and 40% mixture level in duplicate in ten assay runs to evaluate the run-to-run variability of the ViroSeq HIV-1 Genotyping System (refer to results in Table 9).

The data in Table 9 show that the run-to-run %CV is in the range <0.1 to 1.4. Five of the 6% CV values are <0.1 and one is 1.4. The intra-assay %CV is in the range <0.1 to 5.3.Results from this study support the Celera claim that the ViroSeq HIV-1 Genotyping System has a run-to-run variability which is not significant ( 5%).

Clinical Reproducibility Study

The clinical reproducibility of the ViroSeq HIV-1 Genotyping System was measured through a multi-center study to determine the site-to-site and lot-to-lot variation of the assay. Three sites tested 11 patient samples ranging in viral load from 1,800 to 10,500 copies/mL. Each of these 11 samples was tested in duplicate

Table 8. Detection at 2,000 copies/mL

Mixture Level of Mutant(%) Reverse Transcriptase Gene Protease

Gene

20 — L10RD30NI50V

30 K65RK70RY181CM184IY188CT215F

K219E/Q

V82FV82SV82T

40 M41LT69DL74VV75IF77L

K103NV106AF116YV118IY188LM184VL210WT215Y

L10IK20RM36IM46IG48VI54VL63PA71VV82AI84VL90M

Related Viruses Coinfecting Organisms

HIV-2 Cytomegalovirus

HTLV-I Epstein-Barr Virus

HTLV-II Hepatitis B

Hepatitis C

Table 9. Estimates of Coefficient of Variation by Recombinant Virus and Gene

Mutant Virus Gene%CV

Run-to-Run Intra-Assay

GT-4RT <0.1 2.6

Protease <0.1 <0.1

MC-1RT <0.1 3.7

Protease <0.1 1.8

MC-2RT <0.1 2.3

Protease <0.1 2.2

MC-5RT 1.4 1.6

Protease <0.1 <0.1

MC-6RT <0.1 10.9 a

a This value is the result of one sample in which only 3 of 13 mutations were detected. For thissample, the RT-PCR yield did not meet specifications – it was approximately 10 ng, which isless than the required 20 ng minimum RT-PCR yield needed for sequencing reactions.

Protease <0.1 3.3

MC-7RT <0.1 5.3

Protease <0.1 2.2


with three different lots, generating 18 data points per specimen (3 sites x 3 lots x 2 replicates). The detection of all mutations present in a specimen was measured and compared to establish the variability (%CV). The results in Table 10 show that the site-to-site and lot-to-lot variation of the ViroSeq System are negligible.

Results from this study support the Celera claim that the ViroSeq HIV-1 Genotyping System demonstrates site-to site, lot-to-lot, and assay variability that is not significant (<5%).

RT-PCR and Sequencing Success RatesThe ViroSeq System is able to successfully genotype a significant portion ( 98%) of the targeted population samples when sufficient RT-PCR product is available.The 100 adult HIV-1 infected patient specimens were screened to obtain samples representing a geographical cross section consisting of varying viral loads and resistance mutations. 50 of these samples were grouped into a low viral load category (1,800 to 10,500 copies/mL) and 50 were grouped into a high viral load category (above 10,500 copies/mL).For the PACTG 377 study, three laboratories (Johns Hopkins, UCLA, and the University of Medicine and Dentistry of New Jersey) and three operators tested the 196 samples that had viral loads >1084 copies/mL.

Mixture Detection in Patient SamplesThe ViroSeq System is able to accurately detect specific drug resistance mutations in clinical EDTA plasma samples in a low viral load range (1,800 to 10,500 copies/mL) and viral mixture limit ( 40% mutant).This claim is supported by the Clinical Validity Study, which compared the percentage of mutations found in each sample when PCR product was cloned versus the mutations detected by population sequencing using the ViroSeq System. This study shows that mutations representing 40% or more of the quasispecies in low viral load range samples (1,800 to 10,500 copies/mL) can be detected by three clinical sites reproducibly with a concordance value of >98% to the lower limit of the 95% confidence level.

For this study low viral load samples were chosen randomly with respect to age (above 18), sex, and clinical and treatment history. Samples were collected prospectively from HIV-1 infected patients from multiple geographic locations in the targeted population. The detected mutations and their frequency are considered representative of the targeted HIV-1 infected population. Within this population, the ability of the ViroSeq System to detect the selected list of drug resistance mutations is consistently high across all mutations detected. In some cases the assay was able to detect mutations present below the 40% limit, although this was not consistent across all of the sites or with all of mutations.

Data quality control for this clinical validity study consisted of a phylogenetic analysis on 300 samples (50 specimens x 6 replicates each). The phylogenetic tree that was derived showed no evidence of PCR amplicon carryover contamination. This study shows that the AmpErase® Uracil N-glycosylase and recommended containment procedures effectively control PCR contamination.Data were also analyzed for all-base accuracy.

Clinical Utility Study

The ViroSeq® System Software uses drug resistance mutations to generate drug resistance assessments with proven clinical utility.This claim is supported by resequencing samples from the GART study using the ViroSeq System. The GART study had previously demonstrated the clinical utility of several mutations in RT and protease listed in Table 12.14 Additional mutations were factored into the rules-based interpretation algorithm based on recent literature reports.

The Genotyping Antiretroviral Resistance Testing (GART) study was one of the pivotal clinical utility trials. It proved that if physicians had access to genotyping data before altering therapy, they made better treatment decisions and improved the short term patient response to therapy.14 There were two arms to this study – one (GART) allowed treating physicians access to genotyping results and expert panel treatment recommendations based on those results, while the other (nonGART) did not allow access to this information before treatment. These two arms were compared with regards to average viral load decreases at 4, 8, and 12 weeks. The GART arm showed a significant decrease in viral load compared to the nonGART arm, especially when the treating physicians showed a high adherence to expert treatment advice. Because of this protocol and others like it, resistance testing has become the standard of medical care when designing therapeutic strategies for patients experiencing treatment failure. To demonstrate that our assay detects the same resistance mutations detected by the homebrew assay used in the GART study, we have obtained all of the original 153 patient samples from the GART study's original investigators, as well as the original sequencing results from the GART trial. Table 13 shows that the comparison of the genotyping data demonstrates high concordance.

The concordance between the GART homebrew assay and the ViroSeq HIV-1 Genotyping System is quite good (>95%), achieving the level needed to claim the clinical utility of the ViroSeq System. Also, the majority of the samples were successfully analyzed (149/153, or 97%), which allows the original conclusions from the analysis of the data for clinical utility to apply without reanalysis.Therefore, the above study supports the Celera claim that clinical utility is proven for the mutations detected and included in the GART study analysis when detected by the ViroSeq HIV-1 Genotyping System.

Table 10. Clinical Reproducibility Results

%CV Range

Source of Variation RT Protease

Site-to-site <0.1 <0.1

Lot-to-lot <0.1 <0.1

Intra-assay <0.1 <0.1 to 4.7 a

a For most of the samples analyzed, the intra-assay %CV was <0.1. Two samples had values of1.3 and 4.7.

Total <0.1 <0.1

Table 11. The mutations detected in this study include:


M41L E44D A62V K65R D67N S68G T69D K70R

L74V V75I F77L L100I K101E K103N V108I Y115F

F116Y V118I Q151M Y181C Y181I M184V G190A G190S

L210W T215Y T215F K219Q K219E

In Protease

L10I L10F L10V K20M K20R D30N V32I L33F

M36I M46I M46L I47V G48V F53L I54V L63P

A71I A71T A71V G73S V77I V82A V82T I84V

N88D L90M

Table 12. Additional Mutations Detected in the GART Study, But Not in the Original Study List of Analyzed Mutations


M41L A62V K65R D67N T69D K70R L74V V75I

V75T F77L K103N V106A F116Y Q151M Y181C Y181I

M184V Y188C L210W T215Y T215F K219Q K219E

In Protease

L10I L10R L10V K20M K20R L24I D30N M36I

M36L M46I M46L G48V I54V L63P A71T A71V

V77I V82A V82F V82T I84V N88D L90M

Table 13. Sample Statistics for the GART Concordance by Gene

Gene

RT Protease

Mean 0.969 0.954

Median 1.000 1.000

Standard Deviation 0.066 0.086

Standard Error of Mean 0.005 0.007

Minimum 0.652 0.538

Maximum 1.000 1.000


Section 6: Maintenance and Calibration of the Applied Biosystems®

3130 and 3130xl Genetic Analyzers

Required MaterialsFor illustrations and diagrams that explain how to conduct a specific task on the Applied Biosystems® 3130/3130xl Genetic Analyzer, refer to the Applied Biosystems® 3130/3130xl Genetic Analyzers Maintenance, Troubleshooting, and Reference Guide (PN 4352716).

Care of the Applied Biosystems®

3130 and 3130xl Genetic Analyzers

Maintenance

Follow the maintenance schedules below for optimal instrument performance.

IMPORTANT! The Applied Biosystems® 3130 and 3130xl Genetic Analyzers should be installed by a qualified Applied Biosystems field service engineer. Keep the instruments in a room with an ambient temperature of 15 to 30°C (59 to 85°F). Temperatures outside of this range, or high humidity, can cause condensation, decreased performance, and damage to the instrument.

Note: After opening the door of the Applied Biosystems® 3130 or 3130xl Genetic Analyzer, you must allow the autosampler homing sequence to complete before issuing further commands.

Tasks To Perform Before Each Run

Item

Applied Biosystems® 3130xl Genetic Analyzer with:• Data Collection Software v.3.0 • Firmware version 6286200-02 or 6286250-02• PC computer with:



3130XLIMPORTANT! Do not download software from the internet for this intended use.

Applied Biosystems®3130 Genetic Analyzer with:• Data Collection Software v.3.0 • Firmware version 6278250-02• PC computer with:




ABI PRISM® 3130 Capillary Array, 50 cm 4315930

ABI PRISM® 3130xl Capillary Array, 50 cm 4333466








Applied Biosystems® Sequencing Analysis Software v5.3.1 with KB™ Basecaller v1.4

4360967To obtain additional licenses, please contact your Applied Biosystems representative.

IMPORTANT! Do not download software from the internet for this intended use.


BigDye® Terminator Sequencing Standard v1.1 4336791



Contains Formamide.

R 61 May cause harm to the unborn child.R 36/38 Irritating to eyes and skin.S 53 Avoid exposure - Obtain special instructions before use.S 24/25 Avoid contact with skin and eyes.S 35 This material and its container must be disposed of in a safe way.S 36/37/39 Wear suitable protective clothing, gloves and eye/face protection.S 45 In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).

4311320

Ensure that the plate septa are firmly seated and flat.

Ensure that the plate assembly was put together properly.

IMPORTANT! Make sure the plate septa lies flat on the plate. The holes in the plate retainer must align with the holes in the septa or the capillary tips will be damaged.

Ensure that the plate assembly is positioned on the plate deck properly. Plate should sit snug on the deck.

IMPORTANT! Never use warped plates.

Replenish the deionized water and 1X Running Buffer reservoirs on the instrument.

Check for bubbles in the polymer block and polymer block channels. Remove all bubbles with the Bubble Remove Wizard.

Check that the capillary tips are not crushed or damaged.

Check the level of polymer in the polymer bottle to ensure there is enough for all your runs.

Check the lower polymer block to ensure it fits securely on the instrument.

Clean the instrument surfaces.

Check for dried polymer around the lower polymer block and clean as necessary.

Check for leaks around the lower polymer block, array knob and interconnecting tube nuts.

Item


Tasks to Perform Once Per Week

Tasks To Do As Needed

To Clean the Instrument

Resetting the Instrument

Reset the instrument when:• There is a fatal error as indicated by the red status light• The instrument does not respond to the Applied Biosystems® 3130 or 3130xl

Data Collection softwareThere are two ways to reset the instrument.

To Reset With the Reset Button

To Reset by Powering Down

Shutting Down the Instrument

To Perform a Short-Term Shutdown

Flush the water trap.

Clean the lower polymer block.

Replace the polymer using the Replenish Polymer Wizard.

Check the storage conditions of the used arrays.

Check data base space. Delete plate records from the instrument database and archive sample files when the database is getting full (approximately 70 to 75%). Refer to the Applied Biosystems ® 3130/3130xl Genetic Analyzers Maintenance, Troubleshooting, and Reference Guide (PN 4352716).

Restart the computer and instrument.

Maintenance Task Frequency

Run the Water Wash Wizard. Flush the array port during this wizard, whether or not there are bubbles present in the array port.

Every month or as needed

Defragment the hard drive. Every month

Clean the drip trays. As needed

Change the array. After 100 runs

Remove dried polymer from the capillary tips. As needed

Calibrate the autosampler. As needed

1. Press the Tray button on the front of the instrument to move the autosampler to the forward position.

2. Wipe off any liquid on or around the autosampler using a lint-free tissue.

3. Clean out the drip trays with deionized water and lint-free tissue.

4. Clean off any polymer build-up (crystals) on the instrument and the stripper plate with deionized water and lint-free tissue.

IMPORTANT! Never use organic solvents to clean the instrument.

5. Clean the array port knob, plug, or opening threads of these parts with moistened lab wipes.


2. Using a long narrow implement, such as a straightened paper clip, press the Reset button on the front of the instrument.


2. Turn off the instrument by pressing the on/off button on the front of the instrument.

3. Restart the computer.a. Select Start > Turn Off Computer.b. In the dialog box, select Restart and click OK.

IMPORTANT! Wait until the computer has completely restarted before proceeding.

4. Turn on the instrument and wait for the solid green light.

5. Launch the Data Collection software. (Service console application start automatically.)

If the instrument will be unattended for. . .

Perform this shutdown procedure. . .

No more than 1 week with a full buffer reservoir

Short-term.IMPORTANT! The key to a successful short-term shutdown is keeping the capillary array in 1X Running Buffer. This prevents the polymer from drying in the capillaries.

For more than 1 week Long-term.

1. Ensure that the oven and instrument doors are closed.

2. Collect polymer waste:a. Select GA Instruments > ga3130 or ga3130xl > instrument name >

Manual Control.b. In the Send Defined Command drop-down menu, select Autosampler.c. In the Command Name drop-down menu, select Move autosampler to

site.d. In the Value menu, select Waste.e. Click Send Command. Wait for the autosampler to stop moving and

Send Command becomes active, before proceeding.

3. Fill the capillary array with fresh polymer using manual control:a. In the Send Defined Command for drop-down menu, select Polymer

Delivery Pump.b. In the Command Name, select the appropriate Fill <length> cm

capillary array length.c. Click Send Command.The array fill is finished when Send Command

becomes active.d. Return the buffer reservoir to the capillaries.


To Perform a Long-Term ShutdownSelect the Instrument Shutdown Wizard and follow the prompts.

IMPORTANT! Make sure all parts are dry before long-term storage.

When to Replenish the Polymer

IMPORTANT! Always replace polymer that has been on the instrument more than one week. The polymer is good at 25°C for 7 days.

To Replace or Replenish the Polymer

CHEMICAL HAZARD. POP-6 polymer causes eye, skin, and respiratory tract irritation. Wear appropriate protective eyewear, clothing, and gloves.

Capillary Array

When to Change a Capillary Array

Expect your capillary array to last approximately 100 runs.A new capillary array may be needed if there is: • Poor sizing precision• Poor resolution and/or decreased signal intensity• Blocked or broken array

Before You Install a Previously Used Capillary Array

• Clean the front of the detection cell• Check that the cathode bar is dry

To Clean the Detection CellNote: This procedure is unnecessary for new arrays unless you have accidently touched the detection cell.

CHEMICAL HAZARD. Methanol is a flammable liquid and vapor. Exposure may cause eye, skin, and respiratory tract irritation, and central nervous system depression and blindness. Wear appropriate protective eyewear, clothing, and gloves. Refer to the manufacturer’s Safety Data Sheet for additional information.

To Check the Cathode BarWhen putting a used array back on the instrument, be sure that the cathode bar is dry. A wet bar could lead to arcing.

ELECTRICAL SHOCK/FIRE HAZARD. Do not leave liquid in the cathode bar. This can lead to electric shock or even fire if not properly maintained.

To Install or Remove the Capillary Array Using the Wizard

IMPORTANT! Wear gloves while performing the following procedure, and any other time you handle the capillary array, septa, or buffer reservoirs.


4. Cleaning the Reservoirsa. Press the Tray button to move the autosampler forward.b. Open the doors, then remove the:

– Plates– Cathode buffer reservoir and water reservoirs

c. Dispose of remaining fluids and rinse out the reservoirs with deionized water.

Note: Follow your company’s waste disposal practices for appropriate disposal procedures.d. Rinse the cathode reservoir with 1X Running Buffer, and then fill to the

line with 1X Running Buffer (about 16 mL).e. Fill the three water reservoirs to the line with quality deionized water

(about 16 mL). Ensure that the septa fits snug and flush on the tops of the reservoirs in order to prevent damage to the capillary tips.

f. Place a clean reservoir septa on each reservoir, and dry the outside of the reservoirs using a lint-free wipe.

g. Place the reservoirs into position on the autosampler as shown below.

h. Close the instrument doors.Note: Closing the doors returns the autosampler to the home position, placing the tips of the capillaries in buffer.i. Shut down the computer and turn off the instrument.

If polymer on the instrument. . . Then. . .

has been on less than one week and is sufficient in quantity to complete your runsa

a A 3130xl Genetic Analyzer run uses 50 to 80 µL of polymer and a 3130 Genetic Analyzer runuses ~25 to 40 µL of polymer.

Remove all bubbles, and proceed with instrument preparation.

has been on less than one week, and is insufficient in quantity to complete your runs

Add fresh polymer to the polymer supply by following the Replenish Polymer Wizard.

has been on longer than one week Replace with fresh polymer by following the Replenish Polymer Wizard.

is the wrong type (a change between POP-4, POP-6, and/or POP-7 polymers is required)

Replace the installed polymer type with a different type by following the Change Polymer Type Wizard.

Water reservoir(waste)

Cathode reservoir(1X Running Buffer)

Water reservoir(rinse)

Water reservoir (spare)

Home position

1. Select Wizard > Replenish Polymer Wizard.

2. Follow the directions given in the wizard to put fresh polymer on the instrument.

1. Put one drop of methanol on the front surface of the detection cell.

2. Allow the cell to air dry.

Note: Do not use pressurized air to dry the detection cell.


To Update the Capillary Array Information

Use the Update Cap Array Info Wizard to:• Update the capillary array length and serial number information into the database• Correct an entry error after using another wizard

IMPORTANT! The capillary array length defined in the wizard must match the array length you are using.

Maintenance

To Care for the Capillary Array and Work Area• Wear gloves and handle the capillary array gently.• Do not touch the detection cell. If it is dirty, refer to To Clean the Detection

Cell.• Keep the ends of the capillary array wet at all times.• Do not overtighten the capillary array knob.• Clean off any polymer buildup (crystals) on the instrument, including the

capillary electrodes and the stripper plate, with deionized water and lint-free tissue.

To Clean the Capillary Array


To Fill the Capillary Array with Polymer Using Manual Control

To Store a Capillary Array On the InstrumentStore the capillary array on the instrument only when the capillary array will be unused for less than 1 week.To store the capillary array on the instrument, perform a short-term shutdown.

To Store a Capillary Array Off the InstrumentStore the capillary array off of the instrument when the capillary array will be unused for longer than 1 week. Store at 15 to 25°C.

IMPORTANT! Before storing the capillary array for long periods, we recommend filling the capillaries with fresh polymer.


IMPORTANT! Wear gloves while performing the following procedure, and any other time you handle the capillary array, septa, or buffer reservoirs.

1. Close the oven and instrument doors, and then press the Tray button.

2. Select Wizards > Install Array Wizard.

3. Open the oven and instrument doors.

4. Follow the directions given in the wizard to replace or install an array.

IMPORTANT! For the ViroSeq assay, use a 50 cm capillary array. The capillary array length that you are using must match the capillary array length in the instrument database.

Note: Proper capillary array information (e.g., capillary array length and lot number) must be entered.

5. Check that the capillary tips are not crushed or damaged.

6. Click Finish.

7. Close and lock the oven door, then close the instrument door.

1. Flush the capillary array with fresh polymer as instructed in the section To Fill the Capillary Array with Polymer Using Manual Control below.

2. Clean off any polymer buildup (crystals) on the instrument, including the capillary electrodes and the stripper plate, with deionized water and lint-free tissue.

Note: When cleaning the capillary electrodes, be careful not to bend them out of position.

IMPORTANT! Never use organic solvents to clean the instrument.

3. Clean the detection cell.

1. Select GA Instruments > ga3130 or ga3130xl > instrument name > Manual Control.

2. In the Send Defined Command drop-down list, select Polymer Delivery Pump.

3. In the Command Name drop-down list, select Fill 50 cm capillary array.

4. In the Value drop-down list, select 50 cm for array length and POP-6 for polymer.

5. Click Send Command. The array fill is finished when Send Command becomes active.

6. Return the buffer reservoir to the capillaries.

1. Remove the capillary array from the instrument by selecting Install Array Wizard.

2. Select Store Array and follow the prompts.

3. Replace the cover over the detection cell.

4. Fill a buffer reservoir with fresh 1X Running Buffer and cover with a septa strip. Insert the capillary tips into the buffer.

5. Fill the shipping vial with fresh 1X Running Buffer and insert the detection end of the capillary array.

6. Store the capillary array upright.

7. Check the 1X Running Buffer level in the reservoir and tube weekly and replenish the buffer as needed.


Flushing and Filling the Water Trap

OverviewThe polymer delivery pump (PDP) water trap should be flushed with either distilled or deionized water at least once per week to wash out any diluted polymer and to clear bubbles. Leave the trap filled with either distilled or deionized water.

To flush the water seal trap:

Water Wash

When to Use the Water Wash WizardWhen dried polymer is observed in the lower polymer block, use the following procedure.

Polymer Block

Polymer Block Care

Avoiding Damage to the BlocksThe pump and lower polymer blocks can be irreversibly damaged if:• Polymer dries in the polymer channels of the pump or lower polymer block• The inside surface of the channel is scratched• The block is exposed to organic solvent• The block is exposed to temperatures greater than 40 °C

IMPORTANT! Use clean, powder-free, silicone-free latex gloves whenever you handle the polymer blocks or any item in the polymer path to minimize background fluorescence.

When to Clean the Polymer Block Using the Lower Polymer Block Cleaning Kit

In nearly all circumstances, the Water Wash Wizard is very effective in cleaning the polymer delivery pump (PDP), including the lower polymer block. Occasionally, the Water Wash Wizard may not sufficiently clean the polymer block, and the Lower Polymer Block Cleaning Kit (Applied Biosystems PN 4359572) should be used. Refer to the Applied Biosystems Lower Polymer Block Cleaning Kit Protocol (PN 4363356 Rev. A).

Note: • Polymer has dried in the channels of the lower block due to maintenance

oversight.Problems such as electrical arcing or mechanical malfunctions may cause dried polymer to appear in the lower polymer block. Washing with either the Water Wash Wizard or this kit may not correct these problems- replacing the lower polymer block may be necessary.

• Some contaminant in the lower polymer block is suspected of causing problems.• A replacement lower polymer block is to be installed. In this case, follow the

protocol for removing the installed lower polymer block, then clean and install the replacement lower polymer block.

IMPORTANT! Do not expose the polymer blocks to any organic solvents.

IMPORTANT! Use clean, powder-free, silicone-free latex gloves whenever you handle the polymer blocks or any item in the polymer path to minimize background fluorescence.

Removing the Lower Block

CHEMICAL HAZARD. POP-6™ polymer cause eye, skin, and respiratory tract irritation. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.

To remove the lower polymer block if the channel is not completely blocked with dry polymer:

1. Fill the 20 mL, all-plastic Luer lock syringe (supplied in the Lower Polymer Block Cleaning Kit, PN 4359572) with distilled or deionized water. Expel any bubbles from the syringe.

Note: Do not use a syringe smaller than 20 mL. Doing so may generate excessive pressure within the trap.

2. Attach the syringe to the forward-facing Luer fitting at the top of the pump block. Hold the fitting with one hand while threading the syringe onto the fitting with the other hand.

3. Open the Luer fitting by grasping the body of the fitting and turning it and the attached syringe approximately one-half turn counterclockwise.

4. Open the exit fitting at the top left side of the pump block by turning it approximately one-half turn counterclockwise.

5. Hold an empty tube or beaker under the exit fitting to receive approximately 5 mL of waste. Flush the trap by pushing steadily on the syringe plunger.

IMPORTANT! DO NOT USE EXCESSIVE FORCE when pushing the syringe plunger as this may damage the trap seals. It should take approximately 30 seconds to flush 5 mL of either distilled or deionized water through the trap.

Note: Because the water trap volume is approximately 325 L, a relatively small volume of water is required to completely flush the water trap. However, a larger volume improves flushing as long as force and flow rate are within the limits given above.

6. Close the fittings in this order by turning each clockwise until the fittings seal against the block:a. Luer fittingb. Exit fitting

IMPORTANT! Do not over-tighten the fittings. Very little pressure develops within the trap during pump operation, so the fittings require only enough tightening to prevent water leaks. Excessive tightening can damage the fittings.

c. Remove the syringe from the Luer fitting. Hold the fitting with one hand while turning the syringe counterclockwise with the other hand.

1. Select Wizards > Water Wash Wizard.

2. Run the Water Wash Wizard to wash the PDP chamber, lower polymer block, channels, and tubing with distilled or deionized water.

1. Run the Water Wash procedure:a. Select Water Wash Wizard:b. Follow the instructions in panels 1 and 2.c. Exit the wizard after executing the water wash procedure at step 2.d. Click Cancel and then Yes in the dialog box to leave the wizard


Dried Polymer Blocking ChannelTo remove the lower polymer block if the channel is completely blocked with dry polymer.

Cleaning the Lower Polymer Block

Reinstalling the Lower Polymer Block

2. Open the buffer valve from the Manual Control panel of Data Collection by selecting:

3. Verify that the buffer valve is open (valve lever in the up position).

4. Open the oven and detection block doors.

5. If you have not already removed the buffer jar during the wizard, do so now and set it aside to avoid spilling the contents.

6. Remove the array comb nearest to the detection cell.

7. Loosen the array knob about halfway by turning it counterclockwise.

8. Pull the pump and lower polymer blocks forward to the stop position on the alignment pins. The detection cell comes out of the holder

9. Attach the detection cell cover to the detection cell to protect the window.

10. Completely loosen the connecting nut for the interconnect tube at the lower polymer block.

11. Pull the lower polymer block off its mounting pins and away from the interconnect tube. This operation may be slightly awkward because of resistance from the stiff interconnect tube.

1. Follow the steps under Removing the Lower Polymer Block starting with step 2, omitting the Water Wash as described in step 1.

2. Immerse the removed block in warm (40°C or below) water.

3. Inspect the block and replace the warm water as necessary until the block can be cleaned using the procedure that follows (refer to Cleaning the Lower Polymer Block.). If the block cannot be cleared sufficiently for cleaning, a new lower polymer block should be installed.

1. Rinse all the fittings with warm (40 °C or below) purified (distilled or deionized) water to remove dried polymer.

IMPORTANT! Do not use water above 40°C to rinse the fittings or the polymer block.

2. With the lower polymer block in warm water (40 °C or below), move the buffer valve in and out to ensure any encrusted polymer is removed from the guide channel.

IMPORTANT! Do not remove any of the components from the lower polymer block.

IMPORTANT! Do not twist the buffer valve against the seat.

Send Defined Command for: Buffer Valve

Command Name: Close/Open buffer valve

Value: Open

Click Send Command

3. Fill the 20-mL silicone-free plastic syringe (P/N 4324463) with warm water(40°C or below).

4. Fit the 6-mm plastic syringe adaptor (P/N 4322928) onto the 20-mL silicone-free plastic syringe (P/N 4324463).

5. Thread the 6-mm plastic syringe adaptor into the polymer block where theinterconnect tube was originally attached.

6. Force several plastic syringe volumes of warm water (40 °C or below) through the channel.

7. Inspect the channels for dried polymer, which appears as white residue. Wash partially obstructed channels with warm water (40°C or below) until the dried polymer is removed.Verify that all polymer is removed before proceeding.

IMPORTANT! Some time may be required for the warm water to clear the obstruction. Do not use any pointed or sharp objects to clear the channel, even if the channel is completely obstructed with dried polymer.

8. Remove the syringe and syringe adapter.

9. Rinse the lower polymer block and all the fittings a final time with water.

10. Dry the fittings and exterior surfaces of the lower polymer block with lab wipes.

IMPORTANT! Do not use compressed gas to blow water from the channels, valve, or any part of the lower polymer block.

1. With the pump block in the forward stop position, slip the interconnect tube from the pump block loosely into the socket of the lower polymer block while sliding the block into the mounting pins. This operation may be slightly awkward because of resistance from the stiff interconnect tube.

2. Verify that the end of the interconnect tube is flush with the bottom of the connector port. Tighten the interconnect tube connection to the lower polymer block. Check again to make sure that the end of the interconnect tube and the bottom of the lower polymer block connector port are in contact.

3. Remove the detection cell cover from the detection cell window.

4. IMPORTANT! Push the pump and lower polymers blocks back against the rear the pump panel. Verify that the buffer valve pin engages the valve lever.

5. Insert the array comb nearest the detection cell into its holder in the oven.

6. Carefully insert the array detection cell into the detection cell holder, making certain that it is mounted flat and secure in the mounting.

7. Close and secure the detection block door.

8. Tighten the array knob into the pump block.

9. Close and secure oven and instrument door.

10. Run the Water Wash Wizard to flush the system with water and then to replace the water in the PDP with polymer. Since the water bottle is already at the polymer supply position, verify that you have enough water volume when starting the wizard.


To Remove Air Bubbles from the Polymer Block

Spatial and Spectral Calibration

To Perform A Spatial Calibration

When to PerformYou must perform a spatial calibration whenever a new or different capillary array is installed on the instrument, the array is temporarily removed from the detection block, or the instrument is moved. Note: The Applied Biosystems® 3130 and 3130xl Genetic Analyzers should be moved by a qualified Applied Biosystems field service engineer.A spatial calibration maps the position of each capillary detected on the CCD camera. A spatial calibration with filling of the capillaries takes approximately six minutes. During this time, multiple frames of data are collected and summed. The collected data is analyzed and saved as a spatial map.

How To Perform

To Evaluate a Spatial Calibration File and Save the Data

If the Spatial Calibration Fails• Repeat the spatial calibration.• Fill the capillaries with polymer, and then repeat the spatial calibration.• Clean the detection cell, and then repeat the spatial calibration.• Reposition the array window in the detection cell, and then repeat the spatial

calibration.• Try another capillary array.• Contact an Applied Biosystems field service engineer to check laser alignment.

Refer to the Applied Biosystems® 3130/3130xl Genetic Analyzers Maintenance, Troubleshooting, and Reference Guide.

To Display a Spatial Calibration Profile

To Perform A Spectral Calibration

When to PerformYou must perform a spectral calibration:• Whenever you use a new dye set on the instrument• When you change capillary array length• When you change polymer type• Whenever a service engineer realigns/replaces the laser, optics, or CCD camera• If you begin to see a decrease in spectral separation (pull-up and/or pull-down

peaks)

For spectral calibration of the ViroSeq System, use BigDye® Terminator Sequencing Standard v1.1 (Applied Biosystems PN 4336791).

What Happens?Run the spectral standards in all 16 or 4 capillaries. The Data Collection software then:• Collects the data and stores it in 16 or 4 separate temporary files• Analyzes the data and generates a matrix for each capillary• Stores the spectral calibration data for the dye set run

How To PerformA spectral calibration is a run that produces a mathematical description of the overlap in the emission spectra of a given dye set. This mathematical description is called a matrix, and is required for each dye set. The process of applying a matrix to sample data is called multicomponenting.

1. Select Wizards > Bubble Remove Wizard to clear bubbles.

IMPORTANT! Remove bubbles from the interconnect tubing and the channel of the lower polymer block. These areas are part of the electrophoresis current path. Absence of bubbles in the current path is important for problem-free electrophoresis.

2. Replace the buffer if excess polymer is expelled into the anode buffer jar.

1. Select GA Instruments > ga3130 or ga3130xl > instrument name > Spatial Run Scheduler.

2. In the Spatial Protocols section, select one of the following:• If the capillaries contain fresh polymer, select

Protocol > 3130SpatialNoFill_1• Otherwise, select Protocol > 3130SpatialFill_1 The capillaries do not have to be filled each time a spatial calibration is performed.

3. Click Start.

Note: The spatial profile window will be displayed as black when the spatial calibration is started.

1. Evaluate the spatial calibration profile.While viewing the calibration profile, use the following criteria to evaluate the data:

Peak Attribute Criteria

Height Similar heights for all peaks

Orange crosses One orange cross marking the top of every peak. No misplaced crosses. To move a cross:

a. Change the value in the Positions (pixels) box.b. Click outside of that box.c. Click Enter using the keyboard.

Shape • Single sharp peak for each capillary.• Small shoulders are acceptable.

Spacing The difference between adjacent positions is 13 to 16 pixels. Theoretical spacing between capillaries is 15.

2. Accept or reject a spatial calibration as follows:

1. To view a current spatial calibration profile, select GA Instruments > ga3130 or ga3130xl > instrument name > Spatial Run Scheduler.

2. To view a previous spatial calibration profile:a. In the Data Collection Viewer window, click GA Instruments >

ga3130 or ga3130xl > Run History.b. In the Run History, search for the run with the associated spatial

calibration profile you want to view or select Find all. c. Select Spatial Calibration Viewer (under the Run History icon).d. Select the run with the associated spatial calibration profile you want to

view from the Run to view pull-down list.

If the Spatial Calibration Profile. . . Then. . .

Passed Click Accept to write the calibration data to the database and .ini file.

Failed Click Reject, then refer to If the Spatial Calibration Fails.


To Prepare and Load the BigDye® Terminator Sequencing Standard onto the 3130 or 3130xl Genetic Analyzers

To Create a ViroSeq Spectral Instrument Protocol

To Create a Spectral Plate Record

To Start the Calibration

1. Resuspend a tube of the BigDye Terminator Sequencing Standard v1.1 with 170 L of Hi-Di formamide.


2. Vortex the standard 10 to 15 seconds to mix well.

3. Spin the mixture for 10 to 15 seconds at 2,000 g in a microcentrifuge.

4. Dispense 10 µL of the standard into the MicroAmp Optical 96-Well Reaction Plate, loading wells A1 through H2 for the 3130xl Genetic Analyzer or wells A1 through D1 for the 3130 Genetic Analyzer.

5. Cover the plate and centrifuge it for 5 to 10 seconds at 2,000 g.

6. Heat the plate at 95°C for 2 minutes to denature the standards.

7. Quick chill the plate on ice for 2 minutes.

8. Immediately load the plate onto the 3130 or 3130xl Genetic Analyzer.

IMPORTANT! Do not store the plate.

1. Select GA Instruments > ga3130 or ga3130xl > Protocol Manager to open the Protocol Manager window.

2. In the Instrument Protocols pane, click New. The Protocol Editor dialog box opens.

3. Complete the Protocol Editor dialog box by entering information or making selections from drop-down lists:a. Name: ViroSeq_spectral_instrument_protocol.b. Description: enter a description for the protocol (optional).c. Type: Spectrald. Dye Set: E-BigDyeV1e. Polymer: POP6f. Array Length: 50g. Chemistry: Sequencing Standardh. Run Module: Spect50_POP6_1

4. Click OK.


2. Click New to open the New Plate Dialog box.

3. Complete the New Plate dialog box:a. Name: enter a name for the plate.b. Description: enter a description for the plate record (optional).c. Application: Spectral Calibration.d. Plate Type: 96-Well.e. Owner Name: enter a name for the owner.f. Operator Name: enter a name for the operator.g. Click OK.

4. Complete the Spectral Calibration Plate Editor dialog box:a. Sample Name: enter the name for the samples.b. Comment: enter any additional comments or notations (optional).c. Priority: the value 100 automatically displays in the column.d. Instrument Protocol 1: select the

ViroSeq_spectral_instrument_protocol. Refer to To Create a ViroSeq Spectral Instrument Protocol.

e. Click OK.

IMPORTANT! Verify that you have selected the correct spectral instrument protocol file. Selecting the incorrect instrument protocol file will cause the spectral calibration to fail.

1. Select GA instruments > ga3130 or ga3130xl > instrument name > Run Scheduler > Plate View.

2. Search for the spectral plate record. There are two search options:• Find All • AdvancedUnder Find All:• Select Barcode in the Type of Search drop-down list. • If you have a limited number of plates in the database, click Find All. All

plates in the database will display in the plate record section.Under Advanced:a. Select Advanced in the Type of Search drop-down list.b. Use the drop-down list to define search conditions for a category or

multiple categories (Run Name, Results Group Name, Plate Name, etc.)

c. Use the Plate Name for the Plate ID category.d. For each category with a condition selected, type a value (primary search

string) in the Value 1 column.e. Click Search. All plates in the database that match the search criteria

display in the plate record section.

3. Select the plate record, then click the plate position indicator that corresponds to the plate being linked. The plate position indicator changes from yellow to green when linked and the green Run Instrument button

is active.

4. Click the Run Instrument button to start the run. The run time is approximately 95 minutes.

5. At the end of the run, the calibration results are generated. The spectral calibration profile is updated and saved to the instrument database unless all capillaries fail.


Evaluate a Spectral Calibration Profile

To View the Pass/Fail Status After the Spectral Calibration Run

To Evaluate the Spectral Profile and Raw DataNote: Refer to the Applied Biosystems® 3130/3130xl Genetic Analyzers Maintenance, Troubleshooting, and Reference Guide if:

• the spectral calibration fails (no spectral profiles are created), • the peaks are not in the correct order or extraneous peaks are adversely

affecting the spectral profile, or• the peaks are not separate and distinct.

If a Capillary FailsA failed capillary is automatically assigned the spectral profile of its nearest passing capillary to the left. If there are no passing capillaries to the left, it will be assigned the profile of the nearest passing capillary to the right. These capillaries are marked in tan instead of green in the Array Viewer.

IMPORTANT! For ViroSeq analysis, where pull-up and pull-down peaks will cause critical errors, repeat the spectral calibration and use a unique spectral for each capillary.

1. Select GA Instruments > ga3130 or ga3130xl > instrument name > Instrument Status > Event Log.

2. In the Events Messages section of the window, view the status of each capillary.

1. Select GA Instruments > ga3130 or ga3130xl > instrument name > Spectral Viewer.

2. In the Dye Set drop-down list, select E-BigDyeV1.

3. In the plate diagram, select a well on the plate diagram to view the capillary spectral results.

Note: A failing capillary is automatically assigned the spectral profile of its nearest passing capillary.

IMPORTANT! Evaluate the spectral calibration profile for each capillary, even if the Spectral Calibration Results box indicates that they passed.

Capillary status:

Passed (dark green)

Selected (light green)

Failed, Not Selected (light tan)

Failed (tan)

4. Evaluate the spectral profile for the selected capillary.

Note: To magnify an area of interest in the spectral profile or raw data, click-drag the cursor to create a box. Release the mouse button to display the selected region. Press the r key to reset the view.a. Verify that the order of the peaks in the spectral profile from left to right

are blue-green-yellow-red.

b. Verify that the peaks in the spectral profile do not contain gross overlaps, dips, or other irregularities (e.g., wide peaks that overarch other peaks; peaks that are not distinct).

5. Click each well to review the data for each capillary. For a good-quality calibration, each capillary must have a:

– Q-value above 0.95– Condition number within the range 3.0 to 5.0

Example of a 4-dye spectral profile

Blue Green Red Yellow

Peaks are distinct, regular and in the proper order – pass

Red peak is not distinct, regular or in the proper order – fail


If the Calibration FailsTry one or more of the following:• Verify that the correct parameter file and run module were selected. If not, make

the correction and repeat the run.• Verify that fresh reagents were used. If the reagents used were not fresh, stored

improperly, or expired, prepare fresh reagents and repeat the run.

To Activate a Spectral CalibrationIMPORTANT! A run cannot start unless a calibration file that matches the dye set and capillary array length combination to be used for the run, is active.

IMPORTANT! Every new spectral calibration is automatically the active one for that dye set.

Setting an Active Spectral Calibration

To Display a Spectral Calibration Profile

Autosampler

To Calibrate

Calibrate the autosampler if you see:• Poor injection for a small number of capillaries• Low signal strength• No evidence of sample

1. Select GA Instruments > ga3130 or ga3130xl >instrument name > Spectral Viewer.

IMPORTANT! If the Spectral Viewer window is blank and deactivated, then either:

– The spectral calibration for that dye set is not in the database, or, – You changed the array length and you do not have a spectral

calibration file activated for that dye set and array length combination.

2. In the Dye Set drop-down list, select E-BigDyeV1.

3. In the List of Calibrations for Dye Set drop-down list, select a spectral calibration. The spectral profile and raw data is displayed.

4. If the spectral calibration is acceptable, then click Set. Otherwise, run a new spectral calibration.

1. To view a current spectral calibration profile, select GA Instruments > ga3130 or ga3130xl > instrument name > Spectral Viewer.

2. To view a previous spectral calibration profile:a. Select GA Instruments > ga3130 or ga3130xl > Run History.b. In the Run History, search for the run with the associated spectral

calibration profile you want to view or select Find all. c. Select Spectral Calibration Viewer (under the Run History icon).d. Select the run with the associated spectral calibration profile you want to

view from the Run to view pull-down list.

1. Select Wizards > Autosampler Calibration Wizard.

2. Follow the directions in the wizard.


Section 7: Bibliography

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Appendix A: Auditing and Software Access Control 7

This appendix is written for the Administrator who implements auditing and access control to the Data Collection software on the Applied Biosystems 3130/3130xl Genetic Analyzer instruments. For more information, refer to the Applied Biosystems AB Navigator Software Administrator Guide (PN 4359472).

AB NavigatorAB Navigator is the access point for these applications:

• Access Control Administration• Audit History Viewer• Audit Map Configuration

To Start the AB Navigator

IMPORTANT! At a minimum, you must start the first two Data Collection software services in order for AB Navigator to function properly.

Enabling Applications

To Enable Access Control Administration

To Enable Features of the Audit Map Configuration

Audit Map Configuration Tool

The Audit Map Configuration Tool is used to manage audit maps. Audit maps are used to control how auditing is done for a given data type. Some features of the Audit Map Configuration Tool:

• Audit states of an audit map can be set to On, Off, or Silent.• There is no Save command. All changes to audit maps are saved

automatically.Note: Access to the Audit Map Configuration is restricted to those with an Administrator Profile.When auditing is ON and a change occurs, the Reason(s) for Change dialog displays and contains:

• The attribute that was changed, created, or deleted• The old and new values, if applicable, in the top half of the dialog box• A text box to enter the reason for the change:

– When you click OK, changes to the attribute and the audit data are saved

– When you click Cancel, no changes are saved and you return to the previous window.

When auditing is Silent, auditing still occurs in the database but the Reason(s) for Change dialog does not display.

IMPORTANT! Always run with Auditing enabled.

1. Start the Data Collection Software v3.0: Start > All Programs > Applied Biosystems > Data Collection > Run Data Collection 3130 or Run Data Collection 3130xl.

2. Start the AB Navigator application: Start > All Programs > Applied Biosystems > AB Navigator.

3. In the AB Navigator dialog box, type “Administrator” (default login name and password) for the Login Name and Password or type your login name and password if you have changed them and click OK.

IMPORTANT! Record your login name and password. Failure to remember the login and password may require uninstalling and reinstalling the Data Collection software!

4. In the AB Navigator window, click Administration to expand the Audit Map Configuration, Audit History Viewer, and Access Control Administration options.

1. In the AB Navigator window, double-click Access Control Administration.

2. Select Applications > FoundationViewerApp.

3. Select the Challenge check box to activate it. The Login and Password dialog box is now enabled.

4. Select File > Save.

5. Exit Access Control Administration.

1. In the AB Navigator left -pane, double-click Audit Map Configuration.

2. Select the Enabled check box for each audit map to activate it.• DC Plate Record• DC Run Module• DC Results GroupNote: An audit record is generated in Data Collection software when you:

• Create or edit a Plate Record• Create a run module or edit the parameters of a Run Module• Create or edit a Results Group

3. Exit Audit Map Configuration.

1. Double-click the Audit Map Configuration icon in the left pane tree.The System Authentication dialog box displays.

2. Type your password in the System Authentication dialog box

3. Click OK.The Audit Map Configuration window displays.

4. Individually select a cell in the Enabled column in the Audit Map Objects pane for DC Run Module, DC Results Group, and DC Plate Record. After each selection, enter the reason(s) in the Reason(s) for Change dialog box and click OK.


Audit History Viewer

The Audit History Viewer is used to view historical audit data. This tool is used as a read-only viewer for audit records. The tool provides data filtering so that Audit Records can be viewed in different formats.

Note: Access to the Audit History Viewer is restricted to those with an Administrator Profile.Audit Records that you can view with the Audit History Viewer are:

• Date and time the Audit Record was created• The user who triggered the Audit Event• The Attribute that was changed• The old and the new values• The reason for the change

To Start the Audit History Viewer

To View an Audit History

Filter Commands in Audit History

To Use Filter Command in Audit HistoryThe filter allows you to limit the audit history records that are displayed. 1. Click Filter. The Filter Audit Records pane displays. 2. Enter search criteria in the applicable text boxes.3. Click Find Now. You can filter audit records by:

• Name• Date (includes before or after a date or between two dates)• User name• Matching whole words• Case-sensitivity

Access Control Administration

The Access Control Administration tool allows an administrator to manage the creation, deletion, and modifications of users and profiles. It also allows an administrator to restrict or grant users access to features and functions of the software.

IMPORTANT! Do not misplace the administrator password! If the administrator password is lost, access control must be reset, causing the loss of all users and profiles.

The default administration user is always associated with the Administration User Group and cannot be deleted. In addition, only one administrator is allowed to modify access control data at one time.

To Create a New UserIMPORTANT! You must set a default password for each new user.

5. Individually select the object (e.g., DC Run Module) in the Audit Map Objects pane then select an attribute in the Attributes pane. From the State drop-down menu, select ON for each attribute. Enter text in the Reason(s) for Change dialog box and click OK. Repeat this step until all of the respective attributes are in the ON state.

6. Exit Audit Map Configuration.

7. Restart the software.

IMPORTANT! Any changes to an audit map do not take effect until you restart the software

1. Start the AB Navigator. Refer to To Start the AB Navigator section.

2. In the AB Navigator left Viewer window, double-click Audit History Viewer.

3. In the Audit History Viewer dialog box, type Administrator for the Login Name and Password or type your login name and password if you have changed them and click OK. The Audit History Viewer displays.

1. In the Audit Objects pane, navigate to the object of interest

2. Highlight an object and then select View > Details Panel to display audit record details.

Note: Click the column headers to sort the read-only columns.

Toolbar Menu Command Function

File Reload Refreshes the Audit History Viewer with the latest changes.

Report Customize and then print a report of the selected Audit History Record.

Print Preview Customize and then preview a report of the selected Audit History Record.

Page Setup Customize the page setup of the Report printout.

State Change Description Parameter

ON Reason for change required Records old and new values

OFF Reason for Change dialog box does not display

Does not record old or new value changes

SILENT Reason for Change dialog box does not display

Records old and new values

Go To Displays a list of applications that are currently running. Select an application to go to that application.

Exit Application Exits the Audit Map Configuration application.

Exit AB Navigator

Exits the AB Navigator application.

View Filter Displays the filter pane on the top of the frame when selected. It allows the user to specify criteria that limits the amount of audit records in the Audit Record table.

Default Profiles:

Administrator Complete access to Instrument Protocols, Instrument Operation, and Instrument Maintenance

Scientist Complete access to Instrument Protocols, Instrument Operation, and Instrument Maintenance

Technician Access to Instrument Operation and Maintenance

Access Rights:

Instrument ProtocolsIncludes Run Module Operations, Results Group Operations, Analysis Protocol Operations, Instrument Protocol Operations and Re-extraction.

Instrument Operation Includes Plate Operations, Event Log, and Instrument Control Operations.

Instrument Maintenance Includes Spatial Calibration Operations, Manual Instrument Control, and Miscellaneous Operations.

1. Click the New User icon. The New User dialog displays.

2. Click Next. The Configure pane displays.

3. Complete the information in the Create User window, click Set Password. enter the new password and click OK.

4. Click Finish to complete the creation of a new user.

5. Click Save.

Toolbar Menu Command Function

DRAFTOctober 12, 2009 2:57 pm, ViroSeq IFU 3130 Back.fm

© 2001-2009 Celera Corporation. All rights reserved.

NOTICE TO PURCHASER: LIMITED LICENSE

This product is sold under licensing arrangements between Celera Corporation and Life Technologies Corporation. The purchase price of this product includes limited, nontransferable rights under US Patents 5,035,996; 5,683,896; 5,945,313 and 6,287,823 and foreign equivalents owned by Life Technologies Corporation to use only this amount of the product to practice the claims in said patents solely for activities of the purchaser within the field of human diagnostics. Further information on purchasing licenses under the above patents may be obtained by contacting the Licensing Department, Life Technologies Corporation, 1600 Faraday Avenue, Carlsbad, CA 92008.

U.S. Patent No. 6,806,046



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Printed in USA, 10/2009

ViroSeq 2.8 English 5002961-RevA 3130

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Transcript of ViroSeq 2.8 English 5002961-RevA 3130