3500 Series Data Collection Software v3 · The 3500 Series Data Collection Software v3 .1 supports...

USER BULLETIN

For Research Use Only. Not for use in diagnostic procedures.

3500 Series Data Collection Software v3.1Pub. No. MAN0014110 Rev. A.0

SUBJECT: New Features and HID Validation Summary

■ Purpose of this user bulletin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

■ Overview of 3500 Series Data Collection Software v3.1 . . . . . . . . . . . . . . . . . . . . . . 2

■ New features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

■ Upgrades from previous versions of the software . . . . . . . . . . . . . . . . . . . . . . . . . . 6

■ Documentation and support. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

■ HID validation summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

■ Materials and samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

■ Methods and data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

■ Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

■ Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

■ Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Purpose of this user bulletin

This user bulletin:

• Provides an overview of the 3500 Series Data Collection Software v3.1.• Describes new features and upgrades for the 3500 Series Data Collection Software

v3.0 and v3.1 that are relevant for performing Human Identification (HID) applications.

• Describes HID validation of the 3500 Series Data Collection Software v3.1, POP-4™ Polymer for 3500/3500xL Genetic Analyzers stability, and 3500 Series buffer stability. HID validation includes methods, analysis, results, and conclusions.

Note: For information on the 3500 Series Data Collection Software v3.0 HID validation, refer to 3500 Series Data Collection Software v3.0 User Bulletin (Pub. no. MAN0010812).

2 3500 Series Data Collection Software v3.1 User Bulletin

Overview of 3500 Series Data Collection Software v3.1

Overview of 3500 Series Data Collection Software v3.1

Benefits of the software

The 3500 Series Data Collection Software v3.1 supports genotyping applications on the 3500 and 3500xL Genetic Analyzers. It integrates instrument control, data collection, consumable and maintenance quality control, and size- and basecalling functions into a single software program. The software streamlines instrument setup, plate setup, data collection, and preliminary data review.

With its user-friendly navigation and intuitive dashboard design, the 3500 Series Data Collection Software v3.1 lets you assess the quality of your data as it is analyzed on the instrument. By providing immediate access to size- and basecalled data, you can make decisions about the quality of your data as it is generated, without transferring output files into secondary analysis software packages.

The system includes pre-configured protocols and plate templates to support rapid and efficient fragment and sequencing run setup. Additionally, it offers Security, Audit, and Electronic-Signature (E-Sig) features to enable you to comply with 21CFR Part 11 requirements.

Features of the software

• Dashboard: Provides easy access to common operations. Displays instrument status, an overview of consumable usage, and reminders for scheduled tasks.

• Spatial and Spectral Calibrations: Enable instrument calibration for different types of consumables and operating conditions. You can review the calibration results and choose to accept or reject results. Reports can be generated and results can be exported. With the History View, you can view recent calibrations.

• Install Check for Sequencing: Verifies that the instrument meets the sequencing read-length specifications. Easy-to-read reports are provided to review Install Checks. Additionally, with the History View, you can review recent records of performed Install Checks.

• Install Check for Fragment/HID Analysis: Verifies that the instrument meets fragment/HID analysis capability and sizing precision needs. Easy-to-read reports are provided to review the Install Check. Additionally, with the History View, you can review recent records of performed Install Checks.

• Plate Setup: Provides feature-rich plate and table views to make plate setup easy. You can assign and create resources such as Assays, File Name Conventions, and Result Groups, and set well property definitions in one place without having to navigate to different views. You can set convenient defaults for these views in the Preferences, including default plate type, polymer used, and display grid or table view. A variety of features are available to search well contents, assist in well-to-capillary mapping, customize how well attributes are displayed, and so on. Editing features such as undo, redo, fill, cut, copy, and paste make plate setup easy.

• Data Collection and Run Monitoring: Various views are provided to easily link plates, monitor the data collection, and manage injection lists. The Link Plates view allows you to link plates to the plate A and B positions on the instrument. The Injection List view displays the list of injections to be performed with progress status, instrument protocol used, and primary analysis status. Real-time views (Gel View, Trace View) display data as it is being collected. The EPT view plots values of various instrument parameters against time. Features such as zooming and detaching are provided for ease of use.

33500 Series Data Collection Software v3.1 User Bulletin

• Primary Analysis: After data collection, sample files are generated for each capillary and primary analysis is performed on these samples. Any error/warning flags regarding sample quality are displayed showing the quality information about the injection, sample name, and assay used. The primary analysis algorithms (base-calling and size-calling) remain unchanged from 3500 Series Data Collection Software v1.0.

• Library Management: You can easily manage different library items such as plate records, assays, and instrument protocols. Features are provided to create, edit, delete, export, and import library items. Search and filter features enable you to find items of interest easily.

• Instrument Maintenance Wizards: Wizard-style operations are provided to guide you through procedures to maintain the instrument. Wizards are provided for installing the capillary array, removing bubbles from the polymer pump, washing the pump chamber and channels, filling the array with fresh polymer, replenishing the polymer installed on the instrument, changing the type of polymer, and handling long-term shutdown of the instrument.

• RFID Consumables Tracking: RFID (radio frequency identification) consumable tracking evaluates the usage and expiry of consumables at various points before and during the instrument run to inform you about the status of the consumables.

• Calendar Functionality: Recommended calendar/maintenance tasks (factory default) such as cleaning the anode buffer valve pin assembly and periodically restarting the computer are provided. You can also create custom maintenance tasks.

• Review and Quality Check of Primary Analysis Results: Primary analysis results are automatically displayed in the View Results screens and can be reviewed using the View Sequencing Results screen for sequencing applications. Flags indicate the quality of the results, and sample files created during the instrument run are automatically imported into these views for review. During a run, you can re-inject samples after reviewing the results.

• Reports for Calibrations and Install Checks: Easy-to-read reports are provided for spatial and spectral calibrations. Summary and detailed reports are available for all applications.

• Quality Control Reports: Results can be displayed using various reports for sequencing analysis such as: Trace Score, Plate, CRL, CRL Distribution, QV20+, and Signal strength.

• Preferences: Allows you to set system- or user-level preferences for sequencing reports, plot views, tables, date formats, and so on.

• SAE (Security, Audit and E-Signature): The SAE module is a component of the 3500 Series Data Collection Software v3.1. System security controls user access to the software. Auditing tracks changes and actions performed by users. E-Signature determines if users are permitted, prompted, or required to provide a user name and password when performing certain actions.

• Runs on Windows® 7 Operating System: Thermo Fisher Scientific-configured Windows® 7 Professional Operating System Service Pack 1. Do not install on any other operating system.

For more information, refer to the 3500/3500xL Genetic Analyzer with 3500 Series Data Collection Software v3.1 User Guide (Pub. no. 100031809).


New features

New features

Table 1 below and Table 2 on page 5 list the 3500 Series Data Collection Software v3.0 and v3.1 updates and enhancements most relevant for users performing Human Identification (HID) applications. For more information, including other software updates, refer to the:

• 3500 Series Data Collection Software v3.0 User Bulletin (Pub. no. MAN0010812)• 3500 Series Data Collection Software v3.1 Release Notes (Pub. no. 100032109)

IMPORTANT! The primary analysis algorithms (base-calling and size-calling) remain unchanged from 3500 Series Data Collection Software v1.0. There were no changes in v2.0, v3.0, or v3.1 in the core algorithms for instrument control, data collection, spectral multi-componenting, peak detection, sizing, or quality check.

New features for v3.0

Table 1 New features for 3500 Series Data Collection Software v3.0

Feature category Enhancements

Install check and calibration

Calibration/Install check status displays a warning when a required step is missed.

Option to enter notes when accepting or rejecting a calibration run.

Option to perform only one spectral calibration run. The system now requires only sufficient consumables for a single run (rather than three runs) to start the spectral calibration run.

Library user interface

Ability to permanently lock datastore objects to prevent editing.

Improved ability to filter library objects to hide unnecessary or unused objects:• Filter by application type (Sequencing, Fragment, HID, All)• Filter by number of capillaries (8, 24)• Filter by excluding objects with names that match that of the filter

Ability to specify the number of runs retained in the run log.

User interface operation

Performance improvements to streamline startup sequence, reduce response time, and eliminate instances of overlapping “ghost box” windows.

Warning appears at software launch if the instrument door is open.

Option to enter notes when stopping a run prematurely.

Option to terminate the injection list when aborting a run.

Ability to use the plate grid to select individual samples for re-injection and see the corresponding injection number.

Improved ability to set up plates using imported text files that have been modified in Microsoft® Excel® 2007.

Added user confirmation step after reverse plate-link button is pressed (to help prevent inadvertent plate switching).

Progress bar displays elapsed time (rather than percentage) with improved accuracy.


New features for v3.1

Table 2 New features for 3500 Series Data Collection Software v3.1

RFID consumable tracking

Reduced occurrence of RFID hard stops in RUO mode; the only remaining hard stop is for the Conditioning Reagent capacity limit.

Improved accuracy of the consumable expiration dashboard display to include days and hours. Expiration is now set to occur at 11:59 PM local time (rather than noon GMT).

Wizards Improved dashboard and UI layout to make wizards more easily accessible.

Changed the wizard event name to “Wash Pump and Channels” to be consistent with the wizard name.

Updated the Wash Pump and Channels and Install Capillary Array wizards with additional instructions to install a fresh Anode Buffer Container when finishing the wizards.

New Instrument Reactivation wizard assists in preparing the instrument for operation when no capillary array is already installed on the instrument.

Reports Ability to customize report logos using the Preferences menu.

Improved consumables reporting to highlight expired consumables in the annotation view.

New sample file annotation view for fragment analysis sample files.

New utility gathers multiple log files to assist the Field Service Engineer in rapidly gathering troubleshooting information.

Security, Audit, and e-Signature (SAE)

Optimized the amount of information stored in the AuditSummary.aud file to improve performance and reduce the need to create new audit files.

Disabled the ability to delete pre-defined roles.

New “run ended” event added to the action log.

Option to prohibit pasting into the password field.

Option to disable a non-administrative user from editing preferences.

New audit event added to document object deletion.



Polymer and buffer limits†

† On-instrument supported limits are the lower of: 14 days, stated number of injections, or the expiry date.

Increase from 7 to 14 days.

(8-capillary instrument) Increase from 120 to 240 injections.

(24-capillary instrument) Increase from 50 to 100 injections.

Polymer pouch size Support 96-sample pouch.

Note: Validation of the 96-sample pouch is not included in this User Bulletin. The Genetic Analysis Team tested and passed the 96-sample pouch. For the validation studies described in this User Bulletin, the HID Team used the 960-sample pouch for sizing precision and size accuracy, and the 384-sample pouch for genotype concordance and peak height.


Upgrades from previous versions of the software

Upgrades from previous versions of the software

System requirements

The computer provided with the 3500 and 3500xL instruments contains validated software and settings. Do not update the Windows® operating system or firewall settings. Do not rename the computer after the 3500 Series Data Collection Software is installed. For more information, refer to the “Instrument and computer requirements” section of the 3500/3500xL Genetic Analyzer with 3500 Series Data Collection Software v3.1 User Guide (Pub. no. 100031809).

Upgrade fromDC v1.0 to v3.1

An upgrade from Data Collection Software v1.0 to v3.1 requires:

• A certified system provided by Thermo Fisher Scientific for 3500 Series Data Collection Software v3.1, with Windows® 7 Professional Operating System (OS), Service Pack (SP) 1

• Installation by your Field Service Engineer

Note: This ensures successful migration of your “datastore,” including existing spatial calibration, spectral calibration, HID install check, plate records, assays, instrument protocols, size standards, and QC protocols.


An upgrade from Data Collection Software v2.0 to v3.1 can be installed by the user.

IMPORTANT! If you are upgrading from v2.0 to v3.1, follow the detailed instructions in the 3500 Series Data Collection Software v3.1 Release Notes (Pub. no. 100032109). This ensures successful migration of your “datastore,” including existing spatial calibration, spectral calibration, HID install performance check, plate records, assays, instrument protocols, size standards, and QC protocols.


An upgrade from Data Collection Software v3.0 to v3.1 can be installed by the user.

IMPORTANT! If you are upgrading from v3.0 to v3.1, follow the detailed instructions in the 3500 Series Data Collection Software v3.1 Release Notes (Pub. no. 100032109). This ensures successful migration of your “datastore,” including existing spatial calibration, spectral calibration, HID install performance check, plate records, assays, instrument protocols, size standards, and QC protocols.


Documentation and support

Related documentation

For more detailed information on the 3500 Series Genetic Analyzers and related procedures, refer to the following documents:

Unless otherwise noted, portable document format (PDF) versions of the documents listed above are available at www.thermofisher.com/lifescience.

Note: To open the user documentation available from the web site, use the Adobe® Acrobat® Reader® software available from www.adobe.com.

Document title Pub. no.

3500/3500xL Genetic Analyzer with 3500 Series Data Collection Software v3.1 User Guide

100031809

3500 Series Data Collection Software v3.1 User Bulletin: New Features and HID Validation Summary

MAN0014110

3500 Series Data Collection Software v3.1 Release Notes (Download from www.lifetechnologies.com/datacollection3_1.)

100032109

3500 Series Data Collection Software v3.0 User Bulletin: New Features and HID Validation Summary

MAN0010812

User Bulletin: Manage 3500/3500xL instrument data before upgrading to Data Collection Software v3.0

100031135

3500/3500xL Genetic Analyzer User Bulletin: Protocols for the analysis of AmpFlSTR™ PCR Amplification Kit PCR products and validation summary

4469192

http://www.adobe.com




HID validation summary

Customer and technical support

Visit thermofisher.com/techresources for the latest in services and support, including:

• Worldwide contact telephone numbers• Product support, including:

– Product FAQs – Software, patches, and updates

• Order and web support• Product documentation, including:

– User guides, manuals, and protocols– Certificates of Analysis– Safety Data Sheets (SDSs; also known as MSDSs)

Note: For SDSs for reagents and chemicals from other manufacturers, contact the manufacturer.

HID support • In North America – Send an email to [email protected], or call 888-821-4443 option 1.

• Outside North America – Contact your local support office.

HID validation summary

Objectives The objective of the HID validation was to confirm that:

• The performance of the 3500 Series Data Collection Software v3.1 meets these requirements:

– 100% operation accuracy for instrument control, calibration, run setup, and data collection with both legacy and current 6-dye Applied Biosystems™ PCR Amplification Kits

– 100% data concordance of sample files generated on Data Collection Software v3.0 and analyzed by GeneMapper™ ID-X v1.5 software.

– Free of any critical limitations or defects in the general user interface and workflow.

• POP-4™ Polymer for 3500/3500xL Genetic Analyzers and the 3500 Series buffers are stable for up to 14 days on the 3500 and 3500xL instruments for HID fragment analysis. We assessed the sizing precision and size accuracy, genotyping concordance, and peak height of genomic DNA samples with representative Applied Biosystems™ PCR Amplification Kits with the POP-4™ polymer and 3500 Series buffers at 0, 7, and 14 days.


Materials and samples

Instruments and computers

One 3500 Genetic Analyzer and one 3500xL Genetic Analyzer were used in the validation experiments.

Software Internal software tested:

• 3500 Series Data Collection Software v3.1• GeneMapper™ ID-X Software v1.5

External software used for data analysis:

• Microsoft® Excel® software• Minitab® statistical software

Instrument Capillary array Upgrade path tested Computer for DC v3.1

3500 Genetic Analyzer

3500 Genetic Analyzer 8-Capillary Array, 36 cm (Cat. no. 4404683)

Data Collection v3.0 to v3.1 using the datastore migration utility

Dell® OptiPlex® XE2

3500xL Genetic Analyzer

3500xL Genetic Analyzer 24-Capillary Array, 36 cm(Cat. no. 4404687)

Data Collection v3.0 to v3.1 without data migration (fresh install)

Dell® OptiPlex® XE

Make/Model Processor (CPU)/speed Memory (RAM) Operating System (OS)/Service Pack (SP)

Dell® OptiPlex® XE2 Intel® Core™ 2 Duo E8400 3 GHz 4 Gb Windows® 7 Professional OS (SP1), 32-bit

Dell® OptiPlex® XE Intel® Core™ 2 Duo E8400 3 GHz 4 Gb Windows® 7 Professional OS (SP1), 32-bit


Materials and samples

Instrument consumables

The following instrument consumables were used during this validation study:

• POP-4™ Polymer for 3500/3500xL Genetic Analyzers, 960-sample pouches (Cat. no. 4393710) and 384-sample pouches (Cat. no. 4393715)

• Anode Buffer Container (ABC) 3500 Series (Cat. no. 4393927)• Cathode Buffer Container (CBC) 3500 Series (Cat. no. 4408256)

The POP-4™ polymer and 3500 Series buffers were used to perform the stability tests as described in the table below.

Note: The instrument was left idle between Day 7 and Day 14.

Kits The following kits were used during this validation study to perform fragment analysis for STR typing:

• AmpFlSTR™ Identifiler™ Plus PCR Amplification Kit (Cat. no. 4427368)• GlobalFiler™ PCR Amplification Kit (Cat. no. 4476135)• GlobalFiler™ Express PCR Amplification Kit (Cat. no. 4476609)

Not every PCR Amplification Kit was used in every study. Representative kits were used in each study to demonstrate the ability of the 3500 Series instruments to achieve accurate results within the parameters of each test. Representative kits for each study were selected because of dye chemistry, use of a particular size standard, and other similarities among the PCR Amplification Kits.

Day 0 Day 7 Day 14

Fresh polymer and buffer taken from storage at 4°C and stored on the instrument at room temperature

Left stored on the instrument since Day 0; installed on the instrument and used for an instrument run on Day 7

Left installed on the instrument since Day 7; used for an instrument run on Day 14


Samples tested The table below lists the samples tested in each study.

Study Samples PCR Amplification Kits tested Dye set Size standard

Sizing precision and size accuracy

AmpFlSTR™ Identifiler™ Plus and GlobalFiler™ Allelic Ladder runs performed at 0, 7, and 14 days:• 12 runs on the 3500

instrument with 4 injections of 3 sets of 8 ladders

• 4 runs on the 3500xL instrument with 4 injections of 2 sets of 24 ladders

• AmpFlSTR™ Identifiler™ Plus

G5 GeneScan™ 600 LIZ™ Size Standard v2.0

• GlobalFiler™ J6

Genotype concordance

18 positive control replicates from each kit, 1 injection

AmpFlSTR™ Identifiler™ Plus


GlobalFiler™ J6

GlobalFiler™ Express

Peak height 18 positive control replicates from each kit, 1 injection

AmpFlSTR™ Identifiler™ Plus


GlobalFiler™ J6

GlobalFiler™ Express


Methods and data analysis

Methods and data analysis

Instrument operation and UI testing

Instrument operation and UI testing were designed to evaluate the 3500 Series Data Collection Software v3.1 for instrument control.

Concordance testing

Concordance testing with the 3500 Series Data Collection Software v3.1 to evaluate the POP-4™ polymer and 3500 Series buffers at 0, 7, and 14 days:

• Setup of results groups and plate records, control of instrument runs, and collection of sample files

• Multi-componenting and genotyping• Analysis of sample files generated on 3500 Series Data Collection Software v3.1

and analyzed by GeneMapper™ ID-X v1.5 software• Analysis of sizing precision, size accuracy, and peak height scaling

Note: The auto-analysis function will not be supported or tested in this version.

Summary table All samples in this study were amplified and run according to the standard protocols in the applicable Applied Biosystems™ PCR Amplification Kit User Guides. Analysis was performed on the computers and instruments listed on page 9 using POP-4™ Polymer for 3500/3500xL Genetic Analyzers and 3500 Series buffers.

Study Method and analysis


The sizing precision and size accuracy of alleles were measured based on:• 12 runs (entire 96-well plate) on the 3500 instrument with 4 injections of 3 sets of 8 AmpFlSTR™

Identifiler™ Plus and GlobalFiler™ Allelic Ladders• 4 runs (entire 96-well plate) on the 3500xL instrument with 4 injections of 1 set of 24

AmpFlSTR™ Identifiler™ Plus and GlobalFiler™ Allelic Ladders

The runs were performed at 0, 7, and 14 days.

The sizing precision was calculated as the standard deviation of the mean bp size of each allele in the allelic ladder from all capillaries in each CE run. The size accuracy was calculated as the size range between the minimum and maximum size of each allele in the allelic ladder from all capillaries in each CE run.


18 positive control replicates were amplified with the GlobalFiler™, GlobalFiler™ Express, and AmpFlSTR™ Identifiler™ Plus Kits. The runs were performed at 0, 7, and 14 days.

Data were analyzed with GeneMapper™ ID-X Software v1.5. The alleles were compared to the published STR profiles, as provided in the GlobalFiler™ Kit, AmpFlSTR™ Identifiler™ Plus Kit, and NGM Select Kit user guides.

Peak height 18 positive control replicates were amplified with the GlobalFiler™, GlobalFiler™ Express, and AmpFlSTR™ Identifiler™ Plus Kits. The runs were performed at 0, 7, and 14 days.

The peak height was calculated by taking the average of the heterozygous allele peak height of each marker, or by dividing the homozygous allele peak height by 2.


Results

For all kits and all time points tested, all metrics were met as described in Table 3. No deviations were observed during this validation.

The results demonstrate that the 3500 and 3500xL Genetic Analyzers are suitable for HID assays under the following conditions:

• Software – 3500 Series Data Collection Software v3.1• Polymer – POP-4™ Polymer for 3500/3500xL Genetic Analyzers, stored on the

instrument at room temperature for up to 7 days, then installed on the instrument for an additional 7 days

• Buffer – 3500 Series buffers, stored on the instrument at room temperature for up to 7 days, then installed on the instrument for an additional 7 days

Table 3 Expected outcomes and results for the validation studies

Study Expected outcome for all kits and all polymer and buffer time points Result

Allelic Ladder sizing precision

The standard deviation (SD) in sizing within an injections is ≤ 0.15 base pairs (bp) for all alleles in the kit ladder.

Pass

Allelic Ladder size accuracy

The size range is ≤ 0.5 bp per injection for all alleles in the kit ladder.† Pass

Genotype concordance 100% concordance for Day 0, Day 7, and Day 14 allele calls. Pass

Peak height Proper evaluation of low-to-high sample signals and accurate calling of off-scale peaks.

Pass

† The AmpFlSTR™ Identifiler™ Plus Kit sizing precision and size accuracy specifications are from the AmpFlSTR™ Identifiler™ Plus PCR Amplification Kit User Guide (Pub. no. 4440211, Rev. F, pages 82–84) and AmpFlSTR™ Identifiler™ PCR Amplification Kit User Guide (Pub. no. 4323291, Rev. J, pages 69–70). The GlobalFiler™ Kit and GlobalFiler™ Express Kit sizing precision and size accuracy specifications are from the GlobalFiler™ PCR Amplification Kit User Guide (Pub. no. 4477604, Rev. C, pages 60–61).


Results


For each kit in this study, the data were collected on the 3500 and 3500xL instruments (Data Collection Software v3.1) with POP-4™ polymer and 3500 Series buffers at 0, 7, and 14 days.

For all runs in this study, the sizing precision and size accuracy were well within the passing specifications of ≤ 0.15 bp for sizing precision and ≤ 0.5 bp for size accuracy. Minimal variation was observed between data collected at 0, 7, and 14 days. The validation data are provided as follows:

• GlobalFiler™ Kit – See Table 4 below, Figure 1 on page 15, and Figure 2 on page 15.

• AmpFlSTR™ Identifiler™ Plus Kit – See Table 4 below, Figure 3 on page 16, and Figure 4 on page 16.

Additionally, the double allele peaks in GlobalFiler™ Allelic Ladder (D2 and TH01) and AmpFlSTR™ Identifiler™ Plus Allelic Ladder (TH01) were resolved using the 3500 Series Data Collection Software v3.1 at 0, 7, and 14 days. See Figure 5 on page 17 and Figure 6 on page 17.

Table 4 Mean sizing precision and size accuracy of the GlobalFiler™ Allelic Ladder and AmpFlSTR™ Identifiler™ Plus Allelic Ladder, 3500 and 3500xL instruments.

Instrument Allelic ladderPolymer and

buffer – days on instrument

Mean sizing precision

Mean size accuracy

Number of samples

3500 GlobalFiler™ 0 0.03 0.08 4092

7 0.04 0.07 4092

14 0.04 0.09 4092

AmpFlSTR™ IdentiFiler™ Plus

0 0.04 0.08 2436

7 0.04 0.08 2436

14 0.04 0.10 2436

3500xL GlobalFiler™ 0 0.04 0.05 1364

7 0.04 0.06 1364

14 0.04 0.07 1364

AmpFlSTR™ IdentiFiler™ Plus

0 0.04 0.06 812

7 0.04 0.07 812

14 0.04 0.06 812


Figure 1 Sizing precision and size accuracy (range) of the GlobalFiler™ Allelic Ladder across 12 injections for each marker and allele, 3500 instrument. The dotted blue lines are the passing specifications.

Figure 2 Sizing precision and size accuracy (range) of the GlobalFiler™ Allelic Ladder across 4 injections for each marker and allele, 3500xL instrument. The dotted blue lines are the passing specifications.

0.16

0.12

0.08

0.04

0.00

Marker

Yinde

lvW

ATPO

XTH

01SE

33FG

A

DYS39

1

D8S11

79

D7S82

0

D5S81

8

D3S13

58

D2S44

1

D2S13

38

D22S1

045

D21S1

1

D1S16

56

D19S4

33

D18S5

1

D16S5

39

D13S3

17

D12S3

91

D10S1

248

CSF1

PO

0.48

0.36

0.24

0.12

0.00

Sizing Precision0.15

Size Accuracy0.5

0.15

Day0Day7Day14

on-instrumentPOP4

Sizing Precision, Size Range of 3500 GlobalFiler® Allelic Ladder

0.16

0.12

0.08

0.04

0.00

Marker

Yinde

lvW

ATPO

XTH

01SE

33FG

A

DYS39

1

D8S11

79

D7S82

0

D5S81

8

D3S13

58

D2S44

1

D2S13

38

D22S1

045

D21S1

1

D1S16

56

D19S4

33

D18S5

1

D16S5

39

D13S3

17

D12S3

91

D10S1

248

CSF1

PO

0.48

0.36

0.24

0.12

0.00


Size Accuracy0.5

Day0Day7Day14

on-instrumentPOP4

Sizing Precision, Size Range of 3500xl GlobalFiler® Allelic Ladder


Results

Figure 3 Sizing precision and size accuracy (range) of the AmpFlSTR™ Identifiler™ Plus Allelic Ladder across 12 injections for each marker and allele, 3500 instrument. The dotted blue lines are the passing specifications.

Figure 4 Sizing precision and size accuracy (range) of the AmpFlSTR™ Identifiler™ Plus Allelic Ladder across 4 injections for each marker, 3500xL instrument. The dotted blue lines are the passing specifications.

0.16

0.12

0.08

0.04

0.00

MarkervW

ATP

OXTH

01FGA

D8S1

179

D7S8

20

D5S8

18

D3S1

358

D2S1

338

D21S

11

D19S

433

D18S

51

D16S

539

D13S

317

CSF1

PO

0.48

0.36

0.24

0.12

0.00


Size Accuracy0.5

0.15

Day0Day7Day14

on-instrumentPOP4

Sizing Precision, Size Range of 3500 Identifiler® Plus Allelic Ladder

0.16

0.12

0.08

0.04

0.00

MarkervW

ATP

OXTH

01FGA

D8S1

179

D7S8

20

D5S8

18

D3S1

358

D2S1

338

D21S

11

D19S

433

D18S

51

D16S

539

D13S

317

CSF1

PO

0.48

0.36

0.24

0.12

0.00


Size Accuracy0.5

Day0Day7Day14

on-instrumentPOP4

Sizing Precision, Size Range of 3500xl Identifler® Plus Allelic Ladder


Figure 5 Resolution of double allele peaks in the GlobalFiler™ Allelic Ladder D2 and TH01 markers (left) and AmpFlSTR™ Identifiler™ Plus Allelic Ladder TH01 marker (right), 3500 instrument.

Figure 6 Resolution of double allele peaks in the GlobalFiler™ Allelic Ladder D2 and TH01 markers (left) and AmpFlSTR™ Identifiler™ Plus Allelic Ladder TH01 marker (right), 3500xL instrument.

3500 – GF AL – D2

3500 – GF AL – TH01

3500 – IDP AL – THO1

Day0

Day7

Day14

Day0

Day7

Day14

Day0

Day7

Day14

3500xl – GF AL – D2

3500xl – GF AL – TH01

3500xl – IDP AL – THO1

Day0

Day7

Day14

Day0

Day7

Day14

Day0

Day7

Day14


Results


For each kit in this study, the data were collected on the 3500 and 3500xL instruments (Data Collection Software v3.1) with POP-4™ polymer and 3500 Series buffers at 0, 7, and 14 days. The data were analyzed with GeneMapper™ ID-X Software v1.5.

For all runs in this study, each replicate gave the expected number of correct allele calls without any dropout. No variation was observed between data collected at 0, 7, and 14 days. The validation data are provided as follows:

• GlobalFiler™ Kit and GlobalFiler™ Express Kit – See Table 5 below.• AmpFlSTR™ Identifiler™ Plus Kit – See Table 6 on page 19.

Table 5 Allele calls for the GlobalFiler™ Kit and GlobalFiler™ Express Kit with 007 DNA as the target, 3500 and 3500xL instruments.

MarkerAllele position Expected number

of allele calls

Observed number of allele calls

Day 0 Day 7 Day 14

Allele 1 Allele 2 Allele 1 Allele 2 Allele 1 Allele 2 Allele 1 Allele 2 Allele 1 Allele 2

D3S1358 15 16 18 18 18 18 18 18 18 18

vWA 14 16 18 18 18 18 18 18 18 18

D16S539 9 10 18 18 18 18 18 18 18 18

CSF1PO 11 12 18 18 18 18 18 18 18 18

TPOX 8 NA 18 18 18 18 18 18 18 18

Yindel 2 NA 18 18 18 18 18 18 18 18

AMEL X Y 18 18 18 18 18 18 18 18

D8S1179 12 13 18 18 18 18 18 18 18 18

D21S11 28 31 18 18 18 18 18 18 18 18

D18S51 12 15 18 18 18 18 18 18 18 18

DYS391 11 NA 18 18 18 18 18 18 18 18

D2S441 14 15 18 18 18 18 18 18 18 18

D19S433 14 15 18 18 18 18 18 18 18 18

TH01 7 9.3 18 18 18 18 18 18 18 18

FGA 24 26 18 18 18 18 18 18 18 18

D22S1045 11 16 18 18 18 18 18 18 18 18

D5S818 11 NA 18 18 18 18 18 18 18 18

D13S317 11 NA 18 18 18 18 18 18 18 18

D7S820 7 12 18 18 18 18 18 18 18 18

SE33 17 25.2 18 18 18 18 18 18 18 18

D10S1248 12 15 18 18 18 18 18 18 18 18

D1S1656 13 16 18 18 18 18 18 18 18 18

D12S391 18 19 18 18 18 18 18 18 18 18

D2S1338 20 23 18 18 18 18 18 18 18 18


Table 6 Allele calls for the AmpFlSTR™ Identifiler™ Plus Kit with 9947A DNA as the target, 3500 and 3500xL instruments.

MarkerAllele position Expected number

of allele calls

Observed number of allele calls

Day 0 Day 7 Day 14

Allele 1 Allele 2 Allele 1 Allele 2 Allele 1 Allele 2 Allele 1 Allele 2 Allele 1 Allele 2

D8S1179 13 NA 18 18 18 18 18 18 18 18

D21S11 30 NA 18 18 18 18 18 18 18 18

D7S820 10 11 18 18 18 18 18 18 18 18

CSF1PO 10 12 18 18 18 18 18 18 18 18

D3S1358 14 15 18 18 18 18 18 18 18 18

TH01 8 9.3 18 18 18 18 18 18 18 18

D13S317 11 NA 18 18 18 18 18 18 18 18

D16S539 11 12 18 18 18 18 18 18 18 18

D2S1338 19 23 18 18 18 18 18 18 18 18

D19S433 14 15 18 18 18 18 18 18 18 18

vWA 17 18 18 18 18 18 18 18 18 18

TPOX 8 NA 18 18 18 18 18 18 18 18

D18S51 15 19 18 18 18 18 18 18 18 18

AMEL X NA 18 18 18 18 18 18 18 18

D5S818 11 NA 18 18 18 18 18 18 18 18

FGA 23 24 18 18 18 18 18 18 18 18


Results

Peak height For each kit in this study, the data were collected on the 3500 and 3500xL instruments (Data Collection Software v3.1) with POP-4™ polymer and 3500 Series buffers at 0, 7, and 14 days. The data were analyzed with GeneMapper™ ID-X Software v1.5.

For all runs in this study, the peak height measurements were consistent at 0, 7, and 14 days.

Note: The 3500xL instrument showed a higher variability in signal compared to the 3500 instrument. This is consistent with instrument-to-instrument variability and sample preparation variability (for example, the electrophoresis samples were freshly prepared on the day of the experiment from pooled PCR products stored at 4°C).

The validation data are provided as follows:

• GlobalFiler™ Kit – See Table 7 on page 20, Figure 7 on page 21, and Figure 8 on page 21.

• GlobalFiler™ Express Kit – See Table 8 on page 22, Figure 9 on page 23, and Figure 10 on page 23.

• AmpFlSTR™ Identifiler™ Plus Kit – See Table 9 on page 23, Figure 11 on page 24, and Figure 12 on page 24.

Table 7 Average peak heights for the GlobalFiler™ Kit with 007 DNA, 3500 and 3500xL instruments.

MarkerAverage peak height 3500 instrument Average peak height 3500xL instrument

Day 0 Day 7 Day 14 Relative SD (%) Day 0 Day 7 Day 14 Relative SD (%)

D3S1358 2548 2559 2610 1 2761 2771 3070 6

vWA 1993 1981 2033 1 2184 2221 2375 4

D16S539 1764 1777 1838 2 1911 1923 2060 4

CSF1PO 1568 1618 1630 2 1759 1798 1878 3

TPOX 1558 1607 1621 2 1736 1753 1816 2

Yindel 1095 1136 1113 2 1196 1260 1393 8

AMEL 2537 2613 2563 2 2763 2959 3201 7

D8S1179 3326 3271 3346 1 3611 3573 3879 5

D21S11 2237 2219 2285 2 2415 2434 2577 4

D18S51 1961 1982 2030 2 2152 2165 2258 3

DYS391 1373 1421 1440 2 1530 1540 1595 2

D2S441 3351 3442 3357 2 3838 4028 4371 7

D19S433 2985 2975 3000 0 3395 3389 3662 4

TH01 3129 3110 3147 1 3551 3573 3782 3

FGA 2886 2918 2951 1 3324 3323 3470 3

D22S1045 1741 1764 1782 1 1912 1974 2192 7

D5S818 2261 2209 2311 2 2472 2467 2667 4

D13S317 2292 2237 2374 3 2463 2455 2621 4

D7S820 2211 2203 2320 3 2397 2389 2504 3


Figure 7 The peak heights differ by 0 to 3% for the GlobalFiler™ Kit on the 3500 instrument.

Figure 8 The peak heights differ by 2 to 8% for the GlobalFiler™ Kit on the 3500xL instrument.

SE33 2001 2053 2123 3 2190 2192 2268 2

D10S1248 1281 1306 1312 1 1337 1426 1559 8

D1S1656 1504 1474 1543 2 1556 1561 1663 4

D12S391 1497 1478 1558 3 1513 1514 1616 4

D2S1338 3585 3646 3763 2 3707 3741 3876 2




Results

Table 8 Average peak heights for the GlobalFiler™ Express Kit with 007 DNA, 3500 and 3500xL instruments.



D3S1358 2476 2702 2909 8 2418 2854 2921 10

vWA 3653 3966 4257 8 3593 4235 4281 10

D16S539 6149 6711 7090 7 5880 6919 6941 9

CSF1PO 5806 6263 6514 6 5686 6592 6536 8

TPOX 4612 4984 5097 5 4489 5147 5081 7

Yindel 559 608 655 8 550 631 636 8

AMEL 2145 2328 2485 7 2105 2437 2434 8

D8S1179 2124 2296 2469 8 2072 2387 2425 8

D21S11 5678 6153 6548 7 5427 6265 6268 8

D18S51 7434 8025 8368 6 7108 8109 8065 7

DYS391 3348 3601 3706 5 3205 3644 3605 7

D2S441 3342 3610 3842 7 3457 3994 3978 8

D19S433 2584 2786 2980 7 2639 3047 3060 8

TH01 4061 4381 4642 7 4108 4725 4712 8

FGA 3967 4278 4458 6 4000 4581 4514 7

D22S1045 3669 4017 4357 9 3661 4267 4360 9

D5S818 3880 4231 4586 8 3800 4468 4568 10

D13S317 4435 4855 5223 8 4239 4962 5035 9

D7S820 6992 7648 8100 7 6650 7744 7756 9

SE33 8409 9188 9478 6 7925 9113 9093 8

D10S1248 1922 2103 2280 9 1823 2148 2184 10

D1S1656 3794 4140 4492 8 3521 4144 4207 10

D12S391 4705 5137 5509 8 4229 4945 4972 9

D2S1338 7715 8361 8717 6 6952 8021 7970 8


Figure 9 The peak heights differ by 5 to 9% for the GlobalFiler™ Express Kit on the 3500 instrument.

Figure 10 The peak heights differ by 7 to 10% for the GlobalFiler™ Express Kit on the 3500xL instrument.

Table 9 Average peak heights for the AmpFlSTR™ Identifiler™ Plus Kit with 9974 DNA, 3500 and 3500xL instruments.



D8S1179 4876 4709 4683 2 5943 6961 7130 10

D21S11 4001 3939 3933 1 4663 5482 5590 10

D7S820 3253 3201 3195 1 3770 4437 4469 9

CSF1PO 4197 4044 3946 3 5155 5880 5870 7

D3S1358 6659 6374 6209 4 8762 9896 10035 7

TH01 5947 5754 5669 2 7802 8942 9066 8

D13S317 5991 5842 5755 2 7753 8877 8900 8


Results

Figure 11 The peak heights differ by 1 to 4% for the AmpFlSTR™ Identifiler™ Plus Kit on the 3500 instrument.

Figure 12 The peak heights differ by 6 to 10% for the AmpFlSTR™ Identifiler™ Plus Kit on the 3500xL instrument.

D16S539 5940 5785 5657 2 7686 8762 8702 7

D2S1338 5053 4898 4739 3 6529 7357 7313 7

D19S433 5009 4805 4635 4 6629 7550 7566 7

vWA 5317 5128 5034 3 6993 7966 7992 7

TPOX 4520 4364 4265 3 5776 6512 6452 7

D18S51 4326 4171 4030 4 5729 6465 6371 6

AMEL 4481 4320 4213 3 6030 6795 6807 7

D5S818 4143 3991 3867 3 5573 6403 6356 8

FGA 3855 3763 3683 2 5076 5841 5777 8




Conclusions

Based on the validation studies performed, we conclude the following for assays performed on the 3500 and 3500xL Genetic Analyzers with 3500 Series Data Collection Software v3.1 and with POP-4™ polymer and 3500 Series buffers:

• All changes in the 3500 Series Data Collection Software v3.1 passed testing criteria and functioned properly.

• Sizing precision and size accuracy were comparable and well within passing specifications at 0, 7, and 14 days.

• The genotype calls from all genomic DNA samples and positive control samples were 100% concordant at 0, 7, and 14 days.

• There was no significant difference in the peak heights (RSD < 10%) at 0, 7, and 14 days.

Recommendations

This validation demonstrated that the POP-4™ polymer and the 3500 Series buffers can be used on the 3500 and 3500xL Genetic Analyzers under the conditions listed in the table below, without negative impact on the instrument function or data quality:

Note: The instrument was left idle between Day 7 and Day 14.

However, to ensure the best long-term performance of the instrument and to maximize array life, we recommended the following steps if the instrument will not be used for an extended period of time (that is, for 3- to 14-day time periods):

1. Refresh the polymer daily (unless the instrument is used for running samples) by performing the “Fill Array with Polymer” wizard once daily, every day that the instrument is idle.

2. Perform the “Water Wash” wizard every 7 days, followed by a polymer refill.

If the instrument will remain idle for more than 2 weeks, prepare the instrument for long-term shutdown.

Day 0 Day 7 Day 14

Fresh polymer and buffer taken from storage at 4°C and stored on the instrument at room temperature

Left stored on the instrument since Day 0; installed on the instrument and used for an instrument run on Day 7

Left installed on the instrument since Day 7; used for an instrument run on Day 14

For support visit thermofisher.com/support or email [email protected]

thermofisher.com/lifescience

5 October 2015

The information in this guide is subject to change without notice.

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