Applied Biosystems SOLiD 4 Systemtools.thermofisher.com/content/sfs/manuals/cms_089471.pdf ·...

Applied Biosystems SOLiD™ 4 System

Standard and Barcoded Fragment Library Preparation Using the Beckman Coulter Biomek® FX/FXP

AB Demonstrated Protocol

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For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

TRADEMARKS:

The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.

AMPure is a registered trademark of Beckman Coulter Genomics, Biomek is a registered trademark of Beckman Coulter and Covaris is a registered trademark of Covaris, Inc.

© Copyright 2010, Life Technologies Corporation. All rights reserved.

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Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Materials and equipment required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Assumptions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Prepare a standard fragment library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Prepare a barcoded fragment library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

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

Tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Methods and files for the Biomek® FX/FXP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Understanding how the robotic arm transfers samples and reagents . . . . . . . . . . . . . . . . . . . . . . . . . 13

Shear the DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Standard P1 and P2 adaptor plate preparation using the “SOLiD 4 Fragment Library Standard Adaptor Plate Prep” method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Barcoded P1 adaptor plate preparation using the “SOLiD 4 Fragment Library Barcode Adaptor Plate Prep” method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

End-repair, ligate adaptors and purify the DNA using the “SOLiD 4 Fragment Library Prep Pt1” method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Nick-translate and amplify the DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Purify the DNA using the “SOLiD 4 Fragment Library Prep Pt2” method . . . . . . . . . . . . . . . . . . . . . . . 31

Quantitate the purified DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Appendix A: Reagent volumes and preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

SOLiD™ 4 System Standard Fragment Library Preparation Automated with the Biomek® FX/FXP

Preface

1SOLiD™ 4 System Fragment Library PreparationAB Demostrated Protocol

PrefaceApplied Biosystems (AB) protocols for the SOLiD™ 4 System can be described by the following categories:

For the latest services and support information, go to:

www.appliedbiosystems.com

Category Description

AB Supported (S) Testing and validation have been performed by Applied Biosystems for this protocol on this instrument system. The technical support and field application specialists have been trained to support this protocol.

AB Demonstrated (A) Applied Biosystems has tested this protocol but no validation was performed for this instrument system. Certain components of the protocol workflow such as reagent kits and other protocols for preparation of reagents may not be available through Applied Biosystems. Supporting documentation such as application notes may be available from Applied Biosystems and/or third parties. Limited support is available from Applied Biosystems.

Customer Demonstrated (C) The performance of this protocol has not been evaluated on this instrument by Applied Biosystems. However, at least one customer or third party has reported successfully performing this protocol on this instrument. Applied Biosystems cannot guarantee instrument and reagent performance specifications with the use of customer demonstrated protocols. However supporting documentation from Applied Biosystems and/or third parties may be available and Applied Biosystems may provide basic guidelines in connection with this protocol.

Experimental/Not Supported (N) This protocol has not been evaluated and /or tested on this instrument by either Applied Biosystems or its customers. Applied Biosystems cannot warranty that attempting to utilize this protocol will not adversely affect the functionality of the instrument and other Applied Biosystems products. Data generated by this protocol on this instrument may not be fully representative of typical results. Applied Biosystems does not provide any support for this protocol.

www.appliedbiosystems.com

2 SOLiD™ 4 System Fragment Library PreparationAB Demostrated Protocol


Materials and equipment required

Materials and equipment requiredIn addition to the required equipment, kits, and consumables outlined in Appendix A in the Applied Biosystems SOLiD™ 4 System Library Preparation Guide (PN 4445673), the following items are required.

Requiredequipment

Requiredconsumables

Item‡

‡ Applied Biosystems has tested this protocol using this specific material. Substitution may adversely affect system performance.

Quantity Source

Biomek® FX/FXP Dual Arm System with Multichannel Pipettor and Span-8 Pipettors

(with at least 20 open ALPs)

1 Beckman CoulterA31844

96-Channel Disposable tip, Pipette Head, 200 µL

1 Beckman Coulter719368

Span-8 Disposal ALP 1 Beckman Coulter719590

Tip Loader 1 Beckman Coulter719356

Disposal ALP 1 Beckman Coulter719347

Biomek® Software version 3.3.14 1 Beckman CoulterA16170

7 Bar Magnet 1 V&P ScientificVP771MM

Modular Frame for Reservoirs 2 Beckman Coulter372795

24-Position Tube Rack 5 Beckman Coulter373661

11mm Diameter Insert (25/case) 5 cases Beckman Coulter373696

Item‡ Source

Quarter Reservoir, Divided by Width Beckman Coulter372792

Quarter Reservoir Beckman Coulter372790

Half Reservoir Beckman Coulter372786

Biomek® Tips, Barrier, P250, AP96, Presterile

Beckman Coulter717253

Biomek® Tips, Barrier, P20, AP96, Presterile Beckman Coulter717256

Biomek® Tips, Barrier, P1000, Span-8, Conductive, Presterile

Beckman Coulter987925

Deep-well plate 96/500 μL, DNA LoBind Eppendorf951032085


Materials and equipment required


Required reagents

Deep-well plate 96/1000 μL, DNA LoBind Eppendorf951032824

twin.tec PCR Plate 96, skirted Eppendorf951020460

twin.tec PCR Plate 96, semi-skirted Eppendorf951020362

DNA LoBind Tubes, 1.5-mL Eppendorf022431021

DNA LoBind Tubes, 2.0-mL Eppendorf022431048

Corning® 96 Well Clear V-Bottom 2-mL Polypropylene Block, Sterile

Corning3960

MicroAmp® 96 Base (10/pkg) Applied BiosystemsN8010531

MicroAmp® 8-Tube Strip, 0.2-mL Applied BiosystemsN8010580

microTUBE™ (6 x 16 mm), AFA Fiber with snap-caps

Corvaris520045

Nunc® Aluminum Seal Tape for 96-Well Plates

Thermo Scientific232698

‡ Applied Biosystems has tested this protocol using this specific material. Substitution may adversely affect system performance, and may damage a consumable(s) and/or spill a reagent.

Item‡ Source

Item‡ Source

SOLiD™ Fragment Library Oligos Kit Applied Biosystems4401151

SOLiD™ Fragment Library Barcoding Kit 1-96§

Applied Biosystems4449637

SOLiD™ Fragment Library Barcoding Kit Module 1-16












SOLiD™ Fragment Library Construction Reagents


Agencourt AMPure® XP, 450 mL Kit Beckman Coulter GenomicsA63882

95% or 100% Ethanol Any supplier

1X Low TE Buffer Applied Biosystems4389764



Assumptions

AssumptionsThis guide assumes that:

• The Biomek® FX/FXP has the appropriate hardware and software installed, and has been calibrated.

• Users are properly trained in the operation, maintenance, and troubleshooting of the Biomek® FX/FXP.

• Users have access to the Biomek® FX/FXP operating manuals and other applicable Biomek® FX/FXP documentation, and the Applied Biosystems SOLiD™ 4 System Library Preparation Guide (PN 4445673).

• Users have read for important safety information related to the use of the Biomek® FX/FXP.

• Users have read the safety information in Appendix I, “Safety” on page 248 in the Applied Biosystems SOLiD™ 4 System Library Preparation Guide (PN 4445673).

IntroductionA robotic method for preparing and purifying standard fragment and barcoded fragment libraries has been demonstrated at Applied Biosystems. The Biomek®FX/FXP liquid handling systems was chosen for this work due to the flexibility and ease of use. This method will ease the burden of multiple pipetting and purification steps.

Prepare a standard fragment libraryThis protocol is designed for 5 ng to 8 µg of genomic DNA.

Refer to “Prepare a standard fragment library” on page 16 in the Applied Biosystems SOLiD™ 4 System Library Preparation Guide (PN 4445673) for more information.

SOLiD™ Library TaqMan® Quantitation Kit Applied Biosystems4449639

Quanti-iT™ dsDNA HS Assay Kits, 500 Assays

InvitrogenQ32854

Quant-iT™ PicoGreen® dsDNA reagent *2000 assays* *10 x 100µL*

InvitrogenP11495

‡ Applied Biosystems has tested this protocol using this specific material. Substitution may adversely affect system performance, and may damage a consumable(s) and/or spill a reagent.

§ This kit is made up of the following modules: SOLiD™ Fragment Library Barcoding Module 1-16 (PN 4444837), SOLiD™ Fragment Library Barcoding Module 17-32 (PN 4449636), SOLiD™ Fragment Library Barcoding Module 33-48 (PN 4449635), SOLiD™ Fragment Library Barcoding Module 49-64 (PN 4449641), SOLiD™ Fragment Library Barcoding Module 65-80 (PN 4449462), SOLiD™ Fragment Library Barcoding Module 81-96 (PN 4449643)

Item‡ Source


Prepare a barcoded fragment library


Prepare a barcoded fragment libraryThe protocol is designed for 5 ng to 8 µg of genomic DNA.

Refer to “Barcoded Fragment Library Preparation” on page 131 in the Applied Biosystems SOLiD™ 4 System Library Preparation Guide (PN 4445673) for more information.

WorkflowThe workflow comprises both manual and automation steps. In Figure 1, the automation steps are highlighted in yellow.

Figure 1 Fragment library preparation workflow with the steps performed on the Biomek® FX/FXP displayed in yellow

End-repair, size select, ligate and purify the DNA on the Biomek® FX/FXP

Start the method and follow the prompts

Nick-translate, then amplify library using GeneAmp® PCR System 9700

Shear the DNA

Prepare the reagents

Setup the Biomek® FX/FXP

Purify the library on the Biomek® FX/FXP

Start the method and follow the prompts



Quantitate the library by performing quantitative PCR (qPCR) or using a Qubit® fluorometer

Prepare the adaptor plate on the Biomek® FX/FXP

Start the method and follow prompts





Workflow

Shear the DNA This step involves sonicating the input DNA into small fragments with a mean fragment size of 165 bp and a fragment size range of 150 to 180 bp (before adaptor ligation) using the Covaris® S2 System. The conditions have been tested for shearing 5 ng to 8 µg DNA in a total volume of 120 µL. For certain DNA samples, optimizing the shearing protocol may be necessary.

End-repair andligate DNA on the

Biomek® FX/FXP

This step is performed on the Biomek® FX/FXP. End Polishing Enzyme 1 and End Polishing Enzyme 2 are used to convert DNA that has damaged or incompatible 5-protruding and/or 3-protruding ends to 5-phosphorylated, blunt-ended DNA. End Polishing Enzyme 1 and ATP are also included for phosphorylation of the 5-ends of the blunt-ended DNA to allow for subsequent ligation. AMPure® XP beads are used to size select and purify the DNA after the end-repair. P1 and P2 Adaptors1 for fragment libraries or Multiplex P1 and P2 Adaptors for barcoded fragment libraries2 are ligated to the ends of the end-repaired DNA.

The adaptor-ligated DNA is purified twice using AMPure® XP beads. An optional PCR set up step aliquots a PCR master mix for subsequent nick-translation and library amplification performed in a thermal cycler.

Nick-translate, thenamplify the library

The library is nick-translated and then amplified using Library PCR Primer 1 and Library PCR Primer 2 (standard fragment library) or Multiplex Library PCR Primer 1 and Multiplex Library PCR Primer 2 (barcoded fragment library) and Platinum® PCR Amplification Mix.

Purify the library onthe Biomek®

FX/FXP

This step is performed on the Biomek® FX/FXP. After amplification, the PCR amplified library is purified twice using AMPure® XP beads.

Quantitate thelibrary

Quantitate the library by either quantitative PCR (qPCR) or using the Qubit® fluorometer. For qPCR, the SOLiD™ Library TaqMan® Quantitation Kit (PN 4449639) is recommended for accurate library quantitation. For the Qubit® fluorometer, use the dsDNA HS assay or the Quant-iT™ PicoGreen® reagent.

1 The P1 and P2 Adaptors are included in double-stranded form in the SOLiD™ Fragment Library Oligos Kit.

2 Multiplex P1 and P2 Adaptors are included in double-stranded form in the SOLiD™ Fragment Library Barcoding Modules 1-96, or any of the individual 16-barcode SOLiD Fragment Library Barcoding Modules. You can design experiments to use as few as 4 barcodes, as long as at least one of the following full sets of four barcodes are used: Barcodes 1-4, 5-8, 9-12, 13-16, 17-20, 21-24, 25-28, 29-32, 33-36, 37-40, 41-44, 45-48, 49-52, 53-56, 57-60, 61-64, 65-68, 69-72, 73-76, 77-80, 81-84, 85-88, 89-92, or 93-96.


Tips


Tips

Reagents • Thaw reagents on ice before use.

• Always prepare fresh 70% ethanol daily. Ethanol that is old and has absorbed water from the atmosphere, may decrease DNA recovery.

Beckman CoulterBiomek FX/FXP

• Dead volumes have been determined for each reagent reservoir:

– 1.5- or 2.0-mL tubes – 20 µL

– Quarter Reservoir, Divided by Width – 200 µL

– Quarter Reservoir – 400 µL

– Half Reservoir – 800 µL

• Instrument setup steps have been included to ensure all volume is removed when discarding the supernatant.

• Several magnet configurations have been tested. The VP771MM magnet performs best in both time to clear solution and ease of resuspension off magnet.

• Never stop a run in the middle of a method. Always pause to troubleshoot.

• The method was developed assuming a disposal ALP is available. If none is available adjust steps requiring a move to TR1.

• Purge the syringes prior to each run to ensure accurate pipetting.

• Ensure the probes on the Span-8 pod are aligned in the Z-axis prior to using any method. Contact Beckman service for assistance.

• Make sure the deck is properly framed prior to running any method.

• Tips from other vendors may be used but the user must verify that the volumes transferred do not cause liquid to be aspirated into the filter.

• Ensure the system has been backed up prior to making any changes or importing methods.

• Applied Biosystems has tested this protocol using materials specified in “Materials and equipment required” on page 2. Substitution may adversely affect system performance, and may damage a consumable(s) and/or spill a reagent.



Methods and files for the Biomek® FX/FXP


Available methodsand files

The following methods and files are available for importing on the Biomek® FX/FXP:

How to obtain themethods

Contact your Applied Biosystems field application specialist to obtain the SOLID methods.

Import the methods 1. Power on the computer.

2. Start the Biomek software.

3. Import the project.

a. Select Project Import Project.

Table 1 Available methods and files, and their function

Method/file name Function

Methods

SOLiD 4 Fragment Library Preparation.imp Project file for Biomek

SOLiD 4 Fragment Library Standard Adaptor Plate Prep.bmf

Prepare standard adaptor plate

SOLiD 4 Fragment Library Barcode Adaptor Plate Prep.bmf Prepare barcoded adaptor plate

SOLiD 4 Fragment Library Prep Pt1.bmf Perform end-repair, size selection, ligation and purification for up to 96 samples

SOLiD 4 Fragment Library Prep Pt2.bmf Perform post-PCR cleanup for up to 96 samples

Files

SOLiD 4 Fragment Library Setup.xls Generate transfer sheets for setting up adaptor plates and create master mix volume sheet

FXCountDown.exe Timer

BC.jpg User input form images

noBC.jpg

pt1 plates.jpg

pt2 plates.jpg




b. Select the file SOLiD 4 Fragment Library Preparation.imp and click Open.

”

c. Select all project items to import and click OK.

4. Import the methods.

a. Select File Import.




b. Select all the method files and click Open.

”

c. Select all methods to import and click OK.

5. Adjust tip capacity values for the AP96, P250, Barrier, Presterile tips.

a. Select Project Tip Type Editor.




b. In the left side of the dialog box, select P200_Barrier.

c. In the right side of the dialog box, change the Capacity value to 175 µL.

d. Change the Air Capacity value to 180 µL.

e. Click OK.

6. Adjust tip capacity values for the AP96, P20, Barrier Presterile tips.

a. In the left side of the dialog box, select P20_Barrier.

b. In the right side of the dialog box, change the Capacity value to 40 µL.

c. Change the Air Capacity value to 50 µL.

d. Click OK.




7. Adjust multichannel pod time out value.

a. Select Instrument Hardware Setup.

b. Select the multichannel pod (1).

c. Expand the Additional Pod Settings (2).

d. Change the Additional Timeout value to at least 60 seconds.

8. Copy SOLiD™ 4 Fragment Library Setup.xls to the desktop or other user-defined location.

9. Copy FXCountdown.exe and jpeg images to: C:\ProgramFiles\Biomek FX\

The deck layout for all methods has been set up as shown in Figure 2 on page 13.


Safety


Figure 2 Empty deck layout

SafetyRefer to the complete safety alert descriptions in Appendix I, “Safety” on page 248 in the Applied Biosystems SOLiD™ 4 System Library Preparation Guide (PN 4445673).

Understanding how the robotic arm transfers samples and reagents

The Biomek FX/FXP uses a Span-8 pod and P1000 tips to dispense reagents into the wells selected for library purification. Master mixes are aliquoted to plates prior to sample addition to prevent any cross-contamination. The 96-channel pod is used for transferring 96 samples at a time, in-tip mixing, and moving labware around the deck. Tips for the 96-channel pod are reused several times within each procedure, because each tip always enters the same well there is minimal risk of cross-contamination. The pipetting techniques have been optimized to minimize droplets formed on the end of tips.



Shear the DNA

Shear the DNA

1. Dilute the desired amount of DNA to 120 µL in 1X Low TE Buffer in a Covaris™ microTUBE™ (see Table 2).

Note: Increase volume of DNA to be sheared from 100 µL to 120 µL to decrease chances of an air bubble causing problems with shearing.

2. Follow the method for shearing the DNA as described in Chapter 2 or 4 of the Applied Biosystems SOLiD™ 4 System Library Preparation Guide (PN 4445673).

3. Transfer 110 µL of sheared DNA to the desired position in a 500-µL deep-well LoBind plate.

4. Store on ice until ready to use.

Table 2 DNA dilution

Component Amount

DNA 5 ng to 8 µg

1X Low TE Buffer Variable

Total 120

If preparing... Then go to page...

Standard fragment library 18

Barcoded fragment library 135


Standard P1 and P2 adaptor plate preparation using the “SOLiD 4 Fragment Library Standard Adaptor Plate Prep” method



Setup thespreadsheet for

standard samples

1. Open the file “SOLiD™ 4 Fragment Library Setup.xls” in Microsoft® Excel and click Enable Macros.

2. Enter File save path in cell C1. For example “C:\Documents and Settings\Administrator\Desktop\”

All files generated will be saved to this directory, make sure the file path ends with a backslash (\).

Note: See “Time and Date Stamp” on page 36 for more details.

3. Enter starting amount of DNA (in µg) for each library in column C.

Note: Blank cells will be ignored by the macro.

4. Select SOLiD™ 4 Fragment Library Create Standard Adaptor Files.

If you are preparing... Then go to...

Standard samples "Setup the spreadsheet for standard samples" below

Barcoded samples “Setup the spreadsheet for barcoded samples” on page 20




The following files are created and saved in the directory specified in cell C1 of the spreadsheet:

• File 1: “Adaptor Plate Setup DD-MMM-YYYY hhmm.csv”

• File 2: “Reagent Volumes DD-MMM-YYYY hhmm.xls”

IMPORTANT! Make sure all the files have the same time stamp in the filename. If not, manually change the time stamp. If they are not the same an error will occur when validating the Biomek method.

IMPORTANT! If any adaptor dilution requires more than 2100 µL, the adaptor plate prep must be divided into two runs. Enter sample information for the first 48 samples, run the macro and continue. Once finished, refill the water reservoir, adaptor dilutions and replace any used tips. Do not replace the Adaptor plate or dilution plate. Enter the sample information for the remaining samples, run the macro and repeat the method. Do not reuse any wells in the macro. Use the Reagent volumes sheet with all samples to calculate master mixes.

Prepare P1 and P2adaptor reagents

1. Prepare a 6-point 3-fold dilution series for P1 and P2 adaptors (ds) in 2.0-mL tubes.

Note: Refer to File 2 for the total required volume for each dilution.

a. Label 2 sets of six tubes 1:3, 1:9, 1:27, 1:81, 1:243 and 1:729.

b. Use nuclease-free water to dilute the P1 and P2 adapters to the concentrations written on the tubes.

c. Store on ice until ready to load on the deck.

Setup the BiomekFX/FXP

1. Open the Biomek software

a. Select File Open.

b. Select SOLiD 4 Fragment Library Standard Adaptor Plate Prep.

2. Use Figure 3 on page 17 to place the P1 and P2 adapters in 24-position tube rack with 11mm adaptors.




Figure 3 Standard adaptor dilution layout in 24-position tube rack

3. Setup the deck.

a. Place a Half Reservoir in the left side of a Modular Frame for Reservoirs. Fill with 50 mL of nuclease-free water and place at P20.

b. Place the 24-position tube rack containing the P1 and P2 dilution tubes at P14.

c. Place a 500-µL LoBind plate at P13.

d. Place AP96, P250, barrier, presterile tips at P15.

e. Place AP96, P20, barrier, presterile tips at P11 and P12.

Start the run 1. Click User Input Form within the method.

2. Enter the values for the variables.

a. Enter the File Path for the files generated by Microsoft® Excel.

b. Enter the day using the dd-mmm-yyyy format (i.e. 03-Oct-2010) and the time using the hhmm format (i.e. 8 AM is 0800 and 3 PM is 1500).

c. Enter the P1 and P2 adaptor volumes from File 2.




3. Click Finish to validate the method.

4. Select Instrument Home All Axes.

5. Click Run.

6. The final deck layout (Figure 4 on page 19) is displayed. Click OK to confirm and start the method.




Figure 4 Deck layout for standard adaptor prep run

7. A dialog box appears after the run is completed. Click OK to end the method.

8. Remove and properly dispose of leftover reagents and used plasticware.

STOPPING POINT. Cover the prepared adaptor plate and store at 4 °C until ready to use or proceed to “End-repair, ligate adaptors and purify the DNA using the “SOLiD 4 Fragment Library Prep Pt1” method” on page 25.



Barcoded P1 adaptor plate preparation using the “SOLiD 4 Fragment Library Barcode Adaptor Plate Prep” method


Setup thespreadsheet for

barcoded samples

1. Open the file “SOLiD™ 4 Fragment Library Setup.xls” in Microsoft® Excel and click Enable Macros.

2. Enter File save path in cell C1. For example “C:\Documents and Settings\Administrator\Desktop\”

All files generated will be saved to this directory, make sure the file path ends with a backslash (\).

Note: See “Time and Date Stamp” on page 36 for more details.

3. Enter starting amount of DNA (in µg) for each library and the barcode desired in columns C and D, respectively.

Note: Blank cells will be ignored by the macro.

• Any barcode may be used for any sample

• If a barcode is used for more than one sample, ensure that there is sufficient volume for the transfer

4. Select SOLiD™ 4 Fragment Library Create Barcoded Adaptor Files.

If you are preparing... Then go to...

Barcoded samples "Setup the spreadsheet for barcoded samples" below

Standard samples “Setup the spreadsheet for standard samples” on page 15




The following files are created and saved in the directory specified in cell C1 of the spreadsheet:

• File 1: “Barcode Dilution DD-MMM-YYYY hhmm.csv”

• File 2: “Multiplex Diluted Setup DD-MMM-YYYY hhmm.csv”

• File 3: “Multiplex Undiluted Setup DD-MMM-YYYY hhmm.csv”

• File 4: “Reagent Volumes DD-MMM-YYYY hhmm.xls”

IMPORTANT! Make sure all the files have the same time stamp in the filename. If not, manually change the time stamp. If they are not the same an error will occur when validating the Biomek method.

IMPORTANT! If any adaptor dilution requires more than 2100 µL, the adaptor plate prep must be divided into two runs. Enter sample information for the first 48 samples, run the macro and continue. Once finished, refill the water reservoir, adaptor dilutions and replace any used tips. Do not replace the Adaptor plate or dilution plate. Enter the sample information for the remaining samples, run the macro and repeat the method. Do not reuse any wells in the macro. Use the Reagent volumes sheet with all samples to calculate master mixes.

Prepare barcodedadaptor reagents

1. Place stock barcoded tubes in a 24-position tube rack with 11mm adaptors. Each tube rack accommodates 24 barcodes arranged in numerical order across the plate as shown in Figure 5 (rack 1: BC001 in A1 to BC024 in D6, rack 2: BC025 in A1 to BC48 in D6, rack 3: BC049 in A1 to BC072 in D6 and rack 4: BC073 in A1 to BC096 in D6).

Figure 5 Placement of the first 24 barcodes in 24-position tube rack

2. Prepare a 6-point 3-fold dilution series for P1 adaptors (ds) in 2-mL Eppendorf LoBind tubes.

Note: Refer to File 4 for the total required volume for each dilution.

a. Label six tubes 1:3, 1:9, 1:27, 1:81, 1:243 and 1:729.




b. Use nuclease-free water to dilute the P1 adapter to the concentrations written on the tubes.

c. Store on ice until ready to load on the deck.


1. Open the Biomek software.


b. Select SOLiD 4 Fragment Library Barcode Adaptor Plate Prep.

2. Use Figure 6 to place the P1 adapters in 24-position tube rack with 11mm adaptors.

Figure 6 P1 adapter placement in 24-position tube rack

3. Setup the deck.

a. Place the 24-position tube rack with BC001-024 at P4, BC025-048 at P7, BC049-072 at P5 and BC073-096 at P8.

b. Place a half reservoir in the left side of a Modular Frame for Reservoirs. Fill with 50 mL of nuclease-free water and place at P20.

c. Place P1 dilution series at P14.

d. Place 500-µL LoBind plates at P10 and P13.

e. Place AP96, P250, barrier, presterile tips at TL1, P11 and P12.

f. Place AP96, P20, barrier, presterile tips at P9.

g. Place Span-8, P1000, barrier, presterile tips at P15.




Start the run 1. Click User Input Form within the method.

a. Enter the File Path for the files generated by Microsoft® Excel.

b. Enter the day using the dd-mmm-yyyy format (i.e. 03-Oct-2010) and the time using the hhmm format (i.e. 8 AM is 0800 and 3 PM is 1500).

c. Enter the P2 dilution range from File 4.

d. Enter the P1 adaptor volumes from File 4.






4. Click Run.

5. The final deck layout (Figure 7) is displayed, click OK to confirm and start the method.

Note: Individual deck layouts may vary. Make sure to validate the deck positions prior to running the method.

Note: Approximate run time is 0:25:00.

Figure 7 Deck layout for barcoded adaptor prep run

6. A dialog box appears after the run is complete. Click OK to end the method.


STOPPING POINT. Cover the prepared adaptor plate and store at 4 °C until ready to use or proceed to “End-repair, ligate adaptors and purify the DNA using the “SOLiD 4 Fragment Library Prep Pt1” method” on page 25.


End-repair, ligate adaptors and purify the DNA using the “SOLiD 4 Fragment Library Prep Pt1” method



Prepare reagents Refer to “Fragment Library Prep Pt1 master mix and required reagents” on page 36 for volumes and preparation.




b. Select SOLiD 4 Fragment Library Prep Pt1.

2. Set up a Modular Frame with two Quarter Reservoir, Divided by Width and one Quarter Reservoir, and place the end-repair mix, ligation mix, elution buffer and PCR mix as shown in Figure 8.

Figure 8 Reagent location for modular reservoir 1

3. Set up a Modular Frame with one Half Reservoir and place the AMPure beads as shown in Figure 9.


4. Aliquot 1050 µL of 70% ethanol to each well of a 2-mL deep-well plate.

5. Setup the deck.

a. Place AP96, P250, barrier, presterile tips at TL1, P16, P17 and P18.

b. Place Span-8, P1000, barrier, conductive, presterile tips at P15.

c. Place 500-µL deep-well LoBind plates at P1, P4, P7, P8 and P19.




d. Place the PCR tubes/plates at P2, P3, P5 and P6.

• If preparing samples requiring different number of PCR cycles place MicroAmp® tubes in MicroAmp® 96 bases

• If all samples require the same number of PCR cycles twin.tec semi-skirted plates in MicroAmp® 96 bases

• If PCR setup is not desired, leave these positions empty

e. Place the magnet at P10.

f. Place a 2-mL deep-well plate at P11.

g. Place the 500-µL LoBind plate with the sheared DNA on top of magnet at P10.

h. Place a twin.tec skirted plate at P9.

i. Place the Adaptor Plate (prepared in "Barcoded P1 adaptor plate preparation using the “SOLiD 4 Fragment Library Barcode Adaptor Plate Prep” method" or "Standard P1 and P2 adaptor plate preparation using the “SOLiD 4 Fragment Library Standard Adaptor Plate Prep” method") at P12.

j. Place the 2-mL deep-well plate containing 70% ethanol at P20. Remove the foil seal.

Start the run 1. Select wells for library preparation.

a. Select Select Wells for Preparation.

b. Select the appropriate wells for preparation. Highlight to select a group of wells and control-click to select individual wells.




2. Click User Input Form within the method.


a. Enter the number of libraries.

b. Select the Format for PCR from the drop-down menu.

c. Confirm the correct wells are selected.

IMPORTANT! The number of wells selected must match the number of wells being processed.

Note: The graphic below always depicts plates and does not change if tubes are selected.



6. Click Run.




7. The final deck layout (Figure 10, Figure 11 and Figure 12 on page 29) displays, select OK to confirm and start the method.


Note: A timer will appear showing the time remaining for the method. Approximate run time is 2:15:00.

Figure 10 Deck layout for plate PCR

Figure 11 Deck layout for striptube PCR




Figure 12 Deck layout without PCR setup

8. A dialog box displays after the run is complete. Select OK to end the method.


10. Remove the PCR plates from the deck and seal them with foil tape.

11. Do one of the following:

12. If PCR set up was not selected, the adapted library was eluted into the twin.tec plate. Cover the prepared adaptor plate and store at 4°C until ready to use or proceed to page 27 (standard adaptors) or page 140 (barcodes adaptors) of the Applied Biosystems SOLiD™ 4 System Library Preparation Guide (PN 4445673) to set up the PCR reactions.

If PCR set up was... Then go to...

selected “Nick-translate and amplify the DNA” on page 30

not selected proceed to step 12



Nick-translate and amplify the DNA

Nick-translate and amplify the DNA

IMPORTANT! If the PCR was not set up in the last method, proceed to page 27 (standard adaptors) or page 140 (barcodes adaptors) of the Applied Biosystems SOLiD™ 4 System Library Preparation Guide (PN 4445673) to set up the PCR reactions.

1. Program the thermal cycler(s) for the conditions in Table 3. Determine the number of cycles based on the amount of starting input DNA.

IMPORTANT! Minimize the number of cycles to avoid over amplification and production of redundant molecules.

2. Transfer the 96-well plates or strip tubes to the thermal cycler(s) programmed in step 1.

3. Start the run(s). Set the ramp speed to 9600 and the volume to 50 µL.

4. When the PCR reaction is complete, remove the seal, and return to the plate to the Biomek FX/FXP.

STOPPING POINT. Store the plate at 4 °C until ready to use or proceed to “Purify the DNA using the “SOLiD 4 Fragment Library Prep Pt2” method” on page 31.

Table 3 PCR conditions to nick-translate and amplify the library

Stage Step Temp Time

Holding Nick translation 72C 20 min

Holding Denature 95C 5 min

Cycling:

• Standard: 2 to 10 cycles‡

• Barcoded: 3 to 10 cycles§

‡ Starting amount of DNA: number of cycles for standard samples:10 ng to 100 ng: 10 cycles100 ng to 1 µg: 6 to 8 cycles1 µg to 2 µg: 4 to 6 cycles2 µg to 5 µg: 2 to 3 cycles

§ Starting amount of DNA: number of cycles for barcoded samples:10 ng to 100 ng: 10 cycles100 ng to 1 µg: 6 to 8 cycles1 µg to 2 µg: 4 to 6 cycles2 µg to 5 µg: 3 to 6 cycles

Denature 95C 15 sec

Anneal 62C 15 sec

Extend 70C 1 min

Holding Extend 70C 5 min

Holding Holding 4C


Purify the DNA using the “SOLiD 4 Fragment Library Prep Pt2” method



Prepare reagents Refer to “Fragment Library Prep Pt2 master mix and required reagents” on page 38 for volumes and preparation.

Set up the BiomekFX/FXP



b. Select SOLiD 4 Fragment Library Prep Pt2.

2. Set up a Modular Frame with a Quarter Reservoir, Divided by Width and place the elution buffer as shown in Figure 13.


3. Set up a Modular Frame with one Half Reservoir and place the AMPure beads as shown in Figure 14.


4. Aliquot 700 µL of 70% ethanol to each well of a 2-mL deep-well plate.

5. Setup the deck.

a. Place modular reservoir 1 at P14.

b. Place modular reservoir 2 at P13.

c. Place the magnet at P10.

d. Place a 2-mL deep-well plate at P11.




e. Place a twin.tec skirted plate at P9.

f. Place a 1000-µL deep-well LoBind plate at P4.

g. Place AP96, P250, barrier, presterile tips at TL1, P16 and P17.

h. Place Span-8, P1000, barrier, conductive, presterile tips at P15.

i. Remove the foil lids from PCR plates or tubes, and place at P2, P3, P5 and P6.

j. Place 500-µL deep-well LoBind plates at P7, P8 and P19.

k. Place the 2-mL plate containing 70% ethanol at P20.

Start the run 1. Select wells for library preparation.

a. Select Select Wells for Preparation.

b. Select the appropriate wells for preparation. Highlight to select a group of wells and control-click to select individual wells.

2. Click User Input Form within the method.


a. Enter the number of libraries.

b. Select the Format for PCR from the drop-down menu.




c. Confirm the correct wells are selected.

IMPORTANT! The number of wells selected must match the number of wells being processed.

Note: The graphic below always depicts plates and does not change if tubes are selected.



6. Click Run.

7. The final deck layout (Figure 15 on page 34 and Figure 16 on page 34) displays, select OK to confirm and start the method.

Note: A timer will appear showing the time remaining for the method. Approximate run time is 1:05:00.





Figure 15 Deck layout for plate PCR

Figure 16 Deck layout for striptube PCR

8. A dialog box displays after the run is complete. Select OK to end the method.


STOPPING POINT. Store the amplified DNA at 4 ºC, or proceed to "Quantitate the purified DNA."


Quantitate the purified DNA


Quantitate the purified DNA

1. Quantitate the library by either quantitative PCR (qPCR) or using the Qubit® fluorometer. For qPCR, the SOLiD™ Library TaqMan® Quantitation Kit (PN 4449639) is recommended for accurate library quantitation. For Qubit fluorometer, use 1 µL of the library and the dsDNA HS assay or the Quant-iT™ PicoGreen® reagent.

STOPPING POINT. Store the purified DNA at 20 °C, or proceed directly to emulsion PCR.

Appendix A: Reagent volumes and preparation

Excel spreadsheet The file “SOLiD™ 4 Fragment Library Setup.xls” contains a macro to prepare adaptor dilutions, prepare standard and barcoded adaptor plates, and calculate the reagent volumes to prepare master mixes required to run the automated method.

For standard and barcoded adaptors, the macro will calculate the required adaptor based on input DNA. For AMPure XP based size selection the yield ranges from 25% to 35% across an input range of 5 ng to 8 µg. The macro assumes a 30% recovery to perform calculations. If your recovery is significantly different, contact your Applied Biosystems field application specialist.

• For standard adaptors, the molar excess has been optimized for the Biomek. The macro uses these optimized values for calculations.

• For barcoded adaptors, a 9-fold molar excess of P2 to P1 is optimal. The macro uses these optimized values for calculations.

Table 4 Adaptor excess for standard library

Library Input Range Molar Excess

> 1 µg DNA 10

200 ng to 1 µg DNA 30

50 ng to 200 ng DNA 90

< 50 ng DNA 120

Table 5 Adaptor excess for barcoded library

Library Input RangeMolar Excess

P1 P2

> 1 µg DNA 10 90

200 ng to 1 µg DNA 30 270

50 ng to 200 ng DNA 90 810

< 50 ng DNA 120 1080




• The macro calculates the required volumes for each reagent plus the required dead volume.

Time and DateStamp

Files will be saved in the directory specified in cell C1 of the spreadsheet.

• The time and date stamps are recorded on each file at the time of generation. The Biomek programs use the date and time stamps as a variable to select the appropriate files for transfer.

• The date stamp will always be in DD-MMM-YYYY format, for example July 1, 2010 will be 01-Jul-2010

• The time stamp will be in 24 hour hhmm style, for example 8:00 am will be 0800 and 5:00 pm will be 1700

• It is important to type the date and time stamps in the Biomek User Input Forms exactly as they appear in the filename

Note: Depending on the speed of the computer being used to generate the files, there is a small chance that the time stamps will not all be the same. Manually check the files to ensure they are all the same, if not rename the files to obtain a uniform time stamp. If they are not the same an error will occur when validating the Biomek method.

Fragment LibraryPrep Pt1 master

mix and requiredreagents

1. Prepare the end-repair master mix.

a. Determine the total volume by the equation:

b. Combine the appropriate volume of components to prepare the end-repair mix for the number of samples to be processed (Table 6), then mix well.

c. Add the end-repair mix to the top section of a labeled Quarter Reservoir, Divided by Width.

d. Store on ice until ready to load on the deck.

Total Volume (µL) = (Number of Samples × 90) + 200

Table 6 Mix for end-repair of the DNA

Component Volume (µL)

5× End-Polishing Buffer Total Volume × 0.4444

dNTP Mix, 10 mM Total Volume × 0.0889

End Polishing Enzyme 1, 10 U/µL Total Volume × 0.0444

End Polishing Enzyme 2, 5 U/µL Total Volume × 0.1778

Nuclease-free water Total Volume × 0.2444




2. Prepare the ligation master mix.


b. Combine the appropriate volume of components to prepare the ligation mix for the number of samples to be processed (Table 7), then mix well.

c. Add the ligation mix to the bottom section of a labeled Quarter Reservoir, Divided by Width.


3. Prepare the PCR master mix.


b. Combine the appropriate volume of components to prepare the PCR mix for the number of samples to be processed (Table 8), then mix well.

c. Transfer the PCR mix to a labeled quarter reservoir.


4. Prepare the elution buffer.


b. Add elution buffer (10 mM TrisCl, pH 8.5) to a labeled Quarter Reservoir, Divided by Width.


Table 7 Mix for ligation of the DNA


5× Ligase Buffer Total Volume × 0.8

T4 Ligase, 5 U/µL Total Volume × 0.2


Table 8 Mix for PCR of the DNA


Platinum® PCR Amplification Mix Total Volume × 0.95

Library PCR Primer 1, 50 µM Total Volume × 0.025

Library PCR Primer 2, 50 µM Total Volume × 0.025





5. Prepare the Agencourt® AMPure XP beads.


b. Gently shake the bottle of AMPure XP beads.

c. Add the AMPure XP beads to a labeled half reservoir.

6. Prepare 70% ethanol from either 100% or 95% ethanol.

IMPORTANT! Prepare fresh 70% ethanol daily to minimize DNA loss.


b. Combine and mix the following components:

c. Aliquot 1050 µL to each well of a 2-mL deep-well plate.

d. Cover with a foil seal until ready for use.

Fragment LibraryPrep Pt2 master

mix and requiredreagents

1. Prepare the elution buffer.


b. Add elution buffer (10 mM TrisCl, pH 8.5) to the top section of a labeled Quarter Reservoir, Divided by Width.



If starting from... Then use...

100% Ethanol Table 9


Table 9 Mix components for 70% ethanol solution


100% Ethanol Total Volume × 0.7


Table 10 Mix components for 70% ethanol solution


95% Ethanol Total Volume × 0.74






2. Prepare the AMPure XP beads.


b. Gently shake the bottle of Agencourt® AMPure XP beads.

c. Add AMPure XP beads to labeled half reservoir.

3. Prepare 70% ethanol from either 100% or 95% ethanol.

IMPORTANT! Prepare fresh 70% ethanol daily to minimize DNA loss.


b. Combine and mix the following components:

c. Aliquot 700 µL to each well of a 2-mL deep-well plate.

d. Cover with a foil seal until ready for use.



If starting from... Then use...



11/2010

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