trusight hla v2 sequencing panel reference guide … · 2018. 2. 2. · revisionhistory document...

TruSight HLA v2 Sequencing PanelReference GuideReference Guide

Document # 1000000010159 v01 20007429

December 2017

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

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Document # 1000000010159 v01 20007429

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

TruSight HLA v2 Sequencing Panel Reference Guide

Revision History

Document Date Description of Change

Material # 20007429Document #1000000010159 v01

December2017

• Added bullet to Tips and Techniques section about plate storage and tipusage.

• Re-arranged wording in step in Tagment HLA PCR Amplicons section.• Added approximate volume to be removed in “Remove and discard allsupernatant” in 8 locations.

• Added step to “Spin the plate briefly” in the procedure in the Tagment HLAPCR Amplicons section. Also hanged “Transfer 40 ul from each well” to“Transfer 40 ul of the liquid-bead mixture from each well”. Added “keep onice or refrigerated until use” for HTM in consumables list.

• Modified note at the end of the Washing section.• Updated Figure 5 in Elution section to correct DRB naming• Updated Figure 6 in Elution section to correct DRB naming• Removed unnecessary centrifugation step in Elution section.

Material # 20007429Document #1000000010159 v00

May 2016 Initial release.

Document # 1000000010159 v01 20007429

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


Table of Contents

Chapter 1 Overview 1Introduction 1DNA Input Recommendations 1Additional Resources 1

Chapter 2 Protocol 3Introduction 3Tips and Techniques 3Library Prep Workflow 4Generate HLA PCR Amplicons 5Clean Up HLA PCR Amplicons 8Normalize HLA PCR Amplicons 11Tagment HLA PCR Amplicons 12Pool and Clean Up Tagmentation Reaction 13Amplify PCR 16Clean Up PCR 18Pool Final Libraries for MiSeq Sequencing 19

Appendix A Supporting Information 22Introduction 22Acronyms 22Kit Contents 22Consumables and Equipment 24

Technical Assistance 26

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

Chapter 1 Overview

OverviewIntroduction 1DNA Input Recommendations 1Additional Resources 1

IntroductionThis protocol explains how to prepare indexed libraries from input DNA for subsequent cluster generation andDNA sequencing using the reagents provided in the Illumina TruSight HLA v2 Sequencing Panel. The goal ofthis protocol is to isolate and amplify HLA loci with long-range polymerase chain reaction (PCR). Afteramplification, Nextera® tagmentation fragments the DNA and leaves behind a sequence tag. An additionalPCR then adds sequence adapters and indexing primers to generate DNA libraries ready for sequencing onthe Illumina MiSeq systems.

The TruSight HLA v2 Sequencing Panel protocol provides:

u A single assay and workflow to obtain ultrahigh resolution sequencing of 11 HLA Loci (Class I HLA-A, -B,-C; Class II HLA-DRB1/3/4/5, -DQA1, -DQB1, -DPA1, -DPB1)

u Unambiguous, phase-resolved HLA typing

u Sample-to-report includes assay-optimized HLA typing analysis and reporting software

u Multiplexing capabilities of up to 24 samples per TruSight HLA v2 kit using MiSeq v2 chemistry

u Gene coverage including exons and introns allows for phase resolution across the entire gene,dramatically reducing the need to resolve phase ambiguities with follow-up assays

This protocol describes how to process 12 samples in parallel. The TruSight HLA v2 kit includes enoughreagents to process 24 samples in total.

DNA Input Recommendationsu Quantify DNA with a fluorometric method specific for double-stranded DNA (dsDNA), such as the

following:u Qubit BR assayu Quant-iT™ PicoGreen

u Avoid methods that measure total nucleic acid content, such as NanoDrop or other UV absorbancemethods.

u Resuspend and normalize samples in water or Tris-HCl 10 mM to a final concentration of 10 ng/µl. Thisassay requires an input of 400 ng of sample (40 µl at the final normalized concentration of 10 ng/µl).

u The TruSight HLA v2 Sequencing Panel protocol is optimized for 400 ng of purified input DNA. Illuminarecommends that the DNA is intact and not fragmented; at least 50% of the DNA must be greater than20 kb. Fragmented DNA affects the long-range PCR performance.

u Use a positive control when running the library preparation for the first time. Illumina recommends thereference DNA control sample IHW09263 from the International Histocompatibility Working Group.

Additional ResourcesVisit the TruSight HLA v2 Sequencing Panel support page on the Illumina website for documentation,software downloads, training resources, and information about compatible Illumina products.

The following documentation is available for download from the Illumina website.

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Resource Description

TruSight HLA v2 SequencingPanel Protocol Guide (document #1000000010160)

Provides only protocol instructions.The protocol guide is intended for experienced users. For new or less experienced users,see the TruSight HLA v2 Sequencing Panel Reference Guide.

TruSight HLA v2 SequencingPanel Checklist (document #1000000010161)

Provides a checklist of the protocol steps.The checklist is intended for experienced users. For new or less experienced users, seethe TruSight HLA v2 Sequencing Panel Reference Guide.

TruSight HLA v2 SequencingPanel Consumables & Equipment(document # 1000000010162)

Provides an interactive checklist of user-provided consumables and equipment.

Conexio Assign™ v1.0 TruSightHLA Analysis Software (document# 15059520)Conexio Assign™ v2.0 TruSightHLA Analysis Software (document# 1000000010450)

Provides information about the Conexio Assign™ TruSight HLA Analysis Softwaresequencing data analysis tool. Enables you to import sequence data, perform basecalling, edit sequences, and compare a consensus sequence with a library ofsequences of HLA alleles.

TruSight HLA v2 Sample SheetTemplate (document #1000000012322)

A template for creating a MiSeq sample sheet for up to 96 samples. The template iscompatible with Nextera XT indexes and Nextera XT v2 indexes.

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Chapter 2 Protocol

ProtocolIntroduction 3Tips and Techniques 3Library Prep Workflow 4Generate HLA PCR Amplicons 5Clean Up HLA PCR Amplicons 8Normalize HLA PCR Amplicons 11Tagment HLA PCR Amplicons 12Pool and Clean Up Tagmentation Reaction 13Amplify PCR 16Clean Up PCR 18Pool Final Libraries for MiSeq Sequencing 19

IntroductionThis chapter describes the TruSight HLA v2 Sequencing Panel protocol.

u Follow the protocols in the order shown, using the specified volumes and incubation parameters.

u Review Best Practices from the TruSight HLA v2 support page on the Illumina website.

u Before proceeding, confirm kit contents and make sure that you have the required equipment andconsumables.

u Before beginning library preparation, record the samples and indexes you are using in a sample sheet.Use the TruSight HLA v2 Sample Sheet Template (part # 1000000012322) on the TruSight HLA v2support page.

Tips and TechniquesUnless a safe stopping point is specified in the protocol, proceed immediately to the next step.

Avoiding Cross-Contaminationu When adding or transferring samples, change tips between each sample.

u When adding adapters or primers, change tips between each row and each column.

u Remove unused index adapter tubes from the working area.

Sealing the Plateu Always seal the 96-well plate before the following steps in the protocol:

u Shaking stepsu Vortexing stepsu Centrifuge stepsu Thermal cycling steps

u Apply the adhesive seal to cover the plate and seal with a rubber roller.

u Microseal 'B' adhesive seals are effective at -40°C to 110°C. Use Microseal 'B' for shaking, centrifuging,and long-term storage.

u Foil seals are effective at -70°C to 105°C.

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Plate Transfersu When transferring volumes between plates, transfer the specified volume from each well of a plate to the

corresponding well of the other plate.u The protocol is written so that plates can be stored prior to each bead cleanup. If you are proceeding

through protocol without the optional stopping point, you can reduce tip usage by adding the beadmixture to the midi plate prior to the transfer of DNA. This is applicable to the Clean Up HLA PCRAmplicons, Normalize HLA PCR Amplicons, and Clean Up PCR steps.

Centrifugationu Centrifuge at any step in the procedure to consolidate liquid or beads in the bottom of the well, and to

prevent sample loss.

Handling Beadsu Pipette bead suspension slowly.

u When mixing, mix thoroughly.

u If beads are aspirated into the pipette tips, dispense back to the plate on the magnetic stand and waituntil the liquid is clear (~2 minutes).

u When washing beads:u Use the appropriate magnet for the plate.u Dispense liquid so that beads on the side of the wells are wetted.u Keep the plate on the magnet until the instructions specify to remove it.u Do not agitate the plate while on the magnetic stand. Do not disturb the bead pellet.

Washingu For removal of supernatant steps, set pipettes to 200 µl for the most efficient way to capture all aqueous

supernatant. Volumes of supernatant vary but will never be more than 200 µl (pulling air into the pipettedoes not cause issues).

Library Prep WorkflowThe following diagram illustrates the workflow using a TruSight HLA v2 Sequencing Panel kit. Safe stoppingpoints are marked between steps.

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Figure 1 Workflow Diagram

Generate HLA PCR AmpliconsThis PCR step generates 8 HLA amplicons from each sample using 2 PCR plates for 12 samples.

NOTE

Performing this PCR step overnight is recommended due to the length of the PCR program.

Consumablesu HLA Locus-Specific Primers: HLA-A, HLA-B.2, HLA-C, DPA1, DPB1, DQA1, DRB.2, DQB1

u HPM (HLA PCR Mix)

u MasterAmp Extra-Long DNA Polymerase

u Foil adhesive seals

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u PCR-grade water

u Purified DNA, 10 ng/µl (40 µl per sample)

u Troughs, sterile, low-volume

u 96-well PCR plate (2)

u 96-well midi plates (2)

u 15 ml conical tube

About Reagentsu Do not remove MasterAmp Extra-Long DNA Polymerase from storage until needed in the procedure.

Preparation1 Review DNA Input Recommendations on page 1.

2 Prepare a sample sheet using the sample sheet template on the TruSight HLA v2 support page.

3 Label 2 new 96-well PCR plates LRP1 and LRP2.

4 Save the following programs as PCR1 and PCR2 on a thermal cycler with a heated lid (95°C to 100°C).

PCR1: LRP1 Plate PCR2: LRP2 Plate

u 94°C for 3 minutesu 30 cycles of:

u 94°C for 30 secondsu 60°C for 2 minutesu 68°C for 15 minutes

u 68°C for 10 minutesu Hold at 10°C

u 94°C for 3 minutesu 10 cycles of:

u 94°C for 30 secondsu 55°C for 2 minutesu 72°C for 15 minutes

u 20 cycles of:u 94°C for 30 secondsu 60°C for 2 minutesu 72°C for 15 minutes

u 72°C for 10 minutesu Hold at 10°C

Procedure

NOTE

This procedure is time sensitive. For best results, complete the steps within 30minutes.

1 Quantify and normalize DNA to 10 ng/µl in a volume of at least 40 µl in water or 10 mM Tris-HC1,according to the DNA Input Recommendations on page 1.

2 Using an Illumina Index Plate Fixture designated for pre-PCR, add 5 µl of each HLA primer to the LRPplates, as follows.u HLA-A—LRP1 row Au HLA-B.2—LRP1 row Bu HLA-C—LRP1 row Cu DPA1—LRP1 row Du DPB1—LRP1 row Eu DQA1—LRP1 row Fu DRB.2—LRP1 row G

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u DQB1—LRP2 row D (to decrease evaporation risk)

Figure 2 LRP1 Plate Layout

Figure 3 LPR2 Plate

3 Add 5 µl of 10 ng/µl template DNA to the LRP plates, as follows.u Samples 1–12—LRP1 rows A–Gu Samples 1–12—LRP2 row D

4 Combine the following reagents in a 15 ml conical tube to create PCR master mix.Volumes listed for 12 samples include 10% overfill.Using a low-volume sterile trough and multichannel pipette to dispense the master mix is recommended.

PCR Component Per Well Per 12 Samples

HPM (HLA PCR Mix) 25 µl 2640 µl

MasterAmp Extra-Long DNA Polymerase 2 µl 212 µl

PCR-grade water 13 µl 1373 µl

NOTE

HPM is the limiting reagent for repeated uses of the kit. If necessary, spin the tube to consolidate theremaining liquid.

5 Add 40 µl PCR master mix to each well that contains sample. Pipette to mix.

6 Seal the plates with foil seals.

NOTE

Make sure that plates are properly sealed to prevent evaporation during long-range PCR. The cornerwells are the most susceptible to evaporation.

7 Centrifuge at 280 × g for 1 minute.

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8 Place the LRP1 plate on a thermal cycler and run the PCR1 program.

9 Place the LRP2 plate on a thermal cycler and run the PCR2 program.

NOTE

The PCR programs can run overnight.

10 After the PCR reaction is complete, centrifuge the plates at 280 × g for 2 minutes.

11 Label a new midi plate LRC.

12 Transfer 45 µl of samples from the 2 LRP plates to a single LRC plate, as follows.u LRP1 rows A–G to LRC rows A–Gu LRP2 row D to LRC row H

NOTE

Evaporation of up to 15 µl does not negatively affect library preparation.

Figure 4 LRC Plate Layout

SAFE STOPPING POINT

If you are stopping, seal the plates and store at -25°C to -15°C for up to 7 days.

Clean Up HLA PCR AmpliconsThis step uses SPB (Sample Purification Beads) to purify the HLA PCR amplicons from other reactioncomponents. The cleanup step is performed with large volumes in midi plates to maximize yield and minimizecross-contamination.

Consumablesu RSB (Resuspension Buffer)

u SPB (Sample Purification Beads)

u Freshly prepared 80% ethanol (EtOH)

u 96-well midi plates

u Microseal 'B' adhesive seals

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Preparation1 Prepare the following consumables.

Reagent Storage Instructions

RSB -25°C to -15°C Thaw at room temperature. Let stand for 30 minutes to bring to roomtemperature.

SPB 2°C to 8°C Let stand for 30 minutes to bring to room temperature.Vortex vigorously to ensure adequate resuspension.

2 Prepare 50 ml of fresh 80% EtOH.

3 Label a new midi plate LRB.

Procedure

Binding1 Add 31.5 µl SPB to each well of the LRC Plate.

NOTE

Return unused SPB to the original tube to use in later steps

2 Shake at 1800 rpm for 2 minutes.

3 Incubate at room temperature for 2 minutes.

Washing1 Place on the magnetic stand and wait until the liquid is clear (~2 minutes).

2 Remove and discard all supernatant.

3 Wash two times, as follows.

a Add 200 µl freshly prepared 80% EtOH to each well.b Incubate on the magnetic stand for 30 seconds.c Remove and discard all supernatant from each well.

4 Use a 20 µl pipette to remove residual EtOH from each well.

5 Air-dry on the magnetic stand for 2 minutes. Make sure that there is no residual liquid.

Elution1 Add 30 µl RSB to each well.



4 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).

5 Transfer 20 µl supernatant from each well of the LRC plate to the corresponding wells of the LRB plate.

6 [Optional] Check amplicon size using the following options:u Run 5 µl resulting HLA PCR amplicon remaining in the LRC plate on a 0.8% agarose gel at 100 volts.

Use a 1 kB ladder for 30 minutes or until the ladder is resolved to verify the size.

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u Run 1 µl resulting HLA PCR amplicon remaining in the LRC plate on a Bioanalyzer using the AgilentDNA 12000 Kit.

Class Gene Size (kb)

I HLA-A 4.1

HLA-B 2.8

HLA-C 4.2

II DPA1 10.3

DPB1 9.7

DQA1 7.3

DRB 4.6

DQB1 7.1

Figure 5 HLA PCR Amplicon Sizes Using Agarose Gel

NOTE

Gel bands can be faint, especially DPA1, but still genotype correctly.

Figure 6 Example HLA PCR Amplicon Sizes Using Bioanalyzer Gel

SAFE STOPPING POINT

If you are stopping, seal the plates and store at -25°C to -15°C for up to 7 days.

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Normalize HLA PCR AmpliconsThis process normalizes the HLA PCR amplicons, which eliminates the need for manual quantification andnormalization.

NOTE

This procedure is time sensitive.

Consumablesu HTB (HLA Tagmentation Buffer)

u LNA1 (Library Normalization Additives 1)

u LNB1 (Library Normalization Beads 1)

u RSB (Resuspension Buffer)

u 15 ml conical tube


u Troughs, sterile

WARNING

This set of reagents contains formamide, an aliphatic amide that is a probable reproductive toxin. Personalinjury can occur through inhalation, ingestion, skin contact, and eye contact. Dispose of containers and anyunused contents in accordance with the governmental safety standards for your region.

For more information, see the SDS for this kit, at support.illumina.com/sds.ilmn.

About Reagentsu Do not use a P200 pipette to handle LNB1.

u Mix only the amounts of LNA1 and LNB1 required for the current experiment.

u Store remaining LNA1 and LNB1 separately at their respective temperatures.

u Make sure that LNB1 is resuspended before use. Homogeneous resuspension is essential for consistentnormalization.



LNA1 -25°C to -15°C Thaw at room temperature. Let stand for 30 minutes to bring to roomtemperature.Vortex vigorously, and then inspect in front of a light to make sure that allprecipitate has dissolved.

LNB1 2°C to 8°C Let stand for 30 minutes to bring to room temperature.Vigorously vortex at least 1 minute, and then invert to resuspend. Make surethat no pellet is present at the bottom of the tube.

HTB -25°C to -15°C Thaw at room temperature.

Procedure1 Add 4.4 ml LNA1 to a new 15 ml conical tube.

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2 Pipette to mix LNB1 to ensure adequate resuspension.

3 Transfer 400 µl LNB1 to the 15 ml conical tube that contains LNA1.Return unused LNB1 to 2°C to 8°C storage.

4 Invert the tube to mix.

Binding1 If the LRB plate was stored, centrifuge the plate at 280 × g for 2 minutes.

2 Add 45 µl LNB1/LNA1 mixture to each well of the LRB plate.


Washing1 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).


3 Add 45 µl RSB to each well.




7 Use a 20 µl pipette to remove residual liquid from each well.

8 Add 40 µl HTB to each well.


NOTE

Proceed immediately to the next step with the bead mixture.

Tagment HLA PCR AmpliconsThis step uses the TruSight HLA v2 transposome to tagment the amplicon DNA, which is a process thatfragments DNA and then tags the DNA with adapter sequences in a single step.

NOTE

Perform tagmentation using a thermal cycler (Option 1) or a TruTemp microheating system (Option 2).

Do not use a foil seal for the tagmentation step when using Option 2 on a TruTemp microheating system. Forthis step, use Microseal 'B' adhesive seals.

Consumablesu HTM (HLA Tagmentation Mix); keep on ice or refrigerated until use

u PCR 8-tube strip

u [Optional] PCR plate


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Preparation1 Gently invert HTM to mix, and then centrifuge briefly.

2 [Option 1] Preheat a thermal cycler without a heated lid to 58°C.

3 [Option 1] Label a new 96-well PCR plate TAG.

4 [Option 2] Preheat a TruTemp microheating system to 58°C.

5 Label a new PCR plate NTC.

Procedure1 Calculate the total volume of HTM for all reactions, including 10% extra. Divide the volume equally in each

well of a PCR 8-tube strip.

2 [Option 1] Using a thermal cycler:

a Using a prealiquoted PCR 8-tube strip, add 10 μl HTM to each well of the TAG plate, and thenpipette to mix.

b Transfer 40 µl from each well of the LRB plate to the corresponding wells of the TAG plate.c Place on a thermal cycler (58°C) for 12 minutes.d Spin the plate briefly.e Place on a magnetic stand and wait until the liquid is clear (~2 minutes).

3 [Option 2] Using a TruTemp microheating system:

a Using a prealiquoted PCR 8-tube strip, add 10 μl HTM to each well of the LRB plate.b Shake for 1 minute at 1600 rpm.c Place on a TruTemp microheating system set to 58°C for 12 minutes.d Spin the plate briefly.e Place on a magnetic stand and wait until the liquid is clear (~2 minutes).

4 Transfer 50 µl from each well of the TAG plate to the NTC plate.

NOTE

Proceed immediately to the next step.

Pool and Clean Up Tagmentation ReactionThis step pools the tagmented DNA into a single well for each sample. The pools are then purified using SPB.



u 96-well midi plate

u 96-well PCR plate



u Troughs, sterile

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RSB 2°C to 8°C Let stand for 30 minutes to bring to room temperature.

2 Prepare 10 ml of fresh 80% EtOH.

3 Label a new midi plate TPP.

4 Label a new PCR plate NPP.

Procedure

Pooling1 For each sample, pool tagmentation products from the NTC plate into row A of the TPP plate, as follows

(see Figure 7).u HLA-A (10 µl)u HLA-B.2 (10 µl)u HLA-C (10 µl)u DPA1 (10 µl)u DPB1 (10 µl)u DQA1 (10 µl)u DRB.2 (20 µl)u DQB1 (10 µl)Store remaining tagmentation product at -25°C to -15°C for up to 1 week.

Figure 7 TPP Plate, Pooling

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Binding1 Add 63 µl SPB to each well of row A of the TPP plate.

NOTE

A 1:0.7 ratio of tagmented DNA reaction to SPB is critical.

NOTE

Return unused SPB to the original tube to use in later steps





3 Wash 2 times, as follows.

a Add 200 µl freshly prepared 80% EtOH to each sample well.b Incubate on the magnetic stand for 30 seconds.c Remove and discard all supernatant from each well.



Elution1 Add 22.5 μl RSB to each sample well.



4 Place on a magnetic stand and wait for the liquid to clear (~2 minutes).

5 Transfer 20 µl supernatant to the NPP plate, as follows.u Samples 1–6—Row A, columns 1–6u Samples 7–12—Row B, columns 1–6

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Figure 8 NPP Plate, Elution

SAFE STOPPING POINT

If you are stopping, seal the plates and store at -25°C to -15°C for up to 1 day.

Amplify PCRIn this step, the tagmented DNA is amplified and sequencing adapters are added using a limited-cycle PCRprogram. The PCR step adds Index 1 (i7) adapters, Index 2 (i5) adapters, and sequences required for clusterformation. To make sure that libraries produce quality sequencing results, use the recommended number ofPCR cycles, which has been calibrated to the amount of input DNA resulting from the Pool and Clean UpTagmentation Step. Use the index combinations selected during preparation of the sample sheet.

Consumablesu Nextera XT Index Kit (24 Indexes, 96 samples)

u NLM (Nextera Library Amplification Mix)

u TruSeq Index Plate Fixture

u Foil adhesive seals



NLM -25°C to -15°C Thaw at room temperature for 20 minutes.Invert each tube to mix.Keep on ice until needed.

Index adapters (i5 and i7) -25°C to -15°C Thaw at room temperature for 20 minutes.Invert each tube to mix. Centrifuge briefly.

2 Save the following program as IndexAmp on a thermal cycler with a heated lid (100°C).

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u 72°C for 3 minutesu 98°C for 30 secondsu 10 cycles of:

u 98°C for 10 secondsu 60°C for 30 secondsu 72°C for 5 minutes

u 72°C for 5 minutesu Hold at 10°C (overnight maximum)

Procedure1 Arrange the Nextera XT Index Kit in the TruSeq Index Plate Fixture, as follows.

u Index 1 (i7) adapters in columns 1–6.u Index 2 (i5) adapters in rows A and B.

NOTE

If processing 12 samples, use only 2 of the 4 Index 2 (i5) adapters. Make sure to use the same indexesspecified in your sample sheet.

Figure 9 TruSeq Index Plate Fixture

A Columns 1–6: Index 1 (i7) adapters (orange caps)B Rows A and B: Index 2 (i5) adapters (white caps)C NPP plate

2 Place the NPP plate on a TruSeq Index Plate Fixture.

3 Using a multichannel pipette, add 5 µl of each Index 1 (i7) adapter to row A and B. Replace the cap oneach i7 adapter tube with a new orange cap.

4 Using a multichannel pipette, add 5 µl of each Index 2 (i5) adapter to columns 1–6. Replace the cap oneach i5 adapter tube with a new white cap.

5 Add 20 µl NLM to each well. Pipette to mix.

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6 Seal with the foil seal and centrifuge at 280 × g at 20°C for 1 minute.

7 Place the plate on the thermal cycler and run the IndexAmp program.

SAFE STOPPING POINT

If you are stopping, seal the plate and store at 2°C to 8°C for up to 2 days. Alternatively, leave on the thermalcycler overnight.

Clean Up PCRThis step uses SPB (Sample Purification Beads) to purify the library DNA, and provides a size-selection stepthat removes small fragments from the library.




u 96-well midi plate

u 96-well PCR plate


u Troughs, sterile

About Reagentsu Return unused SPB to the original tube to use in later steps.




RSB 2°C to 8°C Let stand for 30 minutes to bring to room temperature.

2 Prepare fresh 80% EtOH.

3 Label a new midi plate NPC.

4 Label a new PCR plate HLP.

Procedure

Binding1 Transfer 45 µl of the PCR reactions from each well of the NPP plate to the corresponding wells of the NPC

plate.

2 Add 31.5 µl SPB to each well.

NOTE

A 1:0.7 ratio of PCR reaction to SPB is critical.

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3 Wash two times, as follows.

a Add 200 µl freshly prepared 80% EtOH to each well.b Incubate on the magnetic stand for 30 seconds.c Remove and discard all supernatant from each well.



Elution1 Add 32.5 µl RSB to each well.




5 Transfer 30 µl supernatant from each well of the NPC plate to the corresponding well of the HLP plate.

SAFE STOPPING POINT

If you are stopping, seal the plates and store at 2°C to 8°C for up to 7 days.

Pool Final Libraries for MiSeq SequencingIn preparation for cluster generation and sequencing, equal volumes of library are combined, denatured inNaOH, and diluted in Hybridization Buffer before MiSeq sequencing. This procedure is a guideline for dilutionfor optimal cluster density. Adjust final dilution on a sample-to-sample basis.

Illumina recommends the following pooling guidelines:

u For a micro MiSeq v2 flow cell—Pool 12–24 samples

u For a nano MiSeq v2 flow cell—Pool 6 samples

Consumablesu HT1 (Hybridization Buffer) provided in the MiSeq Reagent Kit

u RSB (Resuspension Buffer)

u MiSeq reagent cartridge v2 (300 cycles), micro or nano

u 0.1 N NaOH (prepared fresh)

u 1.5 ml Eppendorf Tube

u Deionized water

u Qubit dsDNA BR Assay Kit

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MiSeq reagentcartridge

-25°C to -15°C Thaw in a room temperature water bath for 1 hour, or overnight at2°C to 8°C.

HT1 -25°C to -15°C Thaw at room temperature. Set aside at 2°C to 8°C.

2 Prepare a fresh dilution of 0.1 N NaOH from 2 N NaOH.

3 Label a new 1.5 ml Eppendorf tube PHL.

4 Label a new 1.5 ml Eppendorf tube DHL.

Procedure1 Transfer 7 µl from each well of the HLP plate to the PHL tube.

2 [Optional] Run 1 µl of pooled libraries on a DNA 12000 Bioanalyzer chip to check library size distribution.

Figure 10 Size Distribution of HLA Library

3 Quantify the library with the Qubit BR assay or a fluorometric assay, such as PicoGreen.

4 Determine the library volume to denature using the following formula:Y = 15/xu X is the library concentration (ng/µl) as determined by the fluorometric assayu Y is the library volume (µl) to dilute and denature

5 Transfer the volume determined by Y to the DHL tube.

NOTE

If Y > 10 µl in the calculation, the library yield might be too low. Contact Illumina Technical Support [email protected].

6 Dilute with RSB to a final volume of 10 µl.

7 Add 10 µl 0.1 N NaOH.

8 Vortex and then centrifuge briefly to mix.


10 Add 980 µl HT1 for a final volume of 1000 µl, and then invert to mix.

11 Load 600 µl denatured library from the DHL tube onto the thawed reagent cartridge.

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NOTE

See the MiSeq System Guide (document # 15027617) for information about loading consumables andsetting up a run.

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Appendix A Supporting Information

Supporting InformationIntroduction 22Acronyms 22Kit Contents 22Consumables and Equipment 24

IntroductionThe protocols described in this guide assume that you have reviewed the contents of this appendix,confirmed your kit contents, and obtained all the required consumables and equipment.

Acronyms

Acronym Definition

DHL Diluted HLA Libraries

HLP HLA Library Plate

HPM HLA PCR Mix

HTB HLA Tagmentation Buffer

HTM HLA Tagmentation Mix

IHP Input HLA Plate

ISC Input Sample Clean Up

LNA1 Library Normalization Additives 1

LNB1 Library Normalization Beads 1

LRB Long Range Bead Based Normalization 2

LRC Long Range Clean Up

LRP Long Range PCR

NLM Nextera Library Amplification Mix

NPC Nextera PCR Clean Up

NPP Nextera PCR Plate

NTC Nextera Tagmentation Clean Up

PHL Pool HLA Libraries

RSB Resuspension Buffer

SPB Sample Purification Beads

TPP Tagmentation Pooling Plate

Kit ContentsMake sure that you have all the reagents identified in this section before starting the protocol.

Each sample requires a unique index combination. Illumina offers the following kits:

u Nextera XT Index Kit—24 indexes for processing up to 24 samples at a time

u Nextera XT Index Kit v2 (Set A, B, C, D)—4 unique 96 index kits for higher throughput

Illumina recommends running 12–24 samples using a MiSeq Micro Reagent Kit v2 (flow cell and cartridge).Alternatively, you can run 6 samples using a MiSeq Nano Reagent v2 (flow cell and cartridge).

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Kit Name Catalog #

TruSight HLA v2 Sequencing Panel (24 Samples) 20000215

TruSight HLA v2 Sequencing Panel (24 Samples Automated) 20005170

Nextera XT Index Kit (24 Indexes, 96 Samples) FC-131-1001

[Recommended] TruSeq Index Plate Fixture Kit FC-130-1005

Box 1, Store at -25°C to -15°C

Quantity Reagent Description

1 RSB Resuspension Buffer

1 HLA-A HLA-A Primers

1 HLA-B.2 HLA-B Primers

1 HLA-C HLA-C Primers

1 DRB.2 HLA-DRB Primers

1 DQB1 HLA-DQB1 Primers

1 DQA1 HLA-DQA1 Primers

1 DPB1 HLA-DPB1 Primers

1 DPA1 HLA-DPA1 Primers

5 MasterAmp DNA Polymerase

1 HPM HLA PCR Mix

Box 2, Store at 2°C to 8°C


3 SPB Sample Purification Beads

1 LNB1 Library Normalization Beads

Box 3, Store at -25°C to -15°C


3 RSB Resuspension Buffer

2 LNA1 Library Normalization Additives

2 HTM HLA Tagmentation Mix

2 HTB HLA Tagmentation Buffer

1 NLM Nextera Library Amplification Mix

Auxiliary Reagents, Automated Kit Only, Store at 2°C to 8°C


1 SPB Sample Purification Beads

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Consumables and EquipmentConfirm that all required user-supplied consumables and equipment are present and available prior tostarting the protocol.

The protocol has been optimized and validated using the items listed. Comparable performance is notguaranteed when using alternate consumables and equipment.

Consumables

Consumables Supplier/Description

20 µl pipette filter tips General lab supplier

20 µl multichannel pipettes General lab supplier

20 µl single channel pipettes General lab supplier




200 µl multichannel pipettes General lab supplier


96-well storage plates, round well,0.8 ml (midi plate)

Fisher Scientific, part # AB-0859

Ethanol 200 proof (absolute)for molecular biology (500 ml)

Sigma-Aldrich, part # E7023

96-well PCR plates compatible with thermal cycler General lab supplier

RNase/DNase-free multichannel reagent reservoirs,disposable, 100 ml and 25 ml

VWR, part # 89094-658 (100 ml), 89094-664 (25 ml)

Tris-HCl 10 mM, pH 8.5 Prepared from Tris-HCl saltGeneral lab supplier

Laboratory Grade water General lab supplier

Foil seals Beckman Coulter, part # 538619

Microseal 'B' adhesive seals Bio-Rad, part # MSB-1001

MiSeq Reagent Kit v2 (300 cycles) Illumina

1.5 ml microcentrifuge tubes General lab supplier

2 N NaOH General lab supplier

Equipment

Equipment Supplier/Description

96-well thermal cycler(with heated lid)

See table in Thermal Cycler section.

[Optional] Microheating System—SciGene TruTemp Heating System Illumina, catalog # SC-60-503 (115 V)or catalog # SC-60-504 (220 V)

[Optional] midi plate insert for microheating system* Illumina, catalog # BD-60-601

High-Speed Microplate Shaker (1800 rpm) BioShake iQ, catalog # 1808-0505

Magnetic stand-96 Ambion, part # AM10027

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Equipment Supplier/Description

Microplate centrifuge General lab supplier

Vortexer General lab supplier

ThermalCyclersThe following table lists the recommended settings for the thermal cycler and other comparable models. Ifyour lab has a thermal cycler that is not listed, validate the thermal cycler before performing the protocol.

Thermal Cycler Temp Mode Lid Temp Vessel Type

Bio-Rad C1000 Calculated Heated Plate

Applied Biosystems GeneAmpPCR System 9700 Calculated Heated Plate

ABI veriti Calculated Heated Plate

Eppendorf Nexus Calculated Heated Plate

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Technical AssistanceFor technical assistance, contact Illumina TechnicalSupport.Website: www.illumina.comEmail: [email protected]

IlluminaCustomer Support TelephoneNumbers

Region Toll Free Regional

North America +1.800.809.4566

Australia +1.800.775.688

Austria +43 800006249 +43 19286540

Belgium +32 80077160 +32 34002973

China 400.066.5835

Denmark +45 80820183 +45 89871156

Finland +358 800918363 +358 974790110

France +33 805102193 +33 170770446

Germany +49 8001014940 +49 8938035677

Hong Kong 800960230

Ireland +353 1800936608 +353 016950506

Italy +39 800985513 +39 236003759

Japan 0800.111.5011

Netherlands +31 8000222493 +31 207132960

New Zealand 0800.451.650

Norway +47 800 16836 +47 21939693

Singapore +1.800.579.2745

Spain +34 911899417 +34 800300143

Sweden +46 850619671 +46 200883979

Switzerland +41 565800000 +41 800200442

Taiwan 00806651752

United Kingdom +44 8000126019 +44 2073057197

Other countries +44.1799.534000

Safety data sheets (SDSs)—Available on the Illumina website at support.illumina.com/sds.html.

Product documentation—Available for download in PDF from the Illumina website. Go tosupport.illumina.com, select a product, then select Documentation & Literature.

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http://www.illumina.com/

mailto:[email protected]

http://support.illumina.com/sds.html

http://www.illumina.com/support.ilmn

Il lumina

5200 Illumina Way

San Diego, California 92122 U.S.A.

+1.800.809.ILMN (4566)

+1.858.202.4566 (outside North America)

[email protected]

www.illumina.com

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

© 2017 Illumina, Inc. All rights reserved.

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