TruSight Tumor 26 Reference Guide (15042911 v02)

TruSight Tumor 26 Reference Guide (15042911 v02)

Appendix A  Supporting Information

Supporting Information

Introduction

How Does the Assay Work?

Acronyms

TruSight Tumor 26 Kit Contents

Consumables and Equipment

Index Sequences

28

29

30

31

33

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Introduction

The 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.

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How Does the Assay Work?

For each amplicon, 2 pairs of oligos are designed. One pair is complementary to one strand and another pair to the opposite strand. In separate wells of a 96-well plate, these oligos hybridize to the genomic DNA. Extension and ligation then form DNA templates consisting of the regions of interest flanked by universal primer sequences. Using indexed primers supplied with the kit, DNA templates are then amplified using PCR. The library products are then pooled into a single tube, and then sequenced.

Figure 5 How the TruSight Tumor 26 Assay Works

A

Hybridization of custom oligonucleotide probes

B

Extension and ligation

C

Addition of indexes and sequencing adapters by PCR

D

Final amplicon ready for sequencing

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Acronyms

Acronym

FPB

FPU

HT1

HYP

IAP

LNP

OHS3

ACD1

ACP1

CLP

EBT

ELM3

FPA

PAL

PMM2

QCP

QCT

SGP

SW1

TDP1

UB1

Definition

Amplicon Control DNA 1

Amplicon Control Oligo Pool 1

Clean-up Plate

Elution Buffer with Tris

Extension Ligation Mix 3

TruSight Tumor Oligo Pool A

TruSight Tumor Oligo Pool B

Filter Plate Unit

Hybridization Buffer

HYbridization Plate

Indexed Amplification Plate

Library Normalization Plate

Oligo Hybridization for Sequencing

Reagent 3

Pooled Amplicon Library

PCR Master Mix 2

Quality Control Primers

Quality Control Template

Storage Plate

Stringent Wash 1

TruSeq DNA Polymerase 1

Universal Buffer 1

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TruSight Tumor 26 Kit Contents

The TruSight Tumor 26 Kit contains the following components and is shipped on dry ice unless specified otherwise. When you receive your kit, store the kit components at the specified temperatures and in designated pre-amplification and post-amplification areas.

Kit Name

TruSight Tumor 26

TG* TruSight Tumor 26

Catalog #

FC-130-2001

TG-130-2001

# Samples

48

48

NOTE

*TG-labeled consumables include features intended to help reduce the frequency of revalidation. They are available only under supply agreement and require you to provide a binding forecast. Contact your account manager for more information.

TruSight Tumor 26 Kit

Box 1 - Pre-Amplification

1

1

1

1

1

Quantity

1

1

1

Reagent

ACD1

ACP1

OHS3

ELM3

PMM2

TDP1

SW1

UB1

Description

Amplicon Control DNA 1

Amplicon Control Oligo Pool 1

Oligo Hybridization for Sequencing Reagent 3

Extension Ligation Mix 3

PCR Master Mix 2

TruSeq DNA Polymerase 1

Stringent Wash 1

Universal Buffer 1

Storage Temperature

-25°C to -15°C

-25°C to -15°C

-25°C to -15°C

-25°C to -15°C

-25°C to -15°C

-25°C to -15°C

2°C to 8°C

2°C to 8°C

WARNING

This set of reagents contains formamide, an aliphatic amide that is a probable reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Wear protective equipment, including eye protection, gloves, and laboratory coat. Handle used reagents as chemical waste and discard in accordance with

the governmental safety standards for your region. For environmental, health, and safety information, see the SDS for this kit at support.illumina.com/sds.html

.

Box 2 - Post-Amplification

Quantity

1

4

Reagent

HT1

EBT

Description

Hybridization Buffer

Elution Buffer with Tris

Storage Temperature

-25°C to -15°C

Room temperature

Box 3 - TruSight Tumor Oligo Set, Pre-Amplification, Store at -25°C to

-15°C

Quantity

1

Reagent

QCP

Description

Quality Control Primers

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1

1

Quantity

1

Reagent

FPA

FPB

QCT

Description

TruSight Tumor Oligo Pool A

TruSight Tumor Oligo Pool B

Quality Control Template

TruSight Tumor 26 Index Kit

Box 1, Pre-Amplification, Store at -25°C to -15°C

Quantity

8

12

Description

i5 Index Primers, A501 to A508 i7 Index Primers, A701 to A712

Box 2, Pre-Amplification, Store at Room Temperature

Quantity

32

48

Description

i5 Index Tube Caps, White i7 Index Tube Caps, Orange

Additional Required Components, Pre-Amplification, Store at

Room Temperature

Consumable

TruSeq Custom Amplicon Filter Plate with Lid

TruSeq Index Plate Fixture and Collar Kit (reusable)

Catalog #

FC-130-1006

FC-130-1007

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Consumables and Equipment

Make sure that you have the required user-supplied consumables and equipment before starting the protocol.

The protocol has been optimized and validated using the items listed. Comparable performance is not guaranteed when using alternate consumables and equipment.

Consumables

Consumable

PhiX Control v3

10 N NaOH (prepare from tablets or use a standard solution)

96-well skirted PCR plates, 0.2 ml, polypropylene

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

Supplier

Illumina, FC-110-3001

General lab supplier

Bio-Rad, Part # MSP-9601

Adhesive aluminum foil seal

Agarose gel (2% or 4%)

Agencourt AMPure XP, 60 ml kit

Conical tubes, 15 ml

Deparaffinization Solution

DNA 1000 Kit for Bioanalyzer

Fisher Scientific, Part # AB-0859

Fisher Scientific, Part # AB-0765

Beckman Coulter, Part # 538619

General lab supplier

Beckman Coulter, Part # A63881/A63880

General lab supplier

QIAGEN, part # 19093

Agilent 5067-1504

(for 300 samples)

General lab supplier

General lab supplier

DNA molecular weight markers

Eppendorf microcentrifuge tubes (screw top recommended)

Ethanol, 200 proof for molecular biology

Ice bucket

KAPA SYBR FAST qPCR Master Mix (2X)

(Universal)

Microseal 'B' adhesive seals

PCR 8-tube strips

Solution basin, PVC, nonsterile (trough)

QIAamp DNA FFPE Tissue Kit

General lab supplier

General lab supplier

KAPA Biosystems

Bio-Rad, Part # MSB-1001

General lab supplier

Labcor, Part# 730-001

QIAGEN, Part # 56404

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Equipment

Pre-PCR Equipment

Equipment

37° incubator

Heat block, 96-well

Tabletop centrifuge

Supplier

Forced Air Oven, VWR International or comparable

SciGene, Hybex Microsample Incubator for PCR plate

Note: This model is recommended for this assay. Passive cooling, as opposed to active cooling performed in a PCR thermal cycler, is recommended for maximum target enrichment specificity and uniformity.

General lab supplier (Plate centrifuge that attains designated speeds of protocol)

NOTE

Use a dedicated set of pipettes, pipette tips, vortexer, and centrifuge during pre-amplification steps.

Post-PCR Equipment

Equipment

Post-PCR plate shaker

Tabletop centrifuge

Supplier

Q Instruments BioShake iQ high-speed thermoshaker

(part # 1808-0506) or Q Instruments BioShake XP high-speed lab shaker (part # 1808-0505)

General lab supplier (plate centrifuge that attains designated speeds of protocol)

General lab supplier Gel electrophoresis supplies and apparatus

Bioanalyzer System

Heat block for 1.5 ml centrifuge tubes

Magnetic stand-96

Agilent Technologies

General lab supplier

Invitrogen DynaMag™-96 Side Skirted

NOTE

Use a dedicated set of pipettes, pipette tips, vortexer, heat block, and centrifuge during postamplification steps.

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Thermal Cyclers

The following table lists the recommended settings for the Illumina recommended thermal cycler and other comparable models. If your lab has a thermal cycler that is not listed, validate the thermal cycler before performing the TruSight Tumor 26 protocol.

Thermal Cycler

Bio-Rad DNA Engine

Tetrad 2

MJ Research DNA

Engine Tetrad

(no longer available for purchase)

Eppendorf

Mastercycler Pro S

Temp Mode

Calculated

Calculated

Gradient S,

Simulated Tube

Lid Temp

Heated, Constant at 100°C

Heated

Vessel Type

Polypropylene plates and tubes

Plate

Heated Plate

NOTE

The gDNA qPCR evaluation was optimized on the Illumina Eco Real-Time PCR System and the Bio-Rad CFX396 System. If using other machines, verify the protocol before use.

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Index Sequences

Use the following sequences for entry on your sample sheet:

i7 Index PCR Primer

A709

A710

A711

A712

A501

A502

A503

A504

A701

A702

A703

A704

A705

A706

A707

A708

A505

A506

A507

A508

Index Sequence

ATCACGAC

ACAGTGGT

CAGATCCA

ACAAACGG

ACCCAGCA

AACCCCTC

CCCAACCT

CACCACAC

GAAACCCA

TGTGACCA

AGGGTCAA

AGGAGTGG

TGAACCTT

TGCTAAGT

TGTTCTCT

TAAGACAC

CTAATCGA

CTAGAACA

TAAGTTCC

TAGACCTA

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