TruSight Tumor 170. Reference Guide. For Research Use Only. Not for use in diagnostic procedures. Document # v01 April 2017

Transcription

1 TrSight Tmor 170 Reference Gide Docment # v01 April 2017 For Research Use Only. Not for se in diagnostic procedres. ILLUMINA PROPRIETARY

2 TrSight Tmor 170 Reference Gide This docment and its contents are proprietary to Illmina, Inc. and its affiliates ("Illmina"), and are intended solely for the contractal se of its cstomer in connection with the se of the prodct(s) described herein and for no other prpose. This docment and its contents shall not be sed or distribted for any other prpose and/or otherwise commnicated, disclosed, or reprodced in any way whatsoever withot the prior written consent of Illmina. Illmina does not convey any license nder its patent, trademark, copyright, or common-law rights nor similar rights of any third parties by this docment. The instrctions in this docment mst be strictly and explicitly followed by qalified and properly trained personnel in order to ensre the proper and safe se of the prodct(s) described herein. All of the contents of this docment mst be flly read and nderstood prior to sing sch prodct(s). FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN MAY RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TO USERS OR OTHERS, AND DAMAGE TO OTHER PROPERTY. ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S) DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE) Illmina, Inc. All rights reserved. Illmina, HiSeq, NextSeq, Trsight, and the streaming bases design are registered or pending trademarks of Illmina, Inc. and/or its affiliate(s) in the U.S. and/or other contries. All other names, logos, and other trademarks are the property of their respective owners. Docment # v01 For Research Use Only. Not for se in diagnostic procedres. ii

3 TrSight Tmor 170 Reference Gide Revision History Docment Date Description of Change Docment # v01 Docment # v00 April 2017 March 2017 In the RNA/DNA Inpt Recommendations section, corrected the kits that are sed for RNA sample assessment. In the Normalize Libraries introdction, clarified that manal normalization is not crrently spported by TrSight Tmor 170. Added the Advanced Analytical Technologies Standard Sensitivity RNA Analysis Kit and the Agilent RNA 6000 Nano Kit to the Consmables list. Initial release. Docment # v01 For Research Use Only. Not for se in diagnostic procedres. iii

4 Table of Contents Chapter 1 Overview 1 Introdction 1 RNA/DNA Inpt Recommendations 1 DNA Shearing Recommendations 2 Additional Resorces 2 Chapter 2 Protocol 3 Introdction 3 Warnings and Precations 4 Tips and Techniqes 4 Library Prep Workflow 6 Enrichment Workflow 7 Denatre and Anneal RNA 8 Synthesize First Strand cdna 9 Synthesize Second Strand cdna 10 Clean Up cdna 10 Fragment gdna 12 Perform End Repair and A-Tailing 14 Ligate Adapters 15 Clean Up Ligation 16 Index PCR 17 Perform First Hybridization 19 Perform First Captre 20 Perform Second Hybridization 22 Perform Second Captre 23 Amplify Enriched Library 25 Clean Up Amplified Enriched Library 26 Qantify Libraries 28 Normalize Libraries 28 Prepare for Seqencing 30 Appendix A Spporting Information 34 Introdction 34 Acronyms 34 Kit Contents 35 Consmables and Eqipment 37 Technical Assistance 40 Docment # v01 For Research Use Only. Not for se in diagnostic procedres. iv

5 Chapter 1 Overview Overview Introdction 1 RNA/DNA Inpt Recommendations 1 DNA Shearing Recommendations 2 Additional Resorces 2 Introdction The TrSight Tmor 170 protocol describes an enrichment-based approach to convert DNA and RNA extracted from formalin-fixed paraffin embedded (FFPE) tisse samples into libraries enriched for cancer-related genes that can be seqenced on Illmina seqencing systems. The TrSight Tmor 170 kit allows for preparation of 48 libraries (24 from DNA and 24 from RNA). The kit is optimized to provide high sensitivity and specificity for low-freqency somatic variants across 170 genes. These variants inclde single ncleotide variants (SNVs), insertions, deletions, mltincleotide variants (MNVs), amplifications, fsions, and splice variants. Prodct Description The TrSight Tmor 170 RUO kit consists of library preparation reagents to convert sample ncleic acids to seqenceable libraries. The TrSight Tmor 170 kit also contains the associated TrSight Tmor 170 software. The assay begins with DNA and/or RNA extracted from FFPE tisse as the inpt sample type. The variant calling algorithms are provided in the TrSight Tmor 170 App. The TrSight Tmor 170 App reports mtations (single ncleotide variants, mltincleotide variants, indels, copy nmber variants, splice variants, and gene fsions) in fll coding regions of the targeted genes. RNA/DNA Inpt Recommendations The TrSight Tmor 170 assay is optimized for a defined RNA/DNA inpt range. The optimal range for DNA is from 40 ng to 120 ng total or 3.3 ng/µl to 10 ng/µl. The optimal range for RNA is from 40 ng to 85 ng total or 4.7 ng/µl to 10 ng/µl. Qantify the inpt RNA/DNA before beginning the protocol. To obtain sfficient ncleic acid material, Illmina recommends isolating ncleic acid from a minimm of 2 mm 3 of FFPE tisse. Use a ncleic acid isolation method that prodces high recovery yields, minimizes sample consmption, and preserves sample integrity. The QIAGEN AllPrep DNA/RNA FFPE Kit provides a high yield of ncleic acids compared to other extraction methods tested for this assay. Use a florometric qantification method that ses RNA/DNA binding dyes sch as QantiFlor (RNA) or AccClear (DNA). Dilte starting material in RNase/DNase-free water. For optimal performance, assess DNA and RNA sample qality before sing the TrSight Tmor 170 assay. DNA samples can be assessed sing the Illmina FFPE QC Kit. Use DNA samples that reslt in a delta Cq vale 5. Using samples with a delta Cq > 5 may decrease assay performance. RNA samples can be assessed sing Advanced Analytical Technologies Fragment Analyzer (Standard Sensitivity RNA Analysis Kit) or Agilent Technologies 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit). Use RNA samples that reslt in a DV200 vale of 20%. Using samples with a DV200 vale < 20% may decrease assay performance. Docment # v01 For Research Use Only. Not for se in diagnostic procedres. 1

6 TrSight Tmor 170 Reference Gide Reference Samples [Optional] Use characterized reference materials when rnning the library preparation, sch as HorizonDx HD753 (DNA) and Agilent Universal Reference RNA. The Agilent Universal Reference RNA is an intact RNA sample that shold be processed following the intact RNA procedre in Denatre and Anneal RNA on page 8. Qalified FFPE materials from cell line derived xenografts can be sed as reference samples. Use RNase/DNase-free water as a no template control. Do not seqence the no template control. NOTE Rnning a reference sample or no template control redces the total nmber of nknown samples that can be processed. DNA Shearing Recommendations The TrSight Tmor 170 assay has been optimized to prepare libraries from gdna that has been fragmented to bp (with a peak at ~125 bp). The assay has been optimized sing either the Covaris E220evoltion TM or LE220 Focsed-ltrasonicator with the parameters provided in Fragment gdna on page 12. Fragment size distribtion may vary de to differences in sample qality and the sonication instrmentation sed with this test. If yo are not sing the fragmentation method optimized for TrSight Tmor 170 (Covaris E220evoltion or LE220), conslt the TrSight Tmor 170 spport page. Excessive bbbles or an air gap in the shearing tbe can lead to incomplete shearing. Load the gdna into the Covaris tbe slowly to avoid creating bbbles. Centrifge the Covaris tbe to collect the sample at the bottom of the tbe before shearing. If yo are sing the LE220 Covaris instrment, fill nsed Covaris 8 microtube Strip wells with 52l of water for optimal performance. [Optional] Fragment size distribtion of sheared samples may be assessed sing the Agilent DNA 1000 Kit with the Agilent Bioanalyzer Additional Resorces Visit the TrSight Tmor 170 kit spport page on the Illmina website for docmentation, software downloads, training resorces, and information abot compatible Illmina prodcts, inclding analysis software. The following docmentation is available for download from the Illmina website. Resorce Cstom Protocol Selector TrSight Tmor 170 Checklist (docment # v00) TrSight Tmor 170 Consmables & Eqipment List (docment # v00) Description spport.illmina.com/cstom-protocol-selector.html A wizard for generating cstomized end-to-end docmentation that is tailored to the library prep method, rn parameters, and analysis method sed for the seqencing rn. Provides a checklist of steps for the experienced ser. Provides an interactive checklist of ser-provided consmables and eqipment. Docment # v01 For Research Use Only. Not for se in diagnostic procedres. 2

7 Chapter 2 Protocol Protocol Introdction 3 Warnings and Precations 4 Tips and Techniqes 4 Library Prep Workflow 6 Enrichment Workflow 7 Denatre and Anneal RNA 8 Synthesize First Strand cdna 9 Synthesize Second Strand cdna 10 Clean Up cdna 10 Fragment gdna 12 Perform End Repair and A-Tailing 14 Ligate Adapters 15 Clean Up Ligation 16 Index PCR 17 Perform First Hybridization 19 Perform First Captre 20 Perform Second Hybridization 22 Perform Second Captre 23 Amplify Enriched Library 25 Clean Up Amplified Enriched Library 26 Qantify Libraries 28 Normalize Libraries 28 Prepare for Seqencing 30 Introdction This section describes the TrSight Tmor 170 protocol. Before proceeding, confirm kit contents and make sre that yo have the reqired consmables and eqipment. For more information, please see Kit Contents on page 35. Some reagents in this protocol have color-coded caps to identify the associated sample type. Ble caps identify reagents sed only with genomic DNA (gdna) samples. Red caps identify reagents sed only with RNA or complementary DNA (cdna) samples. Follow the protocol in the order described sing the specified parameters. Before beginning library preparation, record sample concentration (DNA or RNA) and sample qality information. Save this information for later se dring data analysis. RNA and DNA libraries may be prepared simltaneosly. Illmina recommends performing the TrSight Tmor 170 assay workflow according to the following schedle: Day 1: cdna Synthesis from RNA samples, DNA Shearing of gdna samples, Library Preparation, and begin Overnight (First) Hybridization. For more information, please see the Library Prep Workflow on page 6. Day 2: Enrichment, Enriched Library QC Check (Qantify Libraries), Bead-Based Normalization of Enriched Libraries, and Loading of Libraries onto Seqencing Platform (NextSeq 500, NextSeq 550, or HiSeq 2500). For more information, please see the Enrichment Workflow on page 7. If it is not possible to perform the TrSight Tmor 170 assay workflow according to the preceding schedle, several safe stopping points are specified throghot the protocol to accommodate alternative schedles. Docment # v01 For Research Use Only. Not for se in diagnostic procedres. 3

8 TrSight Tmor 170 Reference Gide Warnings and Precations WARNING Some of the reagents in this kit contain potentially hazardos chemicals. Personal injry can occr throgh inhalation, ingestion, skin contact, and eye contact. Wear protective eqipment, inclding eye protection, gloves, and laboratory coat appropriate for risk of exposre. Handle sed reagents as chemical waste and discard in accordance with applicable regional, national, and local laws and reglations. For additional environmental, health, and safety information, see the SDS at spport.illmina.com/sds.html. Tips and Techniqes Unless a safe stopping point is specified in the protocol, proceed immediately to the next step. Avoiding Cross-Contamination Use a nidirectional workflow when moving from pre-amp to post-amp areas. To prevent amplification prodct or probe carryover, avoid retrning to the pre-amp area after beginning work in the post-amp area. When adding or transferring samples, change tips between each well. When adding indexing primers, change tips between each well. Change gloves if gloves come into contact with indexing primers, samples, or probes. Clean work srfaces thoroghly before and after the procedre. Remove nsed indexing primer tbes from the working area. Sealing the Plate Always seal the plate with an appropriate plate seal before the following steps in the protocol: Shaking steps Vortexing steps Centrifge steps Thermal cycling steps Apply the adhesive seal to cover the plate and seal with a rbber roller. Microseal 'B' adhesive seals are effective at -40 C to 110 C, and sitable for skirted or semiskirted PCR plates. Use Microseal 'B' for shaking, centrifging, PCR amplification, and long-term storage. Plate Transfers When transferring volmes between plates, transfer the specified volme from each well of a plate to the corresponding well of the other plate. Centrifgation When instrcted to centrifge the plate, centrifge at 280 g for 1 minte. Handling Reagents Tightly recap all reagent tbes immediately after se to limit evaporation and prevent contamination. Docment # v01 For Research Use Only. Not for se in diagnostic procedres. 4

9 TrSight Tmor 170 Reference Gide Retrn reagents to the recommended storage conditions when they are no longer needed for the procedre. Handling Beads Pipette bead sspensions slowly. Mix thoroghly before pipetting. If beads are aspirated into the pipette tips dring magnetic separation steps, dispense back to the plate on the magnetic stand and wait ntil the liqid is clear (~2 mintes). When washing beads: Use the appropriate magnetic stand for the plate. Dispense liqid so that beads on the side of the wells are wetted. Keep the plate on the magnetic stand ntil the instrctions specify to remove it. Do not agitate the plate while on the magnetic stand. Do not distrb the bead pellet. Docment # v01 For Research Use Only. Not for se in diagnostic procedres. 5

10 TrSight Tmor 170 Reference Gide Library Prep Workflow The following diagram illstrates the recommended library preparation workflow sing a TrSight Tmor 170 kit. RNA and DNA libraries can be prepared simltaneosly. Safe stopping points are marked between steps. Figre 1 TrSight Tmor 170 Workflow (Part 1) Docment # v01 For Research Use Only. Not for se in diagnostic procedres. 6

11 TrSight Tmor 170 Reference Gide Enrichment Workflow The following diagram illstrates the recommended enrichment workflow sing a TrSight Tmor 170 kit. Safe stopping points are marked between steps. Figre 2 TrSight Tmor 170 Workflow (Part 2) Docment # v01 For Research Use Only. Not for se in diagnostic procedres. 7

12 TrSight Tmor 170 Reference Gide Denatre and Anneal RNA Dring this process, prified RNA is denatred and primed with random hexamers in preparation for cdna synthesis. If yo are working with only prified DNA, proceed directly to Fragment gdna on page 12. Consmables EPH3 (Elte, Prime, Fragment High Mix 3 [red cap]) FSM (First Strand Synthesis Mix [red cap]) RVT (Reverse Transcriptase [red cap]) 96-well PCR plate Microseal 'B' adhesive seals CAUTION The following procedres reqire an RNase- and DNase-free environment. Thoroghly decontaminate yor work area with an RNase-inhibiting cleaner. Make sre that yo se RNA-dedicated eqipment. Preparation 1 Prepare the following consmables. Item Storage Instrctions EPH3-25 C to -15 C Thaw and bring to room temperatre. Vortex to resspend. Centrifge briefly. FSM -25 C to -15 C Thaw and bring to room temperatre. Vortex to resspend. Centrifge briefly. RVT -25 C to -15 C Keep on ice. Centrifge briefly. 2 Thaw RNA samples on ice. 3 Qalify and qantify the samples. See RNA/DNA Inpt Recommendations on page 1. 4 Dilte each prified RNA sample to a concentration between 4.7 ng/μl and 10 ng/μl in nclease-free water. 5 Save the following programs on a thermal cycler: For FFPE or fragmented RNA, save the LQ-RNA program. Choose the preheated lid option and set to 100 C Set the reaction volme to 17 μl 65 C for 5 mintes Hold at 4 C For Cell Line or intact RNA, save the HQ-RNA program. Choose the preheated lid option and set to 100 C Set the reaction volme to 17 μl 94 C for 8 mintes Hold at 4 C 6 Label a new 96-well PCR plate CF (cdna Fragments). Docment # v01 For Research Use Only. Not for se in diagnostic procedres. 8

13 TrSight Tmor 170 Reference Gide Procedre 1 Combine the following reagents in a microcentrifge tbe to create FSM+RVT master mix. Master Mix Component Per 3 Samples Per 8 Samples Per 16 Samples Per 24 Samples FSM 27 µl 72 µl 144 µl 216 µl RVT 3 µl 8 µl 16 µl 24 µl Prepare a minimm of 3 samples. Discard any remaining master mix after se. 2 Pipette to mix. 3 Place the FSM+RVT master mix on ice ntil Synthesize First Strand cdna on page 9. 4 Add 8.5 µl of each prified RNA sample to the corresponding well of the CF plate. 5 Add 8.5 µl EPH3 to each well. 6 Apply Microseal B and shake the plate at 1200 rpm for 1 minte. 7 Place on the preprogrammed thermal cycler and rn the LQ-RNA or HQ-RNA program. 8 When the thermal cycler reaches 4 C, proceed immediately to the next step. Synthesize First Strand cdna This process reverse transcribes the RNA fragments primed with random hexamers into first strand cdna sing reverse transcriptase. Consmables FSM+RVT master mix (prepared in Denatre and Anneal RNA on page 8) Microseal 'B' adhesive seals Preparation 1 Save the following program as 1stSS on a thermal cycler with a heated lid: Choose the preheated lid option and set to 100 C Set the reaction volme to 25 µl 25 C for 10 mintes 42 C for 15 mintes 70 C for 15 mintes Hold at 4 C Procedre 1 Remove the CF plate from the thermal cycler. 2 Pipette to mix FSM+RVT master mix before se. 3 Add 8 µl FSM+RVT master mix to each well. 4 Apply Microseal B and shake the plate at 1200 rpm for 1 minte. 5 Place on a thermal cycler and rn the 1stSS program. 6 When the thermal cycler reaches 4 C, proceed immediately to the next step. Docment # v01 For Research Use Only. Not for se in diagnostic procedres. 9

14 TrSight Tmor 170 Reference Gide TIP Ifyo are alsopreparingdnalibraries, yo can begin tofragment gdnawhile the 1stSS program isrnning. Refer tofragment gdna on page 12tobegin. Synthesize Second Strand cdna This process removes the RNA template and synthesizes ds cdna. Consmables SSM (Second Strand Mix [red cap]) Microseal 'B' adhesive seals Preparation 1 Prepare the following consmables. Item Storage Instrctions SSM -25 C to -15 C Thaw and bring to room temperatre. Invert 10 times to mix. Centrifge briefly. 2 Save the following program as 2ndSS on a thermal cycler with a heated lid. If the lid temperatre cannot be set to 30 C, trn off the preheated lid heat option: Choose the preheated lid option and set to 30 C Set the reaction volme to 50 µl 16 C for 25 mintes Hold at 4 C Procedre 1 Remove the CF plate from the thermal cycler. 2 Add 25 µl SSM to each well. 3 Apply Microseal B and shake the plate at 1200 rpm for 1 minte. 4 Place on a thermal cycler and rn the 2ndSS program. 5 When the thermal cycler reaches 4 C, proceed to the next step. Clean Up cdna This process ses SPB to prify the cdna from nwanted reaction components. Consmables Freshly prepared 80% ethanol (EtOH) SPB (Sample Prification Beads) RSB (Resspension Bffer) Microseal 'B' adhesive seals 96-well midi plates (1 2) [Optional] 96-well PCR plate Docment # v01 For Research Use Only. Not for se in diagnostic procedres. 10

15 TrSight Tmor 170 Reference Gide CAUTION Proper plate type is reqired for optimal assay performance. To contine the protocol after this step, se a midi plate. Tostore the samplesafter thisstep, se a PCR plate. See Preparation on page 11 for more information. Abot Reagents Vortex SPB before each se. Vortex SPB freqently to make sre that beads are evenly distribted. Aspirate and dispense SPB slowly de to the viscosity of the soltion. Preparation 1 Prepare the following consmables. Item Storage Instrctions SPB 2 C to 8 C Bring to room temperatre. Before sing SPB, vortex for 1 minte. RSB 2 C to 8 C or -25 C to -15 C Bring to room temperatre. If RSB was stored at -25 C to -15 C, thaw at room temperatre and vortex before se. 2 Label a new 96-well midi plate BIND1. 3 Label a new 96-well midi plate PCF (Prified cdna Fragments). [Optional] To store the plate after this step, se a new 96-well PCR plate. 4 Prepare fresh 80% EtOH. Procedre Bind 1 Remove the CF plate from the thermal cycler. 2 Add 90 µl SPB to each well of the BIND1 plate. 3 Transfer 50 µl of each sample from the CF plate to the corresponding well of the BIND1 plate. 4 Apply Microseal 'B' and shake at 1800 rpm for 2 mintes. 5 Incbate at room temperatre for 5 mintes. Wash 1 Place the BIND1 plate on a magnetic stand for 5 mintes. 2 Remove and discard all spernatant from each well. 3 Wash as follows: a b While on the magnetic stand, add 200µlfresh 80% EtOH. Wait 30 seconds, then remove and discard all spernatant from each well. 4 Repeat step 3 (a b) to wash a second time. Docment # v01 For Research Use Only. Not for se in diagnostic procedres. 11

16 TrSight Tmor 170 Reference Gide NOTE Two washes are reqired for optimal assay performance. 5 Use a P20 pipette with fine tips to remove residal spernatant from each well. Elte 1 Remove the BIND1 plate from the magnetic stand. 2 Add 22 µl RSB to each well. 3 Apply Microseal B and shake at 1500 rpm for 2 mintes. 4 Incbate at room temperatre for 2 mintes. 5 Place on a magnetic stand for 2 mintes. 6 Transfer 20 µl of elate from each well of the BIND1 plate to the corresponding well of the PCF plate. 7 Add 30 µl RSB to each well of the PCF plate, and then pipette to mix (a minimm of 10 times). 8 Proceed to Perform End Repair and A-Tailing on page 14 or apply Microseal 'B' and store. TIP If yo are also preparing DNA libraries, prified cdna fragments and sheared DNA samples can be stored in the same plate. Make sre tolabelwells. See the preparation section in Fragment gdna on page 12for more information. SAFE STOPPING POINT If yo are stopping, apply Microseal 'B' to the PCR plate and briefly centrifge at 280 g. Store at 2 C to 8 C overnight, or at -25 C to -15 C for p to 7 days. Fragment gdna This process optimally fragments gdna to a bp fragment size sing the Covaris Focsed-ltrasonicator. Covaris shearing generates dsdna fragments with 3' or 5' overhangs. If yo are working with only prified RNA, skip this step and proceed directly to Perform End Repair and A-Tailing on page 14. Consmables TEB (TE Bffer) Covaris 8 microtube Strip 96-well midi plate [Optional] 96-well PCR plate CAUTION Proper plate type is reqired for optimal assay performance. To contine the protocol after this step, se a midi plate. Tostore the samplesafter thisstep, se a PCR plate. See Preparation on page 13 for more information. TIP If yo are also preparing cdna libraries from RNA samples, prified cdna fragments and sheared DNA samples can be stored in the PCF plate. Make sre tolabelwells. See Preparation on page 13 for more information. Docment # v01 For Research Use Only. Not for se in diagnostic procedres. 12

17 TrSight Tmor 170 Reference Gide Preparation 1 Prepare the following consmables. Item Storage Instrctions TEB 2 C to 8 C Bring to room temperatre. Invert to mix. 2 Trn on and set p the Covaris instrment according to manfactrer gidelines. This instrment reqires ~1 hor to de-gas. 3 Choose 1 of the following plate options: To process only gdna, se a new 96-well midi plate. To process cdna samples simltaneosly, contine to se the PCF plate from Clean Up cdna on page 10. [Optional] To store sheared gdna after this step, se a 96-well PCR plate. 4 Label (or relabel) the plate LP (Library Preparation). 5 Thaw gdna samples at room temperatre. Invert to mix. 6 See RNA/DNA Inpt Recommendations on page 1 to qalify or qantify samples. 7 Dilte each prified DNA sample to a concentration between 3.3 ng/µl and 10 ng/µl in TEB. Procedre 1 Add 12 µl of each dilted, prified gdna sample into a Covaris 8 microtube Strip. 2 Add 40 µl TEB to each sample. TIP Ifyo are singthe LE220Covarisinstrment, fillthe nsed Covaris8microTUBE Strip wellswith 52µlofwater for optimal performance. 3 Pipette to mix. 4 Seal the microtube Strip with the foil seal. 5 Centrifge briefly. 6 If yo are sing the Covaris E220evoltion or LE220 model, fragment the gdna sing the following settings. If yo are sing a different Covaris model, conslt the TrSight Tmor 170 spport page. Setting E220evoltion LE220 Peak Incident Power 175 watts 450 watts Dty Factor 10% 30% Cycles per Brst Treatment Time 280 seconds 250 seconds Temperatre 7 C 7 C Intensifier yes N/A 7 Transfer 50 µl of each sheared gdna sample into the corresponding wells of the LP plate (or PCF plate if yo are processing cdna simltaneosly). 8 [Optional] If the PCF plate is a midi plate and yo plan to store it after this step, transfer 50 μl of cdna and 50 μl of sheared gdna sample to the corresponding wells of a new 96-well PCR plate. Label the plate LP. Docment # v01 For Research Use Only. Not for se in diagnostic procedres. 13

18 TrSight Tmor 170 Reference Gide TIP AP20pipette with fine tipscan be sed when transferringsheared gdnasample tothe LPplate (pipette 20µl+ 20µl+ 10µl). SAFE STOPPING POINT If yo are stopping, apply Microseal 'B' to the PCR plate and briefly centrifge at 280 g. Store at 2 C to 8 C overnight, or at -25 C to -15 C for p to 7 days. Perform End Repair and A-Tailing This process converts the overhangs reslting from fragmentation into blnt ends sing an End Repair A-Tailing master mix (ERA1). The 3' to 5' exonclease activity of this mix removes the 3' overhangs and the 5' to 3' polymerase activity fills in the 5' overhangs. The 3' ends are A-tailed dring this reaction to prevent them from ligating to each other dring the adapter ligation reaction. Consmables ERA1-A (End Repair A-tailing Enzyme Mix 1) ERA1-B (End Repair A-tailing Bffer 1) Microseal 'B' adhesive seals 1.7 ml microcentrifge tbe [Optional] 96-well midi plate CAUTION Ifa PCR plate wassed tostore the gdnaand/or cdnasamples, follow the plate transfer instrctionsin step 4 ofthe Preparation on page 14. Preparation 1 Prepare the following consmables. Item Storage Instrctions ERA1-A -25 C to -15 C Keep on ice. Centrifge briefly, and then pipette to mix. ERA1-B -25 C to -15 C Thaw and bring to room temperatre. Centrifge briefly, and then pipette to mix. If crystals are present, warm the tbe in yor hands, and then pipette to mix ntil the crystals are dissolved. 2 Bring cdna and sheared gdna to room temperatre. 3 If cdna and gdna samples are stored in separate midi plates, move all samples to the same midi plate. 4 [Optional] If cdna and/or sheared gdna samples are stored in a 96-well PCR plate(s), transfer 50 µl of cdna and/or sheared gdna sample to the corresponding wells of a new, single 96-well midi plate. 5 Label (or relabel) the midi plate LP2 (Library Preparation 2). 6 Preheat 2 Hybex incbators with midi heat block inserts as follows: Preheat a Hybex incbator to 30 C. Preheat a Hybex incbator to 72 C. Docment # v01 For Research Use Only. Not for se in diagnostic procedres. 14

19 TrSight Tmor 170 Reference Gide Procedre 1 Combine the following reagents in a microcentrifge tbe to create ERA1 master mix. Master Mix Component Per 3 Samples Per 8 Samples Per 16 Samples Per 24 Samples ERA1-B 26 µl 69 µl 138 µl 207 µl ERA1-A 10 µl 27 µl 54 µl 81 µl Prepare a minimm of 3 samples. Discard any remaining master mix after se. 2 Pipette to mix (a minimm of 10 times) and place ERA1 master mix on ice. 3 Add 10 µl ERA1 master mix to each sample in the LP2 plate. 4 Apply Microseal B and shake the plate at 1800 rpm for 2 mintes. 5 Incbate in a Hybex incbator at 30 C for 30 mintes. 6 Immediately transfer to another Hybex incbator at 72 C and incbate for 20 mintes. 7 Place the plate on ice for 5 mintes. Ligate Adapters This process ligates adapters to the ends of the cdna and/or gdna fragments. Consmables ALB1 (Adapter Ligation Bffer 1) SUA1 (Short Universal Adapters 1) STL (Stop Ligation Bffer) LIG3 (DNA Ligase 3) Microseal 'B' adhesive seals Abot Reagents ALB1 is highly viscos. Pipette slowly to avoid forming bbbles. Preparation 1 Prepare the following consmables. Item Storage Instrctions ALB1-25 C to -15 C Thaw and bring to room temperatre. Vortex to resspend. Centrifge briefly. SUA1-25 C to -15 C Thaw and bring to room temperatre. Vortex to resspend. Centrifge briefly. STL -25 C to -15 C Thaw and bring to room temperatre. Vortex to resspend. Centrifge briefly. LIG3-25 C to -15 C Keep on ice. Centrifge briefly, and then pipette to mix. Docment # v01 For Research Use Only. Not for se in diagnostic procedres. 15

20 TrSight Tmor 170 Reference Gide Procedre 1 Add 60 µl ALB1 to each well. 2 Add 5 µl LIG3 to each well. 3 Vortex SUA1 a minimm of 10 seconds. 4 Add 10 µl SUA1 to each well. 5 Apply Microseal B and shake the plate at 1800 rpm for 2 mintes. 6 Incbate at room temperatre for 30 mintes. 7 Add 5 µl STL. 8 Apply Microseal B and shake the plate at 1800 rpm for 2 mintes. Clean Up Ligation This process ses SPB to prify the cdna or gdna fragments and remove nwanted prodcts. Consmables Freshly prepared 80% ethanol (EtOH) SPB (Sample Prification Beads) RSB (Resspension Bffer) 96-well PCR plate Microseal 'B' adhesive seals Abot Reagents Vortex SPB before each se. Vortex SPB freqently to make sre that beads are evenly distribted. Aspirate and dispense SPB slowly de to the viscosity of the sspension. Preparation 1 Prepare the following consmables. Item Storage Instrctions SPB 2 C to 8 C Bring to room temperatre. Before sing SPB, vortex for 1 minte. RSB 2 C to 8 C or -25 C to -15 C Bring to room temperatre. If RSB was stored at -25 C to -15 C, thaw at room temperatre and vortex before se. 2 Label a new 96-well PCR plate LS (Library Samples). 3 Prepare fresh 80% EtOH. Procedre Bind 1 Add 112 µl SPB to each well in the LP2 plate. Docment # v01 For Research Use Only. Not for se in diagnostic procedres. 16

21 TrSight Tmor 170 Reference Gide 2 Apply Microseal B and shake at 1800 rpm for 2 mintes. 3 Incbate at room temperatre for 5 mintes. Wash 1 Place the LP2 plate on the magnetic stand for 10 mintes. 2 Remove and discard all spernatant from each well. 3 Wash as follows: a b While on the magnetic stand, add 200µlfresh 80% EtOH. Wait 30 seconds, then remove and discard all spernatant from each well. 4 Repeat step 3 (a b) to wash a second time. NOTE Two washes are reqired for optimal assay performance. 5 Use a P20 pipette with fine tips to remove residal spernatant from each well. Elte 1 Remove from the magnetic stand. 2 Add 27.5 µl RSB to each well. 3 Apply Microseal B and shake at 1500 rpm for 2 mintes. 4 Incbate at room temperatre for 2 mintes. 5 Place on a magnetic stand for 2 mintes. 6 Transfer 25 µl of each elate from the LP2 plate to the corresponding well of the LS plate. Index PCR In this step, the cdna and/or gdna fragments are amplified sing primers that add index seqences for sample mltiplexing. The reslting prodct contains DNA fragments flanked by seqences and adapters reqired for clster generation. Consmables EPM (Enhanced PCR Mix) UPXX (Uniqe Index Primer Mixes), see Kit Contents on page 35 CPXX (Combinatorial Index Primer Mixes), see Kit Contents on page 35 Microseal 'B' adhesive seals Preparation 1 Prepare the following consmables. Docment # v01 For Research Use Only. Not for se in diagnostic procedres. 17

22 TrSight Tmor 170 Reference Gide Item Storage Instrctions EPM -25 C to -15 C Thaw and bring to room temperatre. Vortex to resspend. Centrifge briefly. UPXX -25 C to -15 C Thaw and bring to room temperatre. Vortex to resspend. Centrifge briefly. CPXX -25 C to -15 C Thaw and bring to room temperatre. Vortex to resspend. Centrifge briefly. 2 Assign 1 UPXX index primer mix per RNA library and 1 CPXX index primer mix per DNA library (XX = index primer mix nmber). UPXX index primer mixes can also be sed with DNA libraries. Do not se CPXX index primer mixes with RNA libraries. Low-plex seqencing rns shold se 3 libraries containing 1 of the following UPXX index primer sets to provide sfficient diversity. Choose 1 of the following index primer sets for low-plex rns: [UP01,UP02,UP03] [UP04,UP05,UP06] [UP07,UP08,UP09] [UP10,UP11,UP12] For more information, see Kit Contents on page 35. CAUTION If yo are seqencing mltiple libraries on a single flow cell, assign a different indexing primer mix to each library sample. CAUTION When handling indexing primers, avoid cross-contamination. After an indexing primer mix tbe is opened, discard the originaltbe cap and apply a new tbe cap. CAUTION Make sre to assign UPXX index primer mixes to RNA libraries only. Assigning CPXX index primer mixes to RNA libraries may reslt in decreased performance. 3 In the post-amp area, save the following program as I-PCR on a thermal cycler with a heated lid: Choose the preheated lid option and set to 100 C Set the reaction volme to 50 µl 98 C for 30 seconds 15 cycles of: 98 C for 10 seconds 60 C for 30 seconds 72 C for 30 seconds 72 C for 5 mintes Hold at 10 C Procedre 1 Add 5 µl of indexing primer mix (UPXX or CPXX) to each well in the LS plate. (See Preparation on page 17 for more information abot indexing primer mixes.) 2 Add 20 µl EPM to each well. Docment # v01 For Research Use Only. Not for se in diagnostic procedres. 18

23 TrSight Tmor 170 Reference Gide 3 Apply Microseal B and shake the plate at 1500 rpm for 1 minte. CAUTION Perform the following steps in a post-amp area to prevent amplification prodct carryover. 4 Briefly centrifge at 280 g. 5 Place on the preprogrammed thermal cycler and rn the I-PCR program. 6 After the I-PCR program completes, relabel the plate ALS (Amplified Library Samples). 7 Centrifge briefly. SAFE STOPPING POINT If yo are stopping, apply Microseal 'B' to the PCR plate and briefly centrifge at 280 g. Store at 2 C to 8 C overnight, or at -25 C to -15 C for p to 30 days. Perform First Hybridization Dring this process, a pool of oligos specific to the 170 genes targeted by TrSight Tmor 170 is hybridized to the RNA and/or DNA libraries generated dring Index PCR on page 17. Two hybridization steps are reqired to ensre enrichment of the targeted regions. In this step, the first hybridization is performed overnight (8 hors to 24 hors). Consmables TCA1 (Target Captre Additives 1) TCB1 (Target Captre Bffer 1) OPR1 (Oncology Probes RNA 1 [red cap]) OPD1 (Oncology Probes DNA 1 [ble cap]) 96-well PCR plate Microseal 'B' adhesive seals Abot Reagents Use OPR1 for RNA libraries only (red cap). Use OPD1 for DNA libraries only (ble cap). Preparation 1 Prepare the following consmables. Item Storage Instrctions TCB1 2 C to 8 C Bring to room temperatre. Vortex for 1 minte to resspend. Centrifge briefly. If crystals are present, warm the tbe in yor hands, and then vortex ntil the crystals are dissolved. TCA1-25 C to -15 C Thaw and bring to room temperatre. Vortex to resspend. Centrifge briefly. OPR1-25 C to -15 C Thaw and bring to room temperatre. Vortex to resspend. Centrifge briefly. OPD1-25 C to -15 C Thaw and bring to room temperatre. Vortex to resspend. Centrifge briefly. Docment # v01 For Research Use Only. Not for se in diagnostic procedres. 19

24 TrSight Tmor 170 Reference Gide 2 If the ALS plate was stored at -25 C to -15 C, thaw at room temperatre and centrifge. 3 Label a new 96-well PCR plate HYB1 (Hybridization 1). 4 Save the following program as HYB1 on a thermal cycler with a heated lid: Choose the preheated lid option and set to 100 C Set the reaction volme to 50 µl 95 C for 10 mintes 85 C for 2.5 mintes 75 C for 2.5 mintes 65 C for 2.5 mintes Hold at 57 C Procedre 1 Add 20 µl of each RNA and/or DNA library to the HYB1 plate. 2 Add 15 µl TCB1 to each well. 3 Add 10 µl TCA1 to each well. 4 Add the appropriate probe: For RNAlibraries, add 5 µl of OPR1 (red cap). For DNA libraries, add 5 µl of OPD1 (ble cap). 5 Apply Microseal B and shake the plate at 1800 rpm for 2 mintes. 6 Place on the preprogrammed thermal cycler and rn the HYB1 program. Hybridize at 57 C overnight (minimm of 8 hors to a maximm of 24 hors). Perform First Captre This step ses SMB (Streptavidin Magnetic Beads) to captre probes hybridized to the targeted regions of interest. Three heated washes sing EEW2 remove nonspecific binding from the beads. The enriched library is then elted from the beads and prepared for a second rond of hybridization. Consmables SMB (Streptavidin Magnetic Beads) ET2 (Elte Target Bffer 2) EE2 (Enrichment Eltion 2) HP3 (2 N NaOH) EEW2 (Enhanced Enrichment Wash 2) 96-well midi plate 96-well PCR plate Microseal 'B' adhesive seals Abot Reagents Make sre to se SMB and not SPB for this procedre. Vortex SMB freqently to make sre that beads are sspended. Docment # v01 For Research Use Only. Not for se in diagnostic procedres. 20

25 TrSight Tmor 170 Reference Gide Preparation 1 Prepare the following consmables. Item Storage Instrctions EE2-25 C to -15 C Thaw and bring to room temperatre. Vortex to resspend. Centrifge briefly. EEW2-25 C to -15 C Thaw and bring to room temperatre. Vortex for 1 minte to resspend. SMB 2 C to 8 C Bring to room temperatre. Vortex for 1 minte. If the bead pellet is present, pipette p and down to release the pellet, and then vortex to resspend. ET2 2 C to 8 C Bring to room temperatre. Vortex to resspend. Centrifge briefly. HP3 2 C to 8 C Bring to room temperatre. Vortex to resspend. Centrifge briefly. 2 Preheat a Hybex incbator with midi heat block insert to 57 C. 3 Label a new 96-well midi plate CAP1. 4 Label a new 96-well PCR plate ELU1 (Eltion 1). Procedre Bind 1 Remove the HYB1 plate from the thermal cycler. 2 Add 150 µl SMB to each well of the CAP1 plate. 3 Transfer 50 µl of each library from the HYB1 plate to the corresponding well in the CAP1 plate. 4 Apply Microseal B and shake the plate at 1800 rpm for 2 mintes. 5 Incbate in a Hybex incbator at 57 C for 25 mintes. 6 Place on a magnetic stand for 2 mintes. 7 While on the magnetic stand, se a pipette to remove and discard the spernatant. Wash 1 Wash as follows: a b c d e f g Remove the CAP1 plate from the magnetic stand. Add 200µlEEW2toeach well. Pipette tomix5times. Use clean tipsfor each library. Apply Microseal B and shake the plate at 1800rpm for 4mintes. Ifthe bead pellet isstillpresent, remove the Microsealand pipette tomix, makingsre that allbeadsare resspended. Apply a new Microseal'B'. Incbate in a Hybexincbator at 57 Cfor 5mintes. Place on a magnetic stand for 2mintes. While on the magnetic stand, se a pipette to remove and discard the spernatant from each well. 2 Repeat step 1 (a g) to wash a second time. 3 Repeat step 1 (a g) to wash a third time. Docment # v01 For Research Use Only. Not for se in diagnostic procedres. 21

26 TrSight Tmor 170 Reference Gide NOTE Three washes are reqired for optimal assay performance. 4 Use a P20 pipette with fine tips to remove any residal spernatant from each well. Elte 1 Combine the following reagents in a microcentrifge tbe to create EE2+HP3 Eltion Mix. Eltion Mix Component Per 3 Samples Per 8 Samples Per 16 Samples Per 24 Samples EE2 95 µl 228 µl 456 µl 684 µl HP3 5 µl 12 µl 24 µl 36 µl Prepare a minimm of 3 samples. Discard any remaining eltion mix after se. 2 Vortex briefly to mix. 3 Remove the CAP1 plate from the magnetic stand. 4 Add 17 µl EE2+HP3 Eltion Mix to each sample pellet. 5 Apply Microseal B and shake the plate at 1800 rpm for 2 mintes. 6 Place on a magnetic stand for 2 mintes. 7 Careflly transfer 15 µl of elate from each well of the CAP1 plate into the ELU1 plate. CAUTION Transferring less than the specified volme of elate may affect assay performance. 8 Add 5 µl ET2 to each elate in the ELU1 plate. 9 Apply Microseal B and shake the plate at 1800 rpm for 2 mintes. Perform Second Hybridization This step binds targeted regions of the enriched RNA and/or DNA libraries with captre probes a second time. The second hybridization ensres high specificity of the captred regions. To ensre optimal enrichment of libraries, the second hybridization step shold be performed for or a minimm of 1.5 hors to a maximm of 4 hors. Consmables TCA1 (Target Captre Additives 1) TCB1 (Target Captre Bffer 1) OPR1 (Oncology Probes RNA 1 [red cap]) OPD1 (Oncology Probes DNA 1 [ble cap]) Microseal 'B' adhesive seals Abot Reagents Use OPR1 for RNA libraries only (red cap). Use OPD1 for DNA libraries only (ble cap). Docment # v01 For Research Use Only. Not for se in diagnostic procedres. 22

27 TrSight Tmor 170 Reference Gide Preparation 1 Prepare the following consmables. Item Storage Instrctions TCB1 2 C to 8 C Bring to room temperatre. Vortex for 1 minte to resspend. Centrifge briefly. If crystals are present, warm the tbe in yor hands, and then vortex ntil the crystals are dissolved. TCA1-25 C to -15 C Thaw and bring to room temperatre. Vortex to resspend. Centrifge briefly. OPR1-25 C to -15 C Thaw and bring to room temperatre. Vortex to resspend. Centrifge briefly. OPD1-25 C to -15 C Thaw and bring to room temperatre. Vortex to resspend. Centrifge briefly. 2 Save the following program as HYB2 on a thermal cycler with a heated lid: Choose the preheated lid option and set to 100 C Set the reaction volme to 50 µl 95 C for 10 mintes 85 C for 2.5 mintes 75 C for 2.5 mintes 65 C for 2.5 mintes Hold at 57 C Procedre 1 Add 15 µl TCB1 to each well in the ELU1 plate. 2 Add 10 µl TCA1 to each well. 3 Add the appropriate probe: For RNA libraries, add 5 µl OPR1 (red cap). For DNA libraries, add 5 µl OPD1 (ble cap). 4 Apply Microseal B and shake the plate at 1800 rpm for 2 mintes. 5 Place on the preprogrammed thermal cycler and rn the HYB2 program. Hybridize at 57 C for a minimm of 1.5 hors to a maximm of 4 hors. Perform Second Captre This step ses SMB (Streptavidin Magnetic Beads) to captre probes hybridized to the targeted regions of interest. RSB is sed to rinse the captred libraries and remove nonspecific binding from the beads. The enriched library is then elted from the beads and prepared for seqencing. Consmables SMB (Streptavidin Magnetic Beads) ET2 (Elte Target Bffer 2) EE2 (Enrichment Eltion 2) HP3 (2 N NaOH) Docment # v01 For Research Use Only. Not for se in diagnostic procedres. 23

28 TrSight Tmor 170 Reference Gide RSB (Resspension Bffer) 96-well midi plate 96-well PCR plate Microseal 'B' adhesive seals Abot Reagents Make sre to se SMB and not SPB for this procedre. Vortex SMB freqently to make sre that beads are sspended. Preparation 1 Prepare the following consmables. Item Storage Instrctions EE2-25C to -15C Thaw and bring to room temperatre. Vortex to resspend. Centrifge briefly. SMB 2 C to 8 C Bring to room temperatre. Vortex for 1 minte. If the bead pellet is present, pipette p and down to release the pellet, and then vortex to resspend. ET2 2 C to 8 C Bring to room temperatre. Vortex to resspend. Centrifge briefly. HP3 2 C to 8 C Bring to room temperatre. Vortex to resspend. Centrifge briefly. RSB 2 C to 8 C or -25 C to -15 C Bring to room temperatre. If RSB was stored at -25 C to -15 C, thaw at room temperatre and vortex before se. 2 Preheat a Hybex incbator with midi heat block insert to 57 C. 3 Label a new 96-well midi plate CAP2. 4 Label a new 96-well PCR plate ELU2 (Eltion 2). Procedre Bind 1 Remove the ELU1 plate from the thermal cycler. 2 Add 150 µl SMB to each well of the CAP2 plate. 3 Transfer 50 µl of each library from the ELU1 plate to the corresponding well of the CAP2 plate. 4 Apply Microseal B and shake the plate at 1800 rpm for 2 mintes. 5 Incbate in a Hybex incbator at 57 C for 25 mintes. 6 Place on a magnetic stand for 2 mintes. 7 While on the magnetic stand, se a pipette to careflly remove and discard the spernatant from each well. Wash 1 Remove the CAP2 plate from the magnetic stand. 2 Add 200 µl of RSB to each well. 3 Apply Microseal B and shakethe plate at 1800 rpm for 4 mintes. Docment # v01 For Research Use Only. Not for se in diagnostic procedres. 24

29 TrSight Tmor 170 Reference Gide If If the bead pellet is still present, remove the Microseal and pipette to mix, making sre that all beads are resspended. Apply a new Microseal 'B'. 4 Place on a magnetic stand for 2 mintes. 5 While on the magnetic stand, se a pipette to careflly remove and discard the spernatant. 6 Use a P20 pipette with fine tips to remove any residal spernatant from each well. Elte 1 Combine the following reagents in a microcentrifge tbe to create a fresh EE2+HP3 Eltion Mix. Eltion Mix Component Per 3 Samples Per 8 Samples Per 16 Samples Per 24 Samples EE2 95 µl 228 µl 456 µl 684 µl HP3 5 µl 12 µl 24 µl 36 µl Prepare a minimm of 3 samples. Discard any remaining eltion mix after se. 2 Vortex to mix. 3 Remove the CAP2 plate from the magnetic stand. 4 Add 22 µl EE2+HP3 Eltion Mix to each sample pellet. 5 Apply Microseal B and shake the plate at 1800 rpm for 2 mintes. 6 Place on a magnetic stand for 2 mintes. 7 Transfer 20 µl of elate from each well of the CAP2 plate into the ELU2 plate. CAUTION Transferring less than the specified volme of elate may affect assay performance. 8 Add 5 µl ET2 to each elate in the ELU2 plate. 9 Apply Microseal B and shake the plate at 1800 rpm for 2 mintes. SAFE STOPPING POINT If yo are stopping, apply Microseal 'B' to the PCR plate and briefly centrifge at 280 g. Store at 2 C to 8 C overnight, or at -25 C to -15 C for p to 7 days. Amplify Enriched Library This step ses primers to amplify enriched libraries. Consmables PPC3 (PCR Primer Cocktail 3) EPM (Enhanced PCR Mix) Microseal 'B' adhesive seals Preparation 1 Prepare the following consmables. Docment # v01 For Research Use Only. Not for se in diagnostic procedres. 25

30 TrSight Tmor 170 Reference Gide Item Storage Instrctions EPM -25 C to -15 C Thaw on ice. Vortex to resspend. Centrifge briefly. PPC3-25 C to -15 C Thaw and bring to room temperatre. Vortex to resspend. Centrifge briefly. 2 If the ELU2 plate was stored at -25 C to -15 C, thaw at room temperatre and centrifge. 3 Save the following program as EL-PCR on a thermal cycler with a heated lid: Choose the preheated lid option and set to 100 C Set the reaction volme to 50 µl 98 C for 30 seconds 18 cycles of: 98 C for 10 seconds 60 C for 30 seconds 72 C for 30 seconds 72 C for 5 mintes Hold at 10 C Procedre 1 Add 5 µl PPC3 to each well of the ELU2 plate. 2 Add 20 µl EPM to each well. 3 Apply Microseal B and shake the plate at 1500 rpm for 2 mintes. 4 Briefly centrifge at 280 g. 5 Place on a thermal cycler and rn the EL-PCR program. Clean Up Amplified Enriched Library This step ses SPB (Sample Prification Beads) to prify the enriched library from nwanted reaction components. Consmables Freshly prepared 80% ethanol (EtOH) SPB (Sample Prification Beads) RSB (Resspension Bffer) 96-well PCR plate 96-well midi plate Microseal 'B' adhesive seals Abot Reagents Vortex SPB before each se. Vortex SPB freqently to make sre that beads are evenly distribted. Aspirate and dispense SPB slowly de to the viscosity of the soltion. Preparation 1 Prepare the following consmables. Docment # v01 For Research Use Only. Not for se in diagnostic procedres. 26

31 TrSight Tmor 170 Reference Gide Item Storage Instrctions SPB 2 C to 8 C Bring to room temperatre. Before sing SPB, vortex for 1 minte. RSB 2 C to 8 C or -25 C to -15 C Bring to room temperatre. If RSB was stored at -25 C to -15 C, thaw at room temperatre and vortex before se. 2 Label a new 96-well midi plate BIND2. 3 Label a new 96-well PCR plate PL (Prified Libraries). 4 Prepare fresh 80% EtOH. Procedre Bind 1 Remove the ELU2 plate from the thermal cycler. 2 Add 110 µl SPB to each well of the BIND2 plate. 3 Transfer 50 µl of each library from the ELU2 plate to the corresponding well of the BIND2 plate. 4 Apply Microseal B and shake at 1800 rpm for 2 mintes. 5 Incbate at room temperatre for 5 mintes. Wash 1 Place the BIND2 plate on magnetic stand for 5 mintes. 2 Remove and discard all spernatant from each well. 3 Wash as follows: a b While on the magnetic stand, add 200µlfresh 80% EtOH. Wait 30 seconds, then remove and discard all spernatant from each well. 4 Repeat step 3 (a b) to wash a second time. NOTE Two washes are reqired for optimal assay performance. 5 Use a P20 pipette with fine tips to remove residal spernatant from each well. Elte 1 Remove the BIND2 plate from the magnetic stand. 2 Add 32 µl RSB to each well. 3 Apply Microseal B and shake at 1800 rpm for 2 mintes. 4 Incbate at room temperatre for 2 mintes. 5 Place on a magnetic stand for 2 mintes. 6 Transfer 30 µl of each elate from the BIND2 plate to the corresponding well of the PL plate. Docment # v01 For Research Use Only. Not for se in diagnostic procedres. 27

32 TrSight Tmor 170 Reference Gide SAFE STOPPING POINT If yo are stopping, apply Microseal 'B' to the PCR plate and briefly centrifge at 280 g. Store at 2 C to 8 C overnight, or at -25 C to -15 C for p to 30 days. Qantify Libraries Accrately qantify to make sre that there is sfficient library available for clstering on the flow cell. Use a florometric qantification method (ser-spplied) to assess the qantity of enriched libraries before library normalization. Efficient bead-based library normalization reqires 3 ng/µl of each library. The AccClear Ultra High Sensitivity dsdna Qantitation Kit has been demonstrated to be effective for qantifying libraries in this protocol. Recommended Gidelines 1 The DNA standard provided with the florometric qantification kit, the libraries, and the blank soltion shold all be rn in triplicate. 2 Determine the average relative florescence nit (RFU) for each. 3 Calclate the following vales: Average Standard RFU - Average Blank RFU = Normalized Standard RFU Average Library RFU - Average Blank RFU = Normalized RFU for each library Assess Qantity Assess the reslting Normalized RFU for each library against the following criteria. Florescence Measrement Average Blank RFU > Average Blank RFU (and) < Normalized Standard RFU Normalized Standard RFU Recommendation Repeat library preparation and enrichment if prified DNA or RNA sample meets qantity and qality specifications. Proceed to Normalize Libraries. Note: Using libraries with RFU below the Normalized Standard RFU may not yield adeqate seqencing reslts needed to confidently call variants that may be present in the sample. Proceed to Normalize Libraries. Normalize Libraries This process ses bead-based normalization to normalize the qantity of each library to ensre a niform library representation in the pooled libraries. Manal library normalization is not crrently spported by TrSight Tmor 170. Contact Illmina Technical Spport if yo wold like to manally normalize libraries. Consmables LNA1 (Library Normalization Additives 1) LNB1 (Library Normalization Beads 1) LNW1 (Library Normalization Wash 1) LNS2 (Library Normalization Storage 2) HP3 (2N NaOH) PCR-grade water Docment # v01 For Research Use Only. Not for se in diagnostic procedres. 28