TruSight Tumor 15. Reference Guide. For Research Use Only. Not for use in diagnostic procedures. Document # v06 March 2018

Transcription

1 TrSight Tmor 15 Reference Gide Docment # v06 March 2018 For Research Use Only. Not for se in diagnostic procedres. ILLUMINA PROPRIETARY

2 TrSight Tmor 15 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, AND WILL VOID ANY WARRANTY APPLICABLE TO THE PRODUCT(S). 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. All trademarks are the property of Illmina, Inc. or their respective owners. For specific trademark information, see Notice to Prchaser: Limited License The prchase price of this Prodct incldes a limited, non-transferable license nder U.S. and foreign patents owned by BIO-RAD Laboratories, Inc., to se this Prodct. No other license nder these patents is conveyed expressly or by implication to the prchaser by the prchase of this Prodct. This prodct is sold nder license from Affibody AB, Sweden. Phsion DNA Polymerase is manfactred by Thermo Fisher Scientific. Phsion is trademark or registered trademark of Thermo Fisher Scientific, or its sbsidaries. Docment # v06 For Research Use Only. Not for se in diagnostic procedres. ii

3 TrSight Tmor 15 Reference Gide Revision History Docment Date Description of Change Docment # v06 Docment # v05 Docment # v04 Docment # v03 Docment # v02 Docment # v01 Docment # v00 March 2018 Jne 2016 Febrary 2016 Janary 2016 November 2015 October 2015 September 2015 In Tips and Techniqes: Under Sealing the Plate, pdated Microseal A Under Handling Beads pdated bead prep (bllets 2 and 3) In Clean Up Libraries, pdated the Abot Reagents section Added TrSight Tmor 15 MiniSeq Kit catalog nmber to Kit Contents. Added steps to apply seals before centrifging and shaking when appropriate. Corrected catalog nmber for Veriti Thermal Cycler. Added reaction volmes to the TST15 PCR1 and TST15 PCR2 thermal cycler programs. Added instrctions for mixing TAM (TrSight Tmor Amplification Mix) after thawing. In the Clean Up Libraries section, clarified wording in step 6 to wait ntil the beads bind to the magnets. Added mltichannel pipettes to the Consmables list. Removed instrment-specific instrctions. Information is now available in the denatre and dilte libraries gide and system gide for yor seqencing instrment. Clarified TST15 PCR1 ramp rate reqirements. Corrected TST15 PCR2 cycling description. Corrected agarose gel example well labels. Updated the prodct name to TrSight Tmor 15. Initial release. Docment # v06 For Research Use Only. Not for se in diagnostic procedres. iii

4 Table of Contents Revision History iii Chapter 1 Overview 1 Introdction 1 DNA Inpt Recommendations 2 Additional Resorces 2 Chapter 2 Protocol 4 Protocol Introdction 4 Tips and Techniqes 4 Library Prep Workflow 6 Amplify and Tag Targets 6 Index Targets 9 Clean Up Libraries 11 Check Libraries 12 Pool Libraries 14 Appendix A Spporting Information 16 Introdction 16 Acronyms 16 Kit Contents 16 Consmables and Eqipment 17 Index Seqences 19 Technical Assistance 21 Docment # v06 For Research Use Only. Not for se in diagnostic procedres. iv

5 Chapter 1 Overview Introdction 1 DNA Inpt Recommendations 2 Additional Resorces 2 Introdction The Illmina TrSight Tmor 15 protocol describes a mltiplexed PCR-based approach to prepare libraries for seqencing from DNA extracted from formalin-fixed, paraffin embedded (FFPE) tisse samples. The TrSight Tmor 15 kit reagents allow for preparation of p to 48 indexed, paired-end libraries from 24 DNA samples. The kit is optimized to provide amplicon coverage of 15 genes for highly sensitive analysis of lowfreqency somatic variants from FFPE solid tmor samples. These genes and gene regions inclde single ncleotide variants (SNVs), insertions, deletions (indels), and amplifications that have been associated with cancer. The TrSight Tmor 15 protocol offers: Fast and easy sample preparation Prepare p to 48 libraries from 24 samples (each sample is prepared with Primer Mix A and Primer Mix B) in approximately 7 hors, inclding 2.5 hors of hands-on time Low DNA inpt Higher data qality and robst performance with low inpt of 20 ng (10 ng from each sample for mix A and B) at a minimm concentration for inpt DNA of 2 ng/μl Filtered variant reporting lists relevant variants How Does the Assay Work? The assay contains two separate pools of tagged oligoncleotide primers. These pools are sed in a mltiplex PCR to amplify and target regions of interest from DNA extracted from FFPE samples for a select set of targets. Using index adapters provided in the kit, libraries are indexed and frther amplified, and then combined in a single tbe in preparation of a paired-end seqencing rn. After seqencing is complete, an analysis report provides a specific set of single ncleotide variants (SNV) and small insertions, deletions, and amplifications associated with cancer. Docment # v06 For Research Use Only. Not for se in diagnostic procedres. 1

6 TrSight Tmor 15 Reference Gide DNA Inpt Recommendations The TrSight Tmor 15 kit has been optimized for a specific DNA inpt amont. Qantify the inpt DNA before beginning the protocol. Use 20 ng total (10 ng for Mix A and Mix B per sample) hman genomic DNA (gdna) inpt at a minimm concentration of 2 ng/μl. Use a florometric qantification method that ses dsdna binding dyes sch as AccClear (recommended), Qbit, or PicoGreen. Dilte starting material in RNase/DNase-free water. Use DNA samples that reslt in a delta Cq vale 5. Using samples with a delta Cq > 5 may reslt in low prodct yields (< 20ng/l), high primer-dimer, and seqencing rns with low %Q>30. DNA sample's delta Cq vale can be assessed sing the Illmina FFPE QC Kit. Reference Samples [Optional] Use a characterized reference material when rnning the library preparation, sch as HorizonDx HD701 or HD200 (reqires DNA extraction), or AcroMetrix Oncology Hotspot Control. Use 5 μl RNase/DNase-free water as a no template control for PCR amplification. 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 with each library prep kit. FFPE DNA Extraction Use the following recommendations to extract DNA from FFPE tisse: Use at least 140 mm 2 nonmelanoma tisse with at least 30% tmor Use 1 of the following kits to extract DNA from FFPE tisse: AllPrep DNA/RNA FFPE Kit (QIAGEN) (Recommended) QIAamp DSP DNA FFPE Tisse Kit (QIAGEN) ReliaPrep FFPE gdna Miniprep System (Promega) Additional Resorces Visit the TrSight Tmor 15 kit spport page on the Illmina website for docmentation, software downloads, training resorces, and information abot compatible Illmina prodcts. The following docmentation is available for download from the Illmina website. Resorce Cstom Protocol Selector TrSight Tmor 15 Protocol Gide (docment # ) 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 instrctions for the experienced ser. Docment # v06 For Research Use Only. Not for se in diagnostic procedres. 2

7 TrSight Tmor 15 Reference Gide Resorce TrSight Tmor 15 Checklist (docment # ) TrSight Tmor 15 Consmables & Eqipment List (docment # ) Description Provides a checklist of steps for the experienced ser. Provides an interactive checklist of ser-provided consmables and eqipment. Docment # v06 For Research Use Only. Not for se in diagnostic procedres. 3

8 Chapter 2 Protocol Protocol Introdction 4 Tips and Techniqes 4 Library Prep Workflow 6 Amplify and Tag Targets 6 Index Targets 9 Clean Up Libraries 11 Check Libraries 12 Pool Libraries 14 Protocol Introdction Follow the TrSight Chimerism protocol in the order shown sing the specified parameters. Before proceeding, confirm kit contents and make sre that yo have the reqired consmables and eqipment. See Kit Contents on page 1 and Consmables and Eqipment on page 1. Before beginning library preparation, record information abot yor samples for later se in data analysis. Tips and Techniqes Unless a safe stopping point is specified in the protocol, proceed immediately to the next step. Avoiding Cross-Contamination When adding or transferring samples, change tips between each sample. When adding adapters or primers, change tips between each row and each colmn. Remove nsed index adapter tbes from the working area. 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 indexing primers, change tips between each well. Change gloves if gloves come into contact with Mix A or Mix B primers, indexing primers, or samples. Clean work srfaces thoroghly before and after the procedre. Sealing the Plate Always seal the 96-well plate 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, and long-term storage. Microseal 'A' adhesive film is sed for thermal cycling steps to prevent evaporation. Docment # v06 For Research Use Only. Not for se in diagnostic procedres. 4

9 TrSight Tmor 15 Reference Gide 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 Centrifge at any step in the procedre to consolidate liqid or beads in the bottom of the well, and to prevent sample loss. Handling Beads Do not freeze beads. Pipette bead sspensions slowly. Before se, allow the beads to come to room temperatre. Immediately before se, vortex the beads ntil they are well dispersed. The color of the liqid mst appear homogeneos. Vortex throghot protocol as necessary to keep homogenos. If beads are aspirated into pipette tips, dispense back to the plate on the magnetic stand, and wait ntil the liqid is clear (~2 mintes). When washing beads: Use the specified 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 it is on the magnetic stand. Do not distrb the bead pellet. Docment # v06 For Research Use Only. Not for se in diagnostic procedres. 5

10 TrSight Tmor 15 Reference Gide Library Prep Workflow Figre 1 TrSight Tmor 15 Workflow Amplify and Tag Targets This process amplifies target regions to create 2 separate libraries per sample sing 2 primer mixes, Mix A sing Primer Mix A, and Mix B sing Primer Mix B. Consmables TTM (TrSight Tmor Targeting Mix) TPA (TrSight Tmor Primer Mix A) TPB (TrSight Tmor Primer Mix B) TTE (TrSight Tmor Targeting Enzyme) Sample DNA Docment # v06 For Research Use Only. Not for se in diagnostic procedres. 6

11 TrSight Tmor 15 Reference Gide RNase/DNase-free water 1.7 ml microcentrifge tbes (2) 96-well PCR plate Microseal 'A' film Microseal 'B' adhesive seal NOTE Use Microseal 'A' when sealing the plate before placing it on the thermal cycler. Use Microseal 'B' for other steps that reqire a sealed plate. Preparation 1 Prepare the following consmables: Item Storage Instrctions TTM -25 C to -15 C Thaw at room temperatre. Vortex each tbe to mix. Centrifge briefly. TPA -25 C to -15 C Thaw at room temperatre. Vortex each tbe to mix. Centrifge briefly. TPB -25 C to -15 C Thaw at room temperatre. Vortex each tbe to mix. Centrifge briefly. TTE -25 C to -15 C Centrifge briefly and place on ice or in an enzyme cooler. Sample DNA -25 C to -15 C Thaw at room temperatre. 2 Label two 1.7 ml microcentrifge tbes as Master Mix A and Master Mix B. 3 Save the following program as TST15 PCR1 on a thermal cycler with a heated lid. Choose the preheated lid option and set to 102 C Set the reaction volme to 15 μl 98 C for 3 mintes 16 cycles of: 96 C for 45 seconds 70 C for 1 minte 54 C for 3 mintes with the following ramp rate, depending on the thermal cycler: Thermal Cycler Ramp Rate Bio-Rad C1000 Thermal Cycler Down 0.1 C/s Up 0.1 C/s Bio-Rad S1000 Thermal Cycler Down 0.1 C/s Applied Biosystems GeneAmp PCR System 9700 Down 7% Up Up 7% 0.1 C/s Applied Biosystems Veriti Thermal Cycler Down 4.5% Up 4.0% Eppendorf Mastercycler ep Gradient Down 6% Up 4% Eppendorf Mastercycler ep Gradient-S Down 2% Up 2% Docment # v06 For Research Use Only. Not for se in diagnostic procedres. 7

12 TrSight Tmor 15 Reference Gide 72 C for 15 seconds with the following ramp rate, depending on the thermal cycler: Thermal Cycler Ramp Rate Bio-Rad C1000 Thermal Cycler Down 0.1 C/s Up 0.1 C/s Bio-Rad S1000 Thermal Cycler Down 0.1 C/s Applied Biosystems GeneAmp PCR System 9700 Down 7% Up Up 7% 0.1 C/s Applied Biosystems Veriti Thermal Cycler Down 4.5% Up 4.0% Eppendorf Mastercycler ep Gradient Down 6% Up 4% Eppendorf Mastercycler ep Gradient-S Down 2% 72 C for 5 mintes Hold at 10 C CAUTION Procedre Up 2% Ramp rate is critical for TrSight Tmor 15 performance. Failre to set the ramp rate reslts in low prodct yields and high primer-dimer. 1 Qantify the sample DNA sing a florometric method specific for doble-stranded DNA, sch as AccClear (recommended), Qbit, or PicoGreen. 2 Dilte each sample DNA to 2 ng/µl with RNase/DNase-free water in a final volme of 12.5 µl. 3 Combine the following reagents in separate microcentrifge tbes to create PCR master mixes for TPA and TPB. PCR Component Per Well Per 8 Samples Per 16 Samples Per 24 Samples TTM µl 47 µl 94 µl 141 µl TPA or TPB 6.25 µl 50 µl 100 µl 150 µl TTE µl 3 µl 6 µl 9 µl Prepare a minimm of 4 samples. 4 Pipette to mix. 5 Add 10 µl of each PCR master mix to each well. Master Mix A Rows A and C Master Mix B Rows B and D 6 Add 5 µl of 2 ng/μl DNA to the corresponding well. Samples 1 12 Rows A and B Samples Rows C and D 7 Pipette to mix. 8 Apply the seal and centrifge at 1000 g for 1 minte. Docment # v06 For Research Use Only. Not for se in diagnostic procedres. 8

13 TrSight Tmor 15 Reference Gide 9 Immediately place on a thermal cycler and rn the TST15 PCR1 program. SAFE STOPPING POINT If yo are stopping, seal the plate and store at 2 C to 8 C for p to 3 days. Alternatively, leave on the thermal cycler overnight. Index Targets This process tags amplified DNA and adds Index 1 (i7) adapters, Index 2 (i5) adapters, and seqences reqired for clster formation with each library receiving a niqe combination of Index 1 and Index 2 adapters. Consmables Index 1 (i7) adapters and orange tbe caps Index 2 (i5) adapters and white tbe caps TAM (TrSight Tmor Amplification Mix) 1.7 ml microcentrifge tbes (1 per index adapter tbe) Microseal 'A' film Microseal 'B' adhesive seal NOTE Use Microseal 'A' when sealing the plate before placing it on the thermal cycler. Use Microseal 'B' for other steps that reqire a sealed plate. Preparation 1 Prepare the following consmables: Item Storage Instrctions Index adapters (i7 and i5) -25 C to -15 C Only remove adapters being sed. Thaw at room temperatre for 20 mintes. Vortex each tbe to mix. Centrifge briefly sing a 1.7 ml microcentrifge tbe as a holder/adapter in the centrifge. TAM -25 C to -15 C Thaw at room temperatre for 20 mintes. Vortex to mix. Centrifge briefly. 2 Save the following program as TST15 PCR2 on a thermal cycler with a heated lid. Choose the preheated lid option and set to 102 C Set the reaction volme to 50 μl 98 C for 30 seconds 17 cycles of: 98 C for 20 seconds 60 C for 30 seconds 72 C for 45 seconds 72 C for 5 mintes Hold at 10 C Procedre 1 Arrange Index 1 (i7) adapters in the top row of the TrSeq Index Plate Fixtre. Docment # v06 For Research Use Only. Not for se in diagnostic procedres. 9

14 TrSight Tmor 15 Reference Gide 2 Arrange Index 2 (i5) adapters in rows A-B of the TrSeq Index Plate Fixtre so Mix A and Mix B are given niqe i5/i7 index combinations. For example A501 in row A for Mix A, and A502 in row B for Mix B. 3 Record index adapters sed for each sample for seqencing. 4 Place the plate on the TrSeq Index Plate Fixtre. Figre 2 Index Plate Fixtre A B C Colmns 1 12: Index 1 (i7) adapters (orange caps) Rows A B: Index 2 (i5) adapters (white caps) 96-well plate 5 For samples 1 12, perform the following. a b Using a mltichannel pipette, add 4 µl of each Index 2 (i5) adapter (i501 and i502) for rows A and B respectively. If yo do not have additional samples, replace the cap on each i5 adapter tbe with a new white cap. Using a mltichannel pipette, add 4 µl of each Index 1 (i7) adapter (R701 R709, R749, R711 R712) to each colmn of rows A and B. Replace the cap on each i7 adapter tbe with a new orange cap. 6 For samples 13 24, perform the following. a b c d Move Index 2 (i5) adapters (i501 and i502) to rows C and D respectvely (i501 to row C and i502 to row D). Replace Index 1 (i7) adapters in the top row with the second set (R725 R736). Using a mltichannel pipette, add 4 µl of each Index 2 (i5) adapter across rows C and D. Replace the cap on each i5 adapter tbe with a new white cap. Using a mltichannel pipette, add 4 µl of each Index 1 (i7) adapter (R725 R736) to each colmn of rows C and D. Replace the cap on each i7 adapter tbe with a new orange cap. NOTE Record index arrangement sed for preparing the sample sheet. 7 Add 27 µl TAM to each well. Docment # v06 For Research Use Only. Not for se in diagnostic procedres. 10

15 TrSight Tmor 15 Reference Gide 8 Pipette to mix. 9 Apply the seal and centrifge at 1000 g for 1 minte. 10 Immediately place on a thermal cycler and rn the TST15 PCR2 program. SAFE STOPPING POINT If yo are stopping, seal the plate and store at -25 C to -15 C for p to seven days. Clean Up Libraries This step ses SPB (Sample Prification Beads) to prify the PCR prodcts from other reaction components. Consmables SPB (Sample Prification Beads) RSB (Resspension Bffer) Freshly prepared 80% ethanol (EtOH) 96-well midi plate 96-well PCR plate Microseal 'B' adhesive seals Abot Reagents Prior to se, allow SPB to come to room temperatre. Immediately prior to se, vortex SPB ntil beads are well dispersed. The color of the liqid shold appear homogeneos. Aspirate and dispense SPB slowly de to the viscosity of the soltion. Preparation 1 Prepare the following consmables: Reagent Storage Instrctions SPB 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. RSB 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. 2 Prepare fresh 80% EtOH. 3 Label a new 96-well PCR plate PLP (Prified Library Plate). Procedre 1 Centrifge at 1000 g for 1 minte. 2 Add 40 µl SPB to each well of a new midi plate. 3 Transfer 45 µl spernatant from the PCR plate to the corresponding well of the midi plate. 4 Apply the seal and shake at 1800 rpm for 5 mintes. 5 Incbate at room temperatre for 5 mintes. 6 Place on a magnetic stand and wait ntil the beads bind to the magnet (~2 mintes). Docment # v06 For Research Use Only. Not for se in diagnostic procedres. 11

16 TrSight Tmor 15 Reference Gide 7 Remove and discard all spernatant from each well. 8 Wash two times as follows. a b c Add 200 µl fresh 80% EtOH to each well. Incbate on the magnetic stand for 30 seconds. Remove and discard all spernatant from each well. 9 Using a 20 μl pipette, remove residal 80% EtOH from each well. 10 Air-dry on the magnetic stand for 5 mintes. 11 Add 32 µl RSB to each well. 12 Apply the seal and shake at 1800 rpm for 2 mintes. If the beads are not resspended, pipette to mix or repeat shake at 1800 rpm for 2 mintes. 13 Incbate at room temperatre for 2 mintes. 14 Place on a magnetic stand and wait ntil the liqid is clear (~2 mintes). 15 Transfer 30 µl spernatant from each well to the corresponding well of the PLP plate. 16 Apply the seal and centrifge at 1000 g for 1 minte. SAFE STOPPING POINT If yo are stopping, seal the PCR plate and store at -25 C to -15 C for p to 2 months. Check Libraries Perform the following procedres to qantify libraries and check library qality. Accrately qantify DNA libraries to ensre optimm clster densities on the flow cell. For best reslts, seqence samples with library Mix A and Mix B yields 20 ng/μl. Libraries with lower yield give poor qality and less accrate variant reporting. Consmables RSB (Resspension Bffer) 96-well PCR plate 1.7 ml microcentrifge tbes Preparation 1 Prepare the following consmables: Item Storage Instrctions RSB 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. 2 Label a new 96-well PCR plate NLP (Normalized Library Plate). Procedre 1 Qantify the library sing a florometric method, sch as AccClear (recommended), PicoGreen, or Qbit. Docment # v06 For Research Use Only. Not for se in diagnostic procedres. 12

17 TrSight Tmor 15 Reference Gide 2 Calclate the volme of RSB reqired to adjst the library concentration to 5 ng/µl as follows. a Use the formla C1V1 = C2V2, where C1 is the reslt of library qantification, V1 is 8 µl, and C2 is 5 ng/µl to calclate the vale for V2. b Calclate the amont of RSB (V2-8 µl) reqired to adjst the concentration of each library to 5 ng/µl. 3 Add the reqired volme of RSB to the corresponding well of the NLP plate. If a library is 100 ng/µl, transfer the RSB to a 1.7 ml tbe. 4 Transfer 8 µl of each library from the PLP plate to the corresponding well of the NLP plate or 1.7 ml tbe. 5 Rn an aliqot of each normalized library on either of the following methods: 15 µl on a 2% agarose gel sing a 50 bp DNA ladder 1 µl on a Bioanalyzer sing a DNA 1000 chip The expected PCR prodct is ~350 bp, which indicates sccessfl library amplification. A strong primer dimer signal at ~160 bp can indicate library prep failre. Figre 3 Agarose Gel Example A B C D E No template control Sample libraries (~350 bp) Size standards Targeted amplicons Primer dimer (~160 bp) Docment # v06 For Research Use Only. Not for se in diagnostic procedres. 13

18 TrSight Tmor 15 Reference Gide Figre 4 Bioanalyzer Example A B C Marker Sample libraries (~350 bp) Marker SAFE STOPPING POINT If yo are stopping, seal the plate and store at -25 C to -15 C for p to 14 days. Pool Libraries Use the following protocol to pool libraries. Consmables RSB (Resspension Bffer) 1.7 ml microcentrifge tbes (2) Abot Reagents The PNL tbe can be stored at -25 C to -15 C for p to 7 days. The DNL tbe can be stored at -25 C to -15 C for p to 7 days. Preparation 1 Prepare the following consmables. Item Storage Instrctions RSB 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. 2 Label a new 1.7 ml microcentrifge tbe PNL (Pooled Normalized Libraries). 3 Label a new 1.7 ml microcentrifge tbe DNL (Dilted Normalized Libraries). 4 If the NLP plate was stored frozen, thaw at room temperatre and then centrifge briefly. Pipette to mix. 5 To prepare for the seqencing rn, begin thawing reagents according to the instrctions for yor seqencing instrment. Procedre 1 Apply the seal and centrifge the NLP plate at 1000 g for 1 minte. 2 Transfer 4 µl of each library from the NLP plate to the PNL tbe. Docment # v06 For Research Use Only. Not for se in diagnostic procedres. 14

19 TrSight Tmor 15 Reference Gide 3 Vortex to mix, and then centrifge briefly. 4 Add 41 µl RSB to the DNL tbe. 5 Transfer 9 µl from the PNL tbe to the DNL tbe. 6 Vortex to mix, and then centrifge briefly to create a 4 nm pooled library. Docment # v06 For Research Use Only. Not for se in diagnostic procedres. 15

20 Appendix A Spporting Information Spporting Information Introdction 16 Acronyms 16 Kit Contents 16 Consmables and Eqipment 17 Index Seqences 19 Introdction The protocol described in this gide assmes that yo have reviewed the contents of this section, confirmed workflow contents, and obtained all reqired consmables and eqipment. Acronyms Acronym DAL DNL HP3 HT1 NLP PLP PNL RSB SPB TAM TPA TPB TTE TTM Definition Denatred Amplicon Libraries Dilted Normalized Libraries 2N NaOH Hybridization Bffer Normalized Library Plate Prified Library Plate Pooled Normalized Libraries Resspension Bffer Sample Prification Beads TrSight Tmor Amplification Mix TrSight Tmor Primer Mix A TrSight Tmor Primer Mix B TrSight Tmor Targeting Enzyme TrSight Tmor Targeting Mix Kit Contents Make sre that yo have all the reagents identified in this section before proceeding to the library preparation procedres. Kits are available in the following configrations. Consmable Catalog # TrSight Tmor 15 MiSeq Kit (1 Library Prep, 24 Samples; 3 MiSeq Reagent Kits v3) TrSight Tmor 15 (1 Library Prep, 24 Samples) OP OP TrSight Tmor 15 MiniSeq Kit (1 Library Prep, 24 Samples; 3 MiniSeq Reagent Kits) Docment # v06 For Research Use Only. Not for se in diagnostic procedres. 16

21 TrSight Tmor 15 Reference Gide Box 1 (Pre-PCR) Store at -25 C to -15 C Qantity Reagent Description 1 TTM TrSight Tmor Targeting Mix 1 TTE TrSight Tmor Targeting Enzyme 1 TPA TrSight Tmor Primer Mix A 1 TPB TrSight Tmor Primer Mix B Box 2 (Post-PCR) Store at -25 C to -15 C Qantity Reagent Description 2 TAM TrSight Tmor Amplification Mix 1 HP3 2 N NaOH 2 A501, A502 i5 Index Adapters 24 R701-R709, R711 R712, R749, R725 R736 i7 Index Adapters Box 3 (Post-PCR) Store at 2 C to 8 C Qantity Reagent Description 1 SPB Sample Prification Beads 1 RSB Resspension Bffer Consmables and Eqipment Make sre that yo have the reqired ser-spplied consmables and eqipment before starting the protocol. The protocol has been optimized and validated sing the items listed. Comparable performance is not garanteed when sing alternate consmables and eqipment. Consmables Consmable Spplier 1.7 ml microcentrifge tbes General lab spplier 20 µl mltichannel pipettes General lab spplier 200 µl mltichannel pipettes General lab spplier 20 µl barrier pipette tips General lab spplier 200 µl barrier pipette tips General lab spplier 1000 µl barrier pipette tips General lab spplier 96-well flat clear bottom black microplates Note: Used when qantifying samples with a SpectraMax M5 spectroflorometer. 96-well storage plates, rond well, 0.8 ml (midi plate) Conical centrifge tbes (15 ml or 50 ml) Corning, part # 3904 Fisher Scientific, part # AB-0859 General lab spplier Docment # v06 For Research Use Only. Not for se in diagnostic procedres. 17

22 TrSight Tmor 15 Reference Gide Consmable Ethanol 200 proof (absolte) for moleclar biology (500 ml) 96-well PCR Plates Note: Use a PCR plate compatible with yor thermal cycler (see Thermal Cyclers on page 1) Microseal 'A' film Microseal 'B' adhesive seals PCR-grade water RNase/DNase-free mltichannel reagent reservoirs, disposable RNase/DNase-free water Florescence-based qantification reagents sch as AccClear (recommended), Qbit, or PicoGreen. AccClear Ultra High Sensitivity dsdna Qantitation Kit One of the following (for validating libraries): 2% Agarose gel DNA 1000 Chip [Optional] Screw-cap tbes Spplier Sigma-Aldrich, part # E7023 General lab spplier Bio-Rad, part # MSA-5001 Bio-Rad, part # MSB-1001 General lab spplier VWR, part # General lab spplier Biotim, catalog # General lab spplier Agilent, part # General lab spplier Seqencing Kits [Optional] Eqipment Catalog # PhiX Control v3 MiSeq Reagent Kit v3, 600 Cycles FC MS Eqipment Eqipment One of the following magnetic stands: Magnetic Stand-96 DynaMag-96 Side Skirted Magnet Microcentrifge Microplate centrifge Microplate florometer Vortexer One of the following 96-well thermal cyclers: C1000 Toch Thermal Cycler S1000 Thermal Cycler Veriti Thermal Cycler Spplier/Description Thermo Fisher, catalog # AM10027 Thermo Fisher, catalog # General lab spplier General lab spplier General lab spplier General lab spplier Bio-Rad, part # Bio-Rad, part # Thermo Fisher, catalog # Docment # v06 For Research Use Only. Not for se in diagnostic procedres. 18

23 TrSight Tmor 15 Reference Gide Eqipment One of the following microplate shakers: BioShake iq High-Speed Thermal Mixer BioShake XP High-Speed Mixer VWR Advanced High-Speed Microplate Shaker VWR Signatre High-Speed Microplate Shaker One of the following (for library validation): Gel electrophoresis apparats 2100 Bioanalyzer Desktop System Spplier/Description Q Instrments, model # Q Instrments, model # VWR, catalog # (230 V) VWR, catalog # (110 V/120 V) General lab spplier Agilent, part # G2940CA and part # TrSeq Index Plate Fixtre Kit [Optional] Eqipment [Optional] TrSeq Index Plate Fixtre Kit Note: Recommended for setting p indexed adapters. This part is resable. Spplier Illmina, catalog # FC Index Seqences Use the following seqences to set p yor rn. Index A501 A502 R701 R702 R703 R704 R705 R706 R707 R708 R709 R749 R711 R712 R725 R726 R727 R728 R729 R730 R731 R732 R733 Seqence TGAACCTT TGCTAAGT ATCACG CGATGT TTAGGC TGACCA ACAGTG GCCAAT CAGATC ACTTGA GATCAG GATGCT GGCTAC CTTGTA ACTGAT ATGAGC ATTCCT CAAAAG CAACTA CACCGG CACGAT CACTCA CAGGCG Docment # v06 For Research Use Only. Not for se in diagnostic procedres. 19

24 TrSight Tmor 15 Reference Gide Index R734 R735 R736 Seqence CATGGC CATTTT CCAACA Docment # v06 For Research Use Only. Not for se in diagnostic procedres. 20

25 Technical Assistance For technical assistance, contact Illmina Technical Spport. Website: Illmina Cstomer Spport Telephone Nmbers Region Toll Free Regional North America Astralia Astria Belgim China Denmark Finland France Germany Hong Kong Ireland Italy Japan Netherlands New Zealand Norway Singapore Spain Sweden Switzerland Taiwan United Kingdom Other contries Safety data sheets (SDSs) Available on the Illmina website at spport.illmina.com/sds.html. Prodct docmentation Available for download in PDF from the Illmina website. Go to spport.illmina.com, select a prodct, then select Docmentation & Literatre. Docment # v06 For Research Use Only. Not for se in diagnostic procedres. 21

26 Docment # v06 Illmina 5200 Illmina Way San Diego, California U.S.A ILMN (4566) (otside North America) techspport@illmina.com For Research Use Only. Not for se in diagnostic procedres Illmina, Inc. All rights reserved.