TruSight Tumor 26 Reference Guide
Contents
1. 11 Hybridization of Oligo Pool 14 Remove Unbound Oligos sese esee ns 16 Extension Ligation of Bound Oligos 19 PCR Amplification esses lille ll 20 Verify Library Preparation Optional nnnm 23 DChClean Up e cece eee E E ERa AE E AEE 24 Library Quantification esses n 26 Library Normalization 0000000000 sess n 27 Library Denaturing and Pooling eese e enne 28 Appendix A Supporting Information 31 Introduction esses Rer rarnana 32 AGONY MS eea ex Lr LR o ER E Dee 33 TruSight Tumor 26 Kit Contents eee er Rr rrr 34 Consumables and Equipment essen 36 Index Sequences 39 Technical Assistance TruSight Tumor 26 Reference Guide Vi Material 20000848 Part 15042911 v01 Overview ic A ihdidensd Maeceelabiticdddestesdaeeubieeeted se 2 How Does the Assay Work 3 DNA Input Recommendations 4 Additional Resources l l nro 5 A em A TUE AA aT um IDR TT fi ES m ron reas rco rca wa TruSight Tumor 26 Reference Guide 1 LJe3deuo Overview Introduction TruSight Tumor 26 takes a deeper view of variation in solid tumors including lung colon melanoma gastric and ovarian This step enables clinical researchers to look beyond point mutations within hot spots in single genes for a more comprehensive view of somatic variation TruSight Tumor 26 provides amplicon based library preparation reagents DNA QC sample indexes and oligos targetin2. The protocol guide is intended for experienced users For new 1000000001444 or less experienced users see the TruSight Tumor 26 Reference Guide TruSight Tumor 26 Checklist Provides a checklist of the protocol steps document 1000000001445 The checklist is intended for experienced users For new or less experienced users see the TruSight Tumor 26 Reference Guide IEM TruSight Quick Reference Provide information about creating and editing appropriate Card part 15048138 sample sheets for Illumina sequencing systems and analysis software and record parameters for your sample plate Visit the TruSight Tumor 26 Kit support pages on the Illumina website for additional documentation software downloads and best practices TruSight Tumor 26 Reference Guide 5 SooJnoseMH IGUOIUDDv Material 20000848 Part 15042911 v01 Protocol Introduction EE 8 Tips and Techniques 9 Belle nne Ce e UE 10 Qualification of DNA Extracted from FFPE Samples sss 11 Hybridization of Oligo Pool 14 Remove Unbound Oligos 16 Extension Ligation of Bound Oligos 22 ccc cece cece cece eee e ccc eeeeeeeeeeeeeeees 19 PGR nee EEN 20 Verify Library Preparation Optional 23 POR ClSanUD ccc o a e la do DUE 24 Library Quantification aaa RRR I RR I eme e R eaa R 26 Library Normalization 22 2 II RR RRRRR IRR RRRRIeIeleemerrlll lll 27 Library Denaturing and Pooling IR RR IIR erret ells 28 i T E
3. Wash the FPU plate as follows a Using a multichannel pipette add 50 ul of SW1 to each sample well b Centrifuge at 2 400 x g for 5 minutes Repeat the wash as described in the previous step Discard all the flow through waste in a hazardous waste container and then reassemble the FPU The same midi plate can be reused for the rest of the pre amplification process Using a multichannel pipette add 45 ul of UB1 to each sample well Centrifuge the FPU at 2 400 x g for 5 minutes Material 20000848 Part 15042911 v01 Extension Ligation of Bound Oligos This process connects the hybridized upstream and downstream oligos A DNA polymerase extends the upstream oligo through the targeted region and is ligated to the 5 end of the downstream oligo using a DNA ligase The ligation step forms products containing the targeted regions of interest flanked by sequences required for amplification Consumables ELM3 Extension Ligation Mix 3 Adhesive aluminum foil seal Troughs Procedure 1 Using a multichannel pipette add 45 ul of ELMG to each sample well of the FPU plate The extension ligation reaction takes place on the filter plate membrane If you use care to avoid cross contamination changing tips between columns is not required 2 Seal the FPU plate with adhesive aluminum foil and then cover with the lid 3 Incubate the entire FPU plate unit assembly in the preheated 37 C incubator for 45 minutes 4 During incubation p
4. 3 2 Material 20000848 Part 15042911 v01 Acronyms Acronym Definition ACD1 Amplicon Control DNA 1 ACP1 Amplicon Control Oligo Pool 1 CEP Clean up Plate DAL Diluted Amplicon Library EBT Elution Buffer with Tris ELM3 Extension Ligation Mix 3 FPA TruSight Tumor Oligo Pool A FPB TruSight Tumor Oligo Pool B FPU Filter Plate Unit HI Hybridization Buffer HYE HYbridization Plate IAP Indexed Amplification Plate LNP Library Normalization Plate OHS3 Oligo Hybridization for Sequencing Reagent 3 PAL Pooled Amplicon Library PMM2 PCR Master Mix 2 QCP Quality Control Primers OCT Quality Control Template SGP Storage Plate SW1 Stringent Wash 1 TDP1 TruSeq DNA Polymerase 1 UB1 Universal Buffer 1 TruSight Tumor 26 Reference Guide 33 SWAUOIOVY Supporting Information 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 Catalog Samples TruSight Tumor 26 FC 130 2001 48 TG TruSight Tumor 26 TG 130 2001 48 4 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
5. Box 1 Pre Amplification Quantity Reagent Description Storage Temperature 1 ACD1 Amplicon Control DNA 1 25 C to 15 C 1 ACP1 Amplicon Control Oligo Pool 1 25 C to 15 C 1 OHS3 Oligo Hybridization 25 C to 15 C for Sequencing Reagent 3 1 ELM3 Extension Ligation Mix 3 25 C to 15 C 1 PMM2 PCR Master Mix 2 25 C to 15 C 1 TDP1 TruSeq DNA Polymerase 1 25 C to 15 C 1 sw1 Stringent Wash 1 2 C to 8 C 1 UB1 Universal Buffer 1 2 C to 8 C a y 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 Reagent Description Storage Temperature 1 HI Hybridization Buffer 25 C to 15 C 4 EBT Elution Buffer with Tris Room temperature Box 3 TruSight Tumor Oligo Set Pre Amplification Store at 25 C to 15 C Quantity Reagent Description I QCP Quality Control Primers 3 A Material 20000848 Part 15042911 v01 Quantity Reagent Description 1 TruSight Tumor Oligo Pool A 1 TruSight Tumor Oligo Pool B 1 OCT Quality Control Templ
6. Delta Cq 2 5 to 1 5 1 5 to 0 5 0 5 to 0 5 0 5 to 1 5 1 5 to 4 Dilution 16x 8x 4x 2x No dilution NOTE If preparing libraries from the control DNA in parallel with FFPE DNA samples dilute 5 ul of ACD1 with 45 ul of TE Buffer Add 10 ul to each of 2 control wells for FPA and FPB and or 2 control wells for ACP1 Add 10 ul of each diluted sample to wells on the left half of the HYP plate starting with column 1 Then repeat this process on the right half of the HYP plate starting with column 7 Using a multichannel pipette add 5 ul of FPA to all sample containing wells on the left half of the HYP plate Then add 5 ul of FPB to all sample containing wells on the right half of the HYP plate L NOTE If preparing libraries from ACD1 add 5 ul of FPA to 1 control well and 5 ul of FPB to the second control well If preparing libraries using ACP1 add 5 ul of ACP1 to 2 additional control wells of ACD1 Using a multichannel pipette add 35 ul of OHS3 to each sample in the HYP plate Gently pipette to mix L NOTE Make sure that there are no crystals or precipitate visible in the OHS3 Seal the HYP plate with a heat sealer or an aluminum foil seal Centrifuge at 1 000 x g at 20 C for 1 minute Place the HYP plate in the 95 C heat block and incubate for 1 minute Change the temperature of the same heat block to 40 C and incubate for 14 18 hours E NOTE e Moving the plate from the 95 C heat block to another preheated block se
7. Ice bucket KAPA SYBR FAST qPCR Master Mix 2X Universal Microseal B adhesive seals PCR 8 tube strips Solution basin PVC nonsterile trough OlAamp DNA FFPE Tissue Kit Supplier Illumina FC 110 3001 General lab supplier Bio Rad Part MSP 9601 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 General lab supplier General lab supplier KAPA Biosystems Bio Rad Part MSB 1001 General lab supplier Labcor Part 730 001 QIAGEN Part 4 56404 Material 20000848 Part 15042911 v01 Equipment Pre PCR Equipment Consumable 37 incubator Heat block 96 well Tabletop centrifuge d NOTE 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 Use a dedicated set of pipettes pipette tips vortexer and centrifuge during pre amplification steps Post PCR Equipment Consumable Post PCR plate shaker Tabletop centrifu
8. Prepare PhiX Control Library 28 1 In a microcentrifuge tube add 2 ul of stock 10 nM PhiX library to 8 ul EBT buffer to yield 10 ul of 2 nM PhiX library Add 10 ul of 0 1 N NaOH to 10 ul of 2 nM PhiX library to yield 20 ul of 1 nM PhiX library Vortex the 1 nM PhiX library briefly to mix then spin at 280 x g for 1 minute Incubate the PhiX library for 4 minutes 30 seconds at room temperature to denature Make sure that incubation time does not exceed a maximum of 5 minutes Add 980 ul of prechilled HT1 to the 20 ul denatured PhiX library to make 20 pM PhiX library E NOTE The denatured 20 pM PhiX library can be stored up to 3 weeks at 25 C to 15 C as single use aliquots After 3 weeks cluster numbers tend to decrease Material 20000848 Part 15042911 v01 Prepare Samples for Sequencing 1 2 3 Centrifuge the LNP plate at 1 000 x g at 20 C for 1 minute to collect condensation Determine the samples to be pooled for sequencing If the LNP plate was stored frozen use a P200 multichannel pipette to mix each library to be sequenced Library Denaturing and Pooling When sequencing TruSight Tumor 26 libraries on the MiSeq System Illumina recommends sequencing 4 tumor samples 8 libraries total per run when using v2 chemistry If sequencing a different number of samples adjust the following procedure accordingly 10 11 12 z NOTE Two libraries represent each sample and are generated from the TruSight Tum
9. nnenan ne S a Wi A A B K A TruSight Tumor 26 Reference Guide T Z Je1deuo Protocol Introduction This chapter describes the TruSight Tumor 26 protocol Review Best Practices before proceeding See Additional Resources on page 5 for information on how to access TruSight Tumor 26 Best Practices on the Illumina website Follow the protocols in the order shown using the specified volumes and incubation parameters If you are pooling record information about your samples before beginning library preparation for later use in data analysis Use IEM to create and edit well formed sample sheets for Illumina sequencing systems and analysis software See Additional Resources on page 5 for information on how to download IEM software and documentation from the Illumina website As a troubleshooting aid ACD1 Amplicon Control DNA and ACP1 Amplicon Control Oligo Pool have been included in this kit Using ACD1 instead of gDNA and ACP1 instead of FPA and FPB in the TruSight Tumor 26 assay can help narrow down issues arising from gDNA sample prep or primer contamination Libraries made with these controls cannot be sequenced along side other TruSight Tumor 26 libraries as they require longer cycles 8 Material 20000848 Part 15042911 v01 Tips and Techniques 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 betw
10. 5 0 ul 275 ul 550 ul 2X Universal Diluted QCP 1 0 ul 55 ul 110 ul Nuclease free water 2 0 ul 110 ul 220 ul Mix gently but thoroughly Place the reaction mix on ice and protect it from light until use Add 8 ul of the master mix to each well of the plate According to your plate layout add 2 ul of the QCT dilution the sample dilutions or nuclease free water to each well of the plate Seal the plate using an appropriate seal for your qPCR machine Centrifuge the plate at 250 x g for 1 minute Make sure that the seal is free of any liquid or dust Place the plate on the qPCR machine then close the lid and run the following program Procedure Temperature Time minutes Hot Start 50 C 2 95 C 10 x40 95 C 30 60 C 30 72 C 30 Confirm that the instrument captures images after the 72 C step E NOTE e Set the Cq threshold to a value that avoids inaccurate measurements due to background 100 RFU on the Bio Rad 396CFX System After the final step the thermal cycler analyzes the quantified libraries Make sure that amplification of the NTC occurs at least 10 cycles after OCT amplification Make sure that there is good amplification for the OCT and remove outliers from a triplicate group that are gt 0 5 Cq different from the rest of the group NOTE Four or more outliers per plate indicate technical errors Material 20000848 Part 15042911 v01 20 Exclude replicates exhibiting abnormal amplification curves For
11. ST TA ee EE GATAACAGTAACACACTTCTGTTAACC TTAAGATTACT TGATCCACTGAT TCAACG TACCG TAACGAACG TATCAAT TGAGACTAAATAT TAACG TACCAT TAAGAGCTACCGTCT TCTGT TAACCT TAA EE EE ACCATTAAGAGCTACCGTGCAACTTAACCTTAAGAT TACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATATTAACGTACCAT TAAGAGCTACCGTGCAACGACGAACT T CTGT TAACCT TAAGAT TACTTGA GCTACCGTGCAACGAAAATAACC T TAAGATTACT TGATCCACTGAT TCAACGTACTTCTGTTAACCTTAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAATTGAGACTAAGCTACCGTGCAACGACGAAAAGAATGA GAAAAGAATGATAACAG TAACACAC T TCTGT TAACC T TAAGAT TA va D eM TA CCGTAAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTA ovd AU e i es a GATAACAGTAACACACT TCTGTTAACCT TAAGAT TACTTGATCCACTGAT TCAACG TACCGTAACGAACGTAT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTGCAACGACGAAAAGAAT GATAACAGTAACACACT T CTGT ACCA TANGAGCTACCGTGCANCAGTAACAGACTI GTO TACO TTAMQATIACT TONI COAC IGAEM OGIA CSTM CGM CE TACAT CACAO TARATAT TAA GTACCATTAAGAGU TACT ORA C OA EE GATAACAGTAACACACT TCTGTTAACCT TAAGAT T ee ME Mc Eer CTAAATAT Ti d C e eee ee ILIA GTACCGTAACGAACGTATCATTAAGATTACTIGATCCACTGATTCAACG GTAACGAACGTATCAA GACTAAATAT TAACGTACCATTAAGAGCTACCGTGCAACGACGAAAAGAATGATAACAG TAACACACT TCTGT TAACC T T OENE CACICA CAACGITAAGATTACTIGATGCACTGATI CAACGIACCCTAACGAACGTATCAATT CASC LICTOLIAAGC TAAGATTAC LIGATCCAGT GATT CAACCTACCG TAACGAACGTATCARTTOAGACTAGCAACGACE GAAAAGAATGATAACAGTAACACACT TCTGTTAACCT TAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGAT TACT TGATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAG
12. TORT INCLUDING NEGLIGENCE STRICT LIABILITY OR OTHERWISE 6 ILLUMINA S TOTAL AND CUMULATIVE LIABILITY TO PURCHASER OR ANY THIRD PARTY ARISING OUT OF OR IN CONNECTION WITH THESE TERMS AND CONDITIONS INCLUDING WITHOUT LIMITATION THIS PRODUCT INCLUDING USE THEREOF AND ILLUMINA S PERFORMANCE HEREUNDER WHETHER IN CONTRACT TORT INCLUDING NEGLIGENCE STRICT LIABILITY OR OTHERWISE SHALL IN NO EVENT EXCEED THE AMOUNT PAID TO ILLUMINA FOR THIS PRODUCT 7 Limitations on Illumina Provided Warranties TO THE EXTENT PERMITTED BY LAW AND SUBJECT TO THE EXPRESS PRODUCT WARRANTY MADE HEREIN ILLUMINA MAKES NO AND EXPRESSLY DISCLAIMS ALL WARRANTIES EXPRESS IMPLIED OR STATUTORY WITH RESPECT TO THIS PRODUCT INCLUDING WITHOUT LIMITATION ANY IMPLIED WARRANTY OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE NONINFRINGEMENT OR ARISING FROM COURSE OF PERFORMANCE DEALING USAGE OR TRADE WITHOUT LIMITING THE GENERALITY OF THE FOREGOING ILLUMINA MAKES NO CLAIM REPRESENTATION OR WARRANTY OF ANY KIND AS TO THE UTILITY OF THIS PRODUCT FOR PURCHASER S INTENDED USES 8 Product Warranty All warranties are personal to the Purchaser and may not be transferred or assigned to a third party including an affiliate of Purchaser All warranties are facility specific and do not transfer if the Product is moved to another facility of Purchaser unless Illumina conducts such move a Warranty for Consumables Illumina warrants that Consumables other than custom
13. bp Expect this concentration to correlate with the relative library intensities previously observed on the agarose gel For more information see Verify Library Preparation Optional on page 23 Figure 5 Representative DNA Sample Prep Library Size Distribution 300 330 bp 1500 emm e Fu 1000 700 150 500 100 400 50 300 MM 0 200 150 15 100 200 300 500 700 bp Ir 15 NOTE If gt 1076 of the library product is present in the 150 250 bp range Illumina recommends repeating the PCR Clean Up procedure Use 40 ul of product and 32 ul of AMPure XP beads for the repeat procedure Material 20000848 Part 15042911 v01 Library Normalization This process normalizes the quantity of each library to ensure more equal library representation in your pooled sample Consumables Procedure 1 EBT Elution Buffer with Tris Microseal B adhesive film Midi plate From the Agilent Bioanalyzer run determine the concentration for all samples Add all concentrations in terms of nM for all peaks in the 300 330 bp range or 350 380 bp for ACP1 libraries and record the values The expected concentration range is 4 300 nM Label a MIDI plate LNP_plate Library Normalization Plate Dilute 4 ul of all samples gt 20 nM to 4 nM with the EBT buffer For example if a sample is 254 nM add 4 ul of library to 250 ul of EBT buffer to give 4 nM For any samples lt 20 nM d
14. conditions as your CAT Using a multichannel pipette set to 60 ul transfer 55 ul PCR product from the IAP plate to the CLP plate Shake the CLP plate at 1 800 rpm for 2 minutes Incubate at room temperature without shaking for 10 minutes Place the plate on a magnetic stand for 2 minutes or until the supernatant has cleared Keep the plate on the magnetic stand until step 11 Using a multichannel pipette set to 100 ul carefully remove and discard the supernatant E NOTE i Delays during this step can lead to bead clumping following removal of the supernatant Proceed immediately to next step when all supernatant is removed Material 20000848 Part 15042911 v01 10 11 12 13 14 15 16 17 Wash 2 times as follows a Using a multichannel pipette add 200 ul freshly prepared 80 EtOH Avoid disturbing the beads b Incubate on the magnetic stand for 30 seconds or until the supernatant appears clear c Carefully remove and discard all supernatant from each well Using a multichannel pipette and fine pipette tips remove residual EtOH from each well Remove the CLP plate from the magnetic stand and allow the beads to air dry for 5 minutes Using a multichannel pipette add 40 ul of EBT to each sample If you use care to avoid cross contamination changing tips is not required Shake the CLP on a microplate shaker at 1 800 rpm for 5 minutes After shaking if any samples are not resuspended gently pipette o
15. more information see Illumina sequencing white paper Generating Sequencing Libraries Using DNA from FFPE Samples 21 Subtract the average Cq for the OCT from the average Cq for each sample to yield the ACq values for each sample TruSight Tumor 26 Reference Guide 1 3 3d33 W01 P9I9811X3 VNC 40 VOB On IenO Protocol Hybridization of Oligo Pool During this step a custom pool containing upstream and downstream oligos specific to your targeted regions of interest is hybridized to your genomic DNA samples Y WARNING This set of reagents contains formamide an aliphatic amide that is a probable i 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 NOTE Illumina does not support the use of gDNA samples giving a delta Cq value of gt 4 Consumables Optional ACD1 Amplicon Control DNA1 Optional ACP1 Amplicon Control Oligo Pool 1 FPA TruSight Tumor Oligo Pool A FPB TruSight Tumor Oligo Pool B OHS3 Oligo Hybridization for Sequencing 3 Genomic DNA 96 well skirted PCR plate Optional Adhesive aluminum foil seal if a heat sealer is not available Tro
16. the first time aliquot 5 ul of QCT into different PCR tube strips for long term storage to avoid freeze thawing Place thawed tubes on ice Add 5 ul of QCT to 495 ul of nuclease free water in a microcentrifuge tube Vortex the dilution to mix the sample Add 1 ul of QCP to 9 ul of nuclease free water in a microcentrifuge tube L NOTE Make a larger dilution if qualifying more than one genomic DNA sample Vortex the dilution to mix the sample Add 1 5 ul of QIAGEN extracted genomic DNA to 148 5 ul of nuclease free water in microcentrifuge tubes to make a 100 fold dilution Vortex the dilutions to mix the samples TruSight Tumor 26 Reference Guide 1 1 3d33 W01 p919 J1XH VNC JO VOB OL IEnO Protocol 12 7 10 11 12 13 14 15 16 17 18 19 Determine the plate layout of the qPCR reaction For 10 samples use the following layout 1 2 3 4 5 6 A QCT Sample 1 Sample 3 Sample 5 Sample 7 Sample 9 B QCT Sample 1 Sample 3 Sample 5 Sample 7 Sample 9 C OCT Sample 1 Sample 3 Sample 5 Sample 7 Sample 9 D NTC Sample 2 Sample 4 Sample 6 Sample 8 Sample 10 E NTC Sample 2 Sample 4 Sample 6 Sample 8 Sample 10 F NTC Sample 2 Sample 4 Sample 6 Sample 8 Sample 10 NTC No template control Illumina recommends using nuclease free water Prepare the SYBR master mix reaction as follows The master mix contains extra volume Consumable ul per well ul per 48 well ul per 96 well plate plate KAPA SYBR FAST qPCR Master Mix
17. use of its customer in connection with the use of the product s described herein and for no other purpose This document and its contents shall not be used or distributed for any other purpose and or otherwise communicated disclosed or reproduced in any way whatsoever without the prior written consent of Illumina Illumina does not convey any license under its patent trademark copyright or common law rights nor similar rights of any third parties by this document The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order to ensure the proper and safe use of the product s described herein All of the contents of this document must be fully read and understood prior to using such product 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 2015 Illumina Inc All rights reserved Illumina 24sure BaseSpace BeadArray BlueFish BlueFuse BlueGnome cBot CSPro CytoChip DesignStudio Epicentre ForenSeq Genetic Energy GenomeStudio GoldenGate HiScan HiSeq HiSeq X Infinium iScan iSelect MiSeq MiSeqDx MiSeq FGx NeoPrep NextBio Nextera NextSeq Powered by Illum
18. 000848 Part 15042911 v01 Software whether provided separately installed on or embedded in a Product is licensed to Purchaser and not sold Except as expressly stated in this Section no right or license under any of Illumina s intellectual property rights is or are granted expressly by implication or by estoppel Purchaser is solely responsible for determining whether Purchaser has all intellectual property rights that are necessary for Purchaser s intended uses of this Product including without limitation any rights from third parties or rights to Application Specific IP Ilumina makes no guarantee or warranty that purchaser s specific intended uses will not infringe the intellectual property rights of a third party or Application Specific IP 3 Regulatory This Product has not been approved cleared or licensed by the United States Food and Drug Administration or any other regulatory entity whether foreign or domestic for any specific intended use whether research commercial diagnostic or otherwise This Product is labeled For Research Use Only Purchaser must ensure it has any regulatory approvals that are necessary for Purchaser s intended uses of this Product 4 Unauthorized Uses Purchaser agrees a to use each Consumable only one time and b to use only Illumina consumables reagents with Illumina Hard ware The limitations in a b do not apply if the Documentation or Specifications for this Product state otherwise Purchase
19. 1 minute Handling Beads Pipette bead suspension slowly When mixing mix thoroughly To avoid sample loss confirm that no beads remain in pipette tips after resuspension and mixing steps When washing beads Use the appropriate magnet for the plate Dispense liquid so that beads on the side of the wells are wetted Keep the plate on the magnet until the instructions specify to remove it Do not agitate the plate while on the magnetic stand Do not disturb the bead pellet TruSight Tumor 26 Reference Guide 9 senbiuuo2e pue edit Protocol TruSight Tumor 26 Workflow The following diagram illustrates the workflow using the TruSight Tumor 26 Kit Safe stopping points are marked between steps Figure 2 TruSight Tumor 26 Workflow Qualification of DNA Extracted from FFPE Samples Hands on 1 hour Total 3 hours Reagents QCT QCP Hands on 15 minutes Total 14 18 hours overnight Reagents FPA FPB OHS3 e Hybridization of Oligo Pool Removal of Unbound Oligos Hands on 20 minutes Total 20 minutes Reagents ELM3 SW1 UB1 Extension Ligation of Bound Oligos Hands on 5 minutes Total 45 minutes Reagents ELM3 PCR Amplification Hands on 30 minutes Total 1 hour 45 minutes Reagents PMM2 TDP1 15 Index Primers Safe Stopping Point 17 Index Primers NaOH PCR Clean Up Hands on 20 minutes Total 30 minutes Reagents EBT AMPURE XP beads EtOH oo 9 e Library Quantification Library Norm
20. ACGTACCGTAACGAACGTATCAATTGAGCTICTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGT o AET CAATTGAGACT ANAK YA OAS CAT AAGAGT CTGTTAACCTIAAGA TANG HGATCCAGTOATICHACGIACCETAACGAACCIATCAATIGAGA CTAAATATTAACGTACCATTAAGAGCTACCGTGCAACGAAAAGAATGATAACAGT do e TREAT RA U AT TA TT CTTGATCCACTGATTCAACGTIAAGA DAMM UE SUE CCGIAACGAACGTATCAATTGAGCTTCTGTTAACCT TAAGATTACT TGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT T GAGAC TAGCAACGACC GAAAAGAATGATAACAGTAACACAC T TCTGT TAACCT TAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACT TGATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TGAGACTAAATATTAACGTACCATTAAGAGCTAC GATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGT TGATCCACTGAT TCAACGTACCG TATCAAT TGAGAC TAAATAT TAACGTACCAT TAAGAGC TACCGTCTTCTGTTAACC T TAAGAT TACTIGATCCACTGATTCAACGTACCGI CACTGATTCAACGT YQ US ME Er CGTATCAATTGAGACTAAATATTAACGTACCAT TAAGAGCTACCGTCT TC TGT TAACCT TAAGAT TACT T GATCCAC TGAT TCAACGTACCGTAACG GAAAAGAATGATAACAG TAACACACTTCTGTTAACCTTAAGAT TACTT GATCCACTGATTCAACGTACCGTAAAGAT TACT TGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT T GAGAC TAAATAT TAACGTACCAT TAAGAGCTAC A Lee LA Ae CDD ee RAT TACTA AAT TE EST ADEM Ke UM GUEST TET TA etel GATA T VEA UU CATAL L TTGAGACTAAATATTAACGTTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCG TAACGAACG TATCAATTGAGACTAAATATTAACGTACCAT TAAGAGCT TCTGT TAACCT TAAGAT TACTT GATCCACTGAT TCAACGTACCG T7 TATCAAT TGAGACTAAATAT TAACGTAC T TAACCT TAAGAT TACT TGATCCACTGATTCAACGTACCGTAACGAACGTCTTCTGT TAACCTTAAGAT TACT TGATCCACTGATT CAACGTACCGTAACGAACGTAT CAAT TGAGACTAACGACG AM RAT ACTA
21. ACTAAATATTAACGTACCA IIS ce CCGTGCAACGACGAAAAGAAT GATAACAGTAACA REH IACCAT IAAGAGC TACCGTGCAACAGTAACACACT T AAGATTACTTGATCCACTGAT TCAACGTA GE RA e CTAAATATTAACGTACCAT TAAGAGCTACCGTGCAACGACGAAAAGAAT G TGATAACAGTAACACACT TCTGTTAACCTTAAG ATIACTTGATCCAG LTGAPTCANCGTA CCGTAACGAACGTAT CAAT TGAGACT RAATATIAACGTACCATAAGAGCIACCOTCITOT GTIAACCTTAAGA FRAC I GATCCACTGATIGAAG l peg Hed UU ALTA M eMe T GATTARA TNR EE H ENT TAA TT O ULM AACGTTAAGATTACTTGATCCACTGATTCAACG TACCGTAACGAACG TATCAAT TGAGCTTCTGTTAACCT TAAGAT T P TCAAT TGAGACTAGCAACGAC GAAAAGAATGATAACAGTAACACACTTCTGT TAACCT TAAGAT TACTTGAT SGA CT GATT CAACGTACCGTAAAGAT TACT TGATCCACTGAT TCAACGTACCGTAACGAACG TATCAAT TGAGAC TAAATAT TAACG TACCAT TAAGAGCTAC RM TU P ERIS ME L o el EE e Sup RM M AMA pre USE CU C ee SUE poe A Sy Ns RC eerie EEN ACCGTAACGAACGTA AA ee CTAAATAT TAACGTACCATTAAGAGCTACCG TGCAACGACGAAAAGAA NS e e CTTCTGTTAACCT GAAAAGAATGAT AAACACACTTCTGTTAACCT TAAGATTACTTGATCCACTGATTCAACGTA AAAGAT TACTTGATCCACTGAT TCAACGTACCGTAACGAACG TATCAAT TGAG TTAACGTACCAT TAAGAGCTAC COAT TAAGAGCIACOGT GCAACAGTAACACACTICTGT ARCO TAAGATTACT IGAT COAG TOATTOAACG TACCGTARCGAACG AT CAAT TGAGAG AAATAT TAACG AGAT IAAGAGCTACOS I GCAACGAGGAAAAGAAT GATA TGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGT pecs CGTATCAAT TGAGAC TAAATAT TAACGTACCAT TAAGAGCTACCGTCTTCTGTTAACCTTAAGAT TACTT GATCCACTGATTCAAC CTTGATCCACTGATTCAACGTIAAGAT TACTTGATCCACT GATTCAACGTACCGTAACGAACGTATCAAT TGAGCT TCT GT TAACCT TAAGATTACT TGATCCA
22. CAACGACGAAAAGAATGA SRAAAGANTOATAACAGTAACACACHTCTO TAACCLIAAGAT IAG LIGATCCACT GATT CAACTACCGTAMAGATIAG LIGATCCAGTGATICAASGTACCGIAACGAACGTALCAAITCAGAG TAAATAT TAACGTACCAT TAAGAGCTAC MS Vi UT TEC Ce A ATT ACTA AC NAME GATA TAM AC Vee Ca ACCAT IAAGAGC TACCGTGCAACAGTAACACACT TCTGT TAACCTTAAGAT TACT TGATCCACTGAT TCAACGTACCG TAACGAACGTAT CAAT TGAGAC TAAATAT TAACGTACCAT TAAGAGCTACCGT GCAACGACGAAAAGAAT GAT GATAACAGTAACACACT TCTGTTAACCTTAAGAT TACTTGATCCACTGATTCAACGTACCG TAACGAACGTAT CAATTGAGACT AAATATIAACGTACCATIAAGAGCIACCOTCHOTOTT AACCTTAAGA IAC I GATCCACTGATTICAACL O LLL A CR AAA TE ATTRACT Se S IMs CGTTAAGA R RN AAB H EAL CGTAACGAACG TATCAATTGAGCTICTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGAC TAGCAACGACC GAAAAGAATGATAACAG TAACACAC TL TCTGT TAACCT TAAGAT TACTT GA TOGA CT GATT CAACGTACCGTAAAGATTACT TGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGAC TAAATAT TAACG TACCAT TAAGAGCTAC Kee E ee EE GTACCGT E A GATTCAACGTACCGTAACGAACGIA AA CTAAATAT TAACGTACCAT TAAGAGC TA EE e DACIA TRE RT ARE AAAAGAATGAT AACACACTTCTGTTAACCT TAAGAT TACTTGATCCACTGATTCAACGTACCGTAAAGAT TACT TGATCCACTGAT TCAACGTACCG TAACGAACGTATCAATTGAG TTAACGTACCAT TAAGAGCTAC ACA TANGAGCYACGOTACAACAGTAACACACTICTGTTAACUTTARQA TTIACTIGATCCACTGATTCAACGTAGCOTAACCAACG TAT CAATIGAGAGTABATATTAACGTACCAMAAGAGGTACCCTACAACCACGAAAR SANT GATA GATAACAG TAACACA EAS GI T DL ee CTAAATATTAACGTACCATTAAGAGCTACCGTCTICTGTTAACCTTAAGATTACTIGATCCACTGATTCAAC CTTGATCCACTGATTCAACGTIAAGATTACTTGATCCACTGATTCA
23. CTAC 5 E Illumina 5200 Illumina Way San Diego California 92122 U S A 1 800 809 ILMN 4566 1 858 202 4566 outside North America techsupport illumina com www illumina com
24. CTGAT TCAACG TACCG T ARD DSL CUA GA C RAE fu ICAATTGAGACTAAATATTAACGTACCAT TAAGAGTCTGTTAACCT TAAGA HAC TIGATCC AGT GATT CANCE TACCGTARCOAACOTAI CAATIGAQACT AAATATTAACGTACCATTAAGAGC TACCGTGCAACGAAAAGAATGATAACAGT Pee EAT EPA AREE ONORAT ALTO M INR E L ETAT Vy ce QUIS AACGTTAAGATTACTTGATCCACTGATTCAACG TACCGTAACGAACG TATCAAT TGAGCTTCTGTTAACCTTAAGATTACT TGATCCACTGAT TCAACG TACCGTAACGAACG TATCAAT TGAGAC TAGCAACGAC AAAAGAATGATAACAG TAACACACTTCTGT TAACCT T ASA TATI CAA OA TONA E TACCGTAAAGATPACTTGAT COACH GATTORACG TACCQTARCOAAC DATCART GAGACTAAAATTAACO TACOATTAAGAGCIAG CACTGAT ICAACG TACCAAGAT TACTIGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGAC TAAATAT TAACGTACCAI TAAGAGC TACCGT CT TC TGT TAACCT TAAGAT TACT T GATCCAC TGAT TCAACG TACCG TAACG AAAAGAATGATAACAG TAACACACTTCTGTTAACCTTAAGAT TACTTGATCCACTGAT TCAACGTACCGTAAAGATTACT T GATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGAC TAAATAT TAACGTACCAT TAAGAGCTAC GATAACAGTAACAGACT ICTGTTAACCTIAAGAT TACT GATGCACT GAT TCAACGTACCGIAACGAACGIAT CAAT I GAGACTAAATATTAAGGTACCATIAAGAGCTACCGTCITCTGI TAACGT TAAGATTACTTGATGGACTGATICAAC ATTGAGACTAAATAT TAACGTTGTTAACCTTAA SARI ER Out GATTCAACGTACCGTAACGAACGTATCAATT GAGACTAAATAT TAACGTACCAT TAAGAGCTTCTGT TAACCT TAAGAT TA Me i TATCAAT TGAGACTAAATAT TAACGTACT TAACCT TAAGAT TACT TGATCCACTGATTCAACGTACCGTAACGAACG TCTTCTGT TAACCT TAAGAT TACTT GATCCACTGAT TCAACGTACCG TAACGAACG TATCAAT TGAGAC TAACGACG AGACTAAATAT TAACGTACCAT TAAGAGCTACAACCT TAAGAT TACT TGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT
25. Consumables will conform to their Specifications until the later of i 3 months from the date of shipment from Illumina and ii any expiration date or the end of the shelf life pre printed on such Consumable by Illumina but in no event later than 12 months from the date of shipment With respect to custom Consumables i e Consumables made to specifications or designs made by Purchaser or provided to Illumina by or on behalf of Purchaser Illumina only warrants that the custom Consumables will be made and tested in accordance with Illumina s standard manufacturing and quality control processes Illumina makes no warranty that custom Consumables will work as intended by Purchaser or for Purchaser s intended uses b Warranty for Hardware Illumina warrants that Hard ware other than Upgraded Components will conform to its Specifications for a period of 12 months after its shipment date from Illumina unless the Hardware includes Illumina provided installation in which case the warranty period begins on the date of installation or 30 days after the date it was delivered whichever occurs first Base Hardware Warranty Upgraded Components means Illumina provided components modifications or enhancements to Hardware that was previously acquired by Purchaser Illumina warrants that Upgraded Components will conform to their Specifications for a period of 90 days from the date the Upgraded Components are installed Upgraded Components do not extend the
26. Fixture is not needed The indexes can then be added to the appropriate wells of the IAP plate manually Material 20000848 Part 15042911 v01 Procedure 1 Figure 4 TruSeq Index Plate Fixture A i5 primers white caps B i7 primers orange caps C IAP plate Label a new 96 well PCR plate IAP Indexed Amplification Plate and place the plate on the TruSeq Index Plate Fixture Add 9 ul of each Index 1 i7 adapter to each row Add 9 ul of each Index 2 i5 adapter to each column L NOTE i To avoid index cross contamination discard the original index caps and apply the new caps provided in the kit After use remove all index primer tubes from the working area Prepare the PMM2 TDP1 PCR master mix according to the number of reactions For 96 reactions add 60 ul of TDP1 to 2 58 ml of PMM2 If preparing fewer reactions use the following calculation Number of reactions x 0 625 ul TDP 26 875 ul PMM2 4 NOTE Do not pipette volumes of lt 5 ul of TDPI Invert the PMM2 TDP1 PCR master mix 20 times to mix Do not vortex When the 45 minute extension ligation reaction is complete remove the FPU plate from the incubator To ensure the reaction supernatant drains into the waste plate remove the aluminum foil seal and replace with the filter plate lid Centrifuge the FPU plate at 2 400 x g for 2 minutes Using a multichannel pipette add 25 ul of 0 05 N NaOH to each sample well on the FPU plate If necessary gently pipett
27. Purchaser s use of this Product outside of the scope of research use purposes iii any use of this Product not in accordance with this Products Specifications or Documentation or iv any Excluded Claim Conditions to Indemnification Obligations The parties indemnification obligations are conditioned upon the party seeking indemnification i promptly notifying the other party in writing of such claim or action ii giving the other party exclusive control and authority over the defense and settlement of such claim or action iii not admitting infringement of any intellectual property right without prior written Material 20000848 Part 15042911 v01 consent of the other party iv not entering into any settlement or compromise of any such claim or action without the other party s prior written consent and v providing reasonable assistance to the other party in the defense of the claim or action provided that the party reimburses the indemnified party for its reasonable out of pocket expenses incurred in providing such assistance e Third Party Goods and Indemnification Illumina has no indemnification obligations with respect to any goods originating from a third party and supplied to Purchaser Third party goods are those that are labeled or branded with a third party s name Purchaser s indemnification rights if any with respect to third party goods shall be pursuant to the original manufacturer s or licensor s indemnity Upon written
28. Store the additional DAL at 25 C to 15 C for up to 3 days Longer storage can lead to suboptimal cluster densities Centrifuge the DAL tube at 1 000 x g at 20 C for 1 minute to collect contents Incubate the DAL tube in a heat block at 96 C for 2 minutes TruSight Tumor 26 Reference Guide 2 9 Bulijoo y pue Bulinjeuag AISIOn Protocol 30 13 After the incubation invert the DAL tube to mix Incubate immediately in the ice water bath for 5 minutes then transfer contents to the template position in the MiSeq reagent cartridge 4 NOTE The heat denaturation and cooling steps must occur immediately before loading the DAL into the MiSeq reagent cartridge to ensure efficient template loading onto the flow cell 14 Proceed to library sequencing as instructed in the MiSeg System User Guide Material 20000848 Part 15042911 v01 Supporting Information Introduction A nu ede oan elus e du ebd 32 e elek E 33 Trusight Tumor 26 e EE 34 Consumables and Equipment sse RR esses lli 36 Index Sequences E 39 n d AP OPT TAR SS Es m A DN D Ki AN d NV 3 A U gt SA me rico f E E tf Keen gees REA ATGCGGCA sprengen prota TT e itm uis TruSight Tumor 26 Reference Guide 31 v xipueddaw Supporting Information 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
29. TGAGACTAAATAT TAACG TACCAT TAAGAGC TACCGTGCAACGACGAAAAGAAT GATAACAGTAACAC ies El AACACACTTCTGTIAACCTTAAGATTACTTGATCCACTGAT I CAACGTACCG TAACGAACG TATCAAT TGAGAC TAAATAT TAACG TACCAT TAAGAGC TACCG TCT TCTGT TAACCT TAAGAT TACT TGATCCACTGAT TCAAC IACCATTAAGAGCTACCGT GCAACTTAACCTIAAGAT TACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGAC TAAATAT ITAACGTACCAT TAAGAGCTACCGTGCAACGACGAAC LTCTGTTAACCT TAAGAT TACT TGA TO E RM E EC GAAAAG GAAAAGAATGATAACAG TAACACAC T TCTGT TAACCTTAAGAT TACTTGATCCACTGATTCAACGTACCG TAAAGATTACTTGATO TGATAACAGTAACACACT TCTGTTAACCT TAAGAT TACTTGATCCACTGAT TCAACGTACCGTAACGAACG TATCAAT TGAGACTA IACCAT TAAGAGCTACCGTGCAACAGTAACACACT TCTGT TAACCTTAAGAT TACTTGATCCACTGAT TCAACGTACCG TAACGA TGATAACAGTAACACACT TCTGTTAACCTIAAGAT TACTTGATCCACTGATTCAACGTA SE RR pe ZGTACCGTAACGAACGTATCAT TAAGAT TACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGACTAAA t TOCATA CAACGITAAGALTACTTGA CCACTGATICAACGTACCGIAACGAACGTATCAATTGAGCTTC TOT TAAG GAAAAGAATGATAACAGTAACACACT TCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGAT TACTTGATG O Si For Research Use Only Not for use in diagnostic procedures ILLUMINA PROPRIETARY Material 4 20000848 Document 15042911 vO1 September 2015 Customize a short end to end workflow guide with the Custom Protocol Selector support illumina com custom protocol selector html This document and its contents are proprietary to Illumina Inc and its affiliates Illumina and are intended solely for the contractual
30. alization Hands on 30 minutes Total 50 minutes Reagents EBT Safe Stopping Point Library Denaturing and Pooling Hands on 10 minutes Total 10 minutes Reagents HT1 PhiX NaOH TE Buffer Pre Amp Post Amp 1 O Material 20000848 Part 15042911 v01 Qualification of DNA Extracted from FFPE Samples During this step a qPCR reaction determines the amplifiability of your FFPE extracted gDNA samples By comparing the amplifiability of FFPE DNA relative to that of the OCT non FFPE reference gDNA a ACq value is calculated for each sample The ACq value is then used to predict sample performance in the TruSight Tumor 26 assay The exact amount of FFPE DNA input varies according to the quality of the extracted DNA Consumables Preparation 1 Procedure OCT Quality Control Template QCP Quality Control Primer Genomic DNA 48 or 96 well plate Adhesive seal dependent on qPCR machine KAPA SYBR FAST qPCR Master Mix 2X Universal Nuclease free water PCR 8 tube strips if using OCT for the first time Prepare the following consumables Item Storage Instructions OCT 25 C to 15 C Thaw at room temperature for up to 30 minutes OCP 25 C to 15 C Thaw at room temperature for up to 30 minutes Genomic DNA 25 C to 15 C Thaw at room temperature for up to 30 minutes KAPA SYBR FAST 25 C to 15 C Thaw at room temperature for up to 30 minutes qPCR Master Mix 2X Universal If using QCT for
31. ate TruSight Tumor 26 Index Kit Box 1 Pre Amplification Store at 25 C to 15 C Quantity Description 15 Index Primers A501 to A508 12 i7 Index Primers A701 to A712 Box 2 Pre Amplification Store at Room Temperature Quantity Description 32 15 Index Tube Caps White 48 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 TruSight Tumor 26 Reference Guide 3 5 1U91UO2 YM 93 Joun 1yBisna Supporting Information 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 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 DNA molecular weight markers Eppendorf microcentrifuge tubes screw top recommended Ethanol 200 proof for molecular biology
32. d indemnify or hold harmless Purchaser for any Illumina Infringement Claim to the extent such infringement arises from i the use of this Product in any manner or for any purpose outside the scope of research use purposes ii the use of this Product in any manner not in accordance with its Specifications its Documentation the rights expressly granted to Purchaser hereunder or any breach by Purchaser of these terms and conditions iii the use of this Product in combination with any other products materials or services not supplied by Illumina iv the use of this Product to perform any assay or other process not supplied by Illumina or v Illumina s compliance with specifications or instructions for this Product furnished by or on behalf of Purchaser each of i v is referred to as an Excluded Claim Indemnification by Purchaser Purchaser shall defend indemnify and hold harmless Illumina its affiliates their non affiliate collaborators and development partners that contributed to the development of this Product and their respective officers directors representatives and employees against any claims liabilities damages fines penalties causes of action and losses of any and every kind including without limitation personal injury or death claims and infringement of a third party s intellectual property rights resulting from relating to or arising out of i Purchaser s breach of any of these terms and conditions ii
33. e UB1 2 C to 8 C Set aside at room temperature Material 20000848 Part 15042911 v01 2 Assemble the filter plate assembly unit FPU in the following order from top to bottom Figure 3 Filter Plate Unit Assembly Lid Filter plate Adapter collar midi plate UO0U gt 3 Apply the FPU barcode plate sticker to the filter plate 4 Prewash the FPU plate membrane as follows 2 NOTE i Prewash only the wells to be used in the current assay If using a previously opened filter plate use only unused wells Do not reuse wells that have been used in a previous assay a Using a multichannel pipette add 50 ul of SW1 to each well b Cover the FPU plate with the filter plate lid c Centrifuge the FPU at 2 400 x g at 20 C for 5 minutes 5 Preheat the incubator to 37 C 6 After the overnight incubation confirm that the heat block has cooled to 40 C E NOTE e If the heat block fails to cool to 40 C overnight repeat library preparation TruSight Tumor 26 Reference Guide 1 T SOBIIO punoqun now y Protocol Procedure 18 L NOTE E Cover the FPU plate with the filter plate lid during each centrifugation step Remove the HYP plate from the heat block and centrifuge at 1 000 x g at 20 C for 1 minute to collect condensation Using a multichannel pipette set to 60 ul transfer the entire volume of each sample to the corresponding prewashed wells of the FPU plate Centrifuge the FPU at 2 400 x g at 20 C for 5 minutes
34. e a largely intact DNA template that can be denatured The process of preparing FFPE samples negatively impacts DNA quality by fragmenting cross linking and otherwise damaging DNA through various chemical modifications As a result it is essential to assess the extent of the damage and where possible improve the procedures for fixation of tissue extraction of DNA from FFPE This adjustment can partially compensate for damage and improve results from the TruSight Tumor 26 assay with FFPE DNA DNA Extraction Recommendations Illumina recommends using the QIAGEN Supplementary Protocol Purification of Genomic DNA from FFPE Tissue using the QlAamp DNA FFPE Tissue Kit and Deparaffinization Solution This protocol extracts the highest amount of amplifiable DNA from an FFPE tissue block with the following modifications Extract gDNA from 8 separate 5 um FFPE tissue sections per extraction Deparaffinize with 320 ul of QIAGEN Deparaffinization Solution Lyse samples with 40 ul of Proteinase K in a thermal mixer at 56 C overnight at 1000 rpm to improve genomic DNA yields Digest with Proteinase K in a thermal mixer overnight at 1000 rpm Decrease elution volume to 30 ul to maximize DNA concentration A Material 20000848 Part 15042911 v01 Additional Resources The following documentation is available for download from the Illumina website Resource Description TruSight Tumor 26 Protocol Provides only protocol instructions Guide document
35. e on the thermal cycler overnight 2 p Material 20000848 Part 15042911 v01 Verify Library Preparation Optional 1 After PCR combine 5 ul of amplified product with 15 ul of DEPC DI H20 2 Run on a 4 TBE agarose gel along with 100 bp ladder to confirm the presence of the 300 330 bp library product Alternatively the products can be run on a Bioanalyzer If generating libraries with ACPI expect the product to be present at 350 380 bp TruSight Tumor 26 Reference Guide y 3 euonido uoneledalg Meqi UA Protocol PCR Clean Up This process uses AMPure XP beads to purify the PCR products from the other reaction components Consumables Preparation e oO H BB Procedure 24 EBT Elution Buffer with Tris AMPure XP beads 96 well midi plates Freshly prepared 80 ethanol EtOH Microseal B adhesive film Troughs Bring the AMPure XP beads to room temperature Prepare fresh 80 ethanol from absolute ethanol Label a new midi plate CLP_Plate_ID Clean up Plate Label a new 96 well PCR plate SGP Storage Plate Centrifuge the IAP plate at 1 000 x g at 20 C for 1 minute to collect condensation Invert AMPure XP beads 10 times Vortex vigorously and then invert again 10 times A NOTE Immediately proceed to the next step to avoid settling of the beads Using a multichannel pipette add 55 ul of AMPure XP beads to each well of the CLP plate A NOTE The ACD1 ACP1 control can be processed using the same
36. e to mix TruSight Tumor 26 Reference Guide 2 1 uoneoriduwy HOd Protocol 4 Incubate the FPU plate at room temperature for 5 minutes 5 During incubation use a multichannel pipette to transfer 22 ul of the PMM2 TDP1 PCR master mix to each well of the IAP plate containing index primers 6 Transfer samples eluted from the FPU plate to the IAP plate as follows NOTE g Slightly tilt the FPU plate to ensure complete aspiration and to avoid air bubbles a Seta multichannel P20 pipette to 20 ul and pipette to mix the contents in the first column of the FPU plate b Transfer 20 ul from the FPU plate to the corresponding column of the IAP plate c Gently pipette to mix d Transfer the remaining columns from the FPU plate to the IAP plate in a similar manner e After all the samples have been transferred discard the waste collection midi plate of the FPU Store the metal adapter collar 7 Centrifuge the IAP plate at 1 000 x g at room temperature for 1 minute 8 Transfer the IAP plate to the post amplification area 9 On the thermal cycler set the reaction volume to 60 ul and the temperature ramp speed to maximum 10 Perform PCR on a thermal cycler using the following program 95 C for 3 minutes 27 cycles of 95 C for 30 seconds 62 C for 30 seconds 72 C for 60 seconds 72 C for 5 minutes Hold at 10 C SAFE STOPPING POINT If you are stopping seal the plate and store at 2 C to 8 C for up to 2 days Alternatively leav
37. een each sample When adding adapters or primers change tips between each row and each column Remove unused index adapter tubes from the working area Sealing the Plate Always seal the 96 well plate before the following steps in the protocol Shaking steps Vortexing steps Centrifuge steps Thermal cycling steps Apply the adhesive seal to cover the plate and seal with a rubber roller Microseal B adhesive seals are effective at 40 C to 110 C and suitable for skirted or semiskirted PCR plates Use Microseal B for shaking centrifuging and long term storage Foil seals are effective at 70 C to 105 C and suitable for skirted or semiskirted plates Microseal A adhesive film is effective for thermal cycling and easy to cut when using fewer than 96 wells Plate Transfers When transferring volumes between plates transfer the specified volume from each well of a plate to the corresponding well of the other plate If beads are aspirated into the pipette tips dispense back to the plate on the magnetic stand and wait until the liquid is clear 2 minutes When multiple plates are used in a step such as a plate 1 and a plate 2 transfer volumes from the existing plate 1 to the new plate 1 Transfer volumes from the existing plate 2 to the new plate 2 Centrifugation Centrifuge at any step in the procedure to consolidate liquid or beads in the bottom of the well and to prevent sample loss To pellet beads centrifuge at 280 x g for
38. g identified regions of interest Sufficient reagents are supplied for 48 samples and the indexes provided enable sample indexing of 4 samples per sequencing run TruSight Tumor 26 harnesses the paired end read capability speed and high data quality of the MiSeq System providing on instrument variant calling software and cloud based annotation and filtering software The TruSight Tumor 26 protocol offers High Accuracy Low Frequency Variant Detection Highly accurate somatic variant analysis at limit of detection below 5 allele frequency across 174 amplicons with 1000x minimum coverage of each region Optimized for formalin fixed paraffin embedded FFPE tissues Optimized for Formalin Fixed Paraffin Embedded FFPE Tissues Exceptional sample success rate with minimal DNA input for accurate base calling even in degraded FFPE samples Deep Coverage of Variants Involved with Solid Tumors Coverage of exon coding regions for analysis of molecular heterogeneity in highly relevant content selected from CAP and NCCN guidelines and late stage clinical trials 2 Material 20000848 Part 15042911 v01 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 pr
39. ge Gel electrophoresis supplies and apparatus Bioanalyzer System Heat block for 1 5 ml centrifuge tubes Magnetic stand 96 1 NOTE Supplier O Instruments BioShake iO high speed thermoshaker part 1808 0506 or O Instruments BioShake XP high speed lab shaker part 1808 0505 General lab supplier plate centrifuge that attains designated speeds of protocol General lab supplier Agilent Technologies General lab supplier Invitrogen DynaMag 96 Side Skirted Use a dedicated set of pipettes pipette tips vortexer heat block and centrifuge during post amplification steps TruSight Tumor 26 Reference Guide 3 jueuudinbz3 pue sejqewinsuoy Supporting Information 38 Thermal Cyclers The following table lists the recommended settings for the Illumina recommended thermal cycler as well as 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 Temp Mode Lid Temp Vessel Type Bio Rad DNA Engine Calculated Heated Constant Polypropylene plates Tetrad 2 at 100 C and tubes MJ Research DNA Calculated Heated Plate Engine Tetrad no longer available for purchase Eppendorf Gradient S Heated Plate Mastercycler Pro S Simulated Tube y 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 bef
40. ible fields of use the Core IP Application Specific IP and Core IP are separate non overlapping subsets of all IIlumina owned or controlled intellectual property By way of non limiting example Illumina intellectual property rights for specific diagnostic methods for specific forensic methods or for specific nucleic acid biomarkers sequences or combinations of biomarkers or sequences are examples of Application Specific IP Consumable s means Illumina branded reagents and consumable items that are intended by Illumina for use with and are to be consumed through the use of Hardware Documentation means Illumina s user manual for this Product including without limitation package inserts and any other documentation that accompany this Product or that are referenced by the Product or in the packaging for the Product in effect on the date of shipment from Illumina Documentation includes this document Hardware means Illumina branded instruments accessories or peripherals Illumina means Illumina Inc or an Illumina affiliate as applicable Product means the product that this document accompanies e g Hardware Consumables or Software Purchaser is the person or entity that rightfully and legally acquires this Product from Illumina or an Illumina authorized dealer Software means Illumina branded software e g Hardware operating software data analysis software All Software is licensed and not sold and may be subject to addi
41. illumina Trusight Tumor 26 Heference Guide 1a NV LYS CST A A AIT CAT PAGO R TAG eda cata AN TB OCC TORT e TATGAATTOAQITRARDAT DAC GTACCATTAAGAGCTAC TGATAACAG IAACACACT TCTGTTAACCT TAAGAT TACTTGT TGATCCACTGATTCAACGTACCG TATCAAT TGAGAC TAAATAT TAACG TACCAT TAAGAGC TACCGTCTTCTGTTAACCTTAAGAT TACTTGATCCACTGATTCAACGTACCG CAC T S T T A TT DEE GAAAAGAATGATAAC AG TAACACACTTCTGTTAACCT T LDL ACCG Eer Ae ACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTAC TGATAACAGTAACACACT TCTGTTAACCTTAAGAT T ACT GATCCACTGATTCAACGTACCGTAACGAACG TATCAAT TGAGAC TAAATATTAACGTACCAT TAAGAGCTACCGTCTTCTGT TAACCT TAA CATH ACT TGATCCACTGATTCAAC IFIGAGACTAAATAT AGG a TAACCT IAAGAT TACT GATCCACTGAI IGAAGG TACCGTAACGAACG IAT CAAT IGAGACTAAATATIAACGTACCAT IAAGAGC LT T L a E a S El ee O BOOT Ne Ge LY EEN M G re CCGTAACGAACGTCTICTGTIAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAA O EE e T Der CTAAATA CGTACCAT TAAGAGCTACAACCT T TTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACG TACCAT TAAGAGC TACCGTGCAACGACGAAAAGAA GTAACAC IGATAACAGTAACAGAGT e TI GATOCACTGA TOMOS TACOGTAAQGARCO E E er 2 e eer GATTACTTGATCCACT ATTE AE UAE RAGA ACTA OAA H AAAT AT AER ACCATTAAGAGCTACCGTGCAACGACGAACTTCTGTTAACCT TAAGAT TACT T ion GCTACCGTGCAACGAAAATAA AAGATTACTTGATCCACTGATICAACG AACCTTAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAATTGAGAC TAAGC TACCGTGCAACGACGAAAAGAA GARAAGAATGATARCAGTAACACACTICIGT IAAGC LTAAGAT TACT TGATCGAG GAT EE RAG ARRON CTICTGTTAACCTT EE Eh ACCGTAACGAACGTATCAATTGAG
42. ilute so that the volume of the final 4 nM working stock is at least 20 ul SAFE STOPPING POINT If you are stopping seal the plate and store at 25 C to 15 C for up to 7 days TruSight Tumor 26 Reference Guide 2 T uonezi euJoN Aeq Protocol Library Denaturing and Pooling Preparation In preparation for cluster generation and sequencing equal volumes of normalized library are combined diluted in Hybridization Buffer and heat denatured before sequencing on the MiSeq System PhiX is used as an internal control for sequencing Consumables 1 EBT Elution Buffer with Tris HT1 Hybridization Buffer 2 ul 10 nM PhiX Library Laboratory grade water 2 5 L ice bucket Microcentrifuge tubes screw cap recommended PCR 8 tube strip Stock 1 0 N NaOH diluted to 0 1 N NaOH Prepare the following consumables Item Storage Instructions HT1 25 C to 15 C Thaw at room temperature and then place in an ice bath to chill If the LNP plate was stored frozen thaw at room temperature Set a heat block with a microcentrifuge tube insert to 96 C To prepare a fresh dilution of 0 1 N NaOH add 100 ul stock 1 N NaOH toa microcentrifuge tube containing 900 ul laboratory grade water d NOTE Using freshly diluted NaOH is essential to denature samples for cluster generation on the MiSeq Preparing a volume of 1 ml prevents small pipetting errors from affecting the final NaOH concentration Invert the tube several times to mix
43. imer 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 sequenced on the MiSeq System Figure 1 How the TruSight Tumor 26 Assay Works Custom Custom Probe 1 Region of interest Probe 2 4440M Aessy y seo MOH Custom Custom Probe 1 Probe 2 P7 X Index 1 Index 2 P5 B P7 Index 1 Index2 P5 Hybridization of custom oligonucleotide probes Extension and ligation Addition of indexes and sequencing adapters by PCR Final amplicon ready for sequencing on the MiSeq System G 000 E TruSight Tumor 26 Reference Guide 3 Overview DNA Input Recommendations Formalin fixed paraffin embedded FFPE human tissues are a valuable source of material for molecular analysis and clinical studies Several processes and protocols now exist for the extraction and purification of nucleic acids from FFPE samples The assays used to evaluate DNA and RNA have evolved from simple monoplex PCR to higher plexity products As a result the quality and amount of nucleic acid extracted from FFPE material becomes more critical to the success of these assays The TruSight Tumor 26 assay can be used to generate sequencing libraries that are highly multiplexed at both the target and sample level The high level of assay complexity is enabled by combining an oligo extension ligation process with universal PCR Both of these reactions requir
44. ina SureMDA TruGenome TruSeq TruSight Understand Your Genome UYG VeraCode verifi VeriSeq the pumpkin orange color and the streaming bases design are trademarks of Illumina Inc and or its affiliate s in the U S and or other countries All other names logos and other trademarks are the property of their respective owners Patent pending for methods performed by components in this kit For Research Use Only not for any clinical or therapeutic use in humans or animals a This product includes GoTaq Hot Start Polymerase manufactured by Promega A Corporation for distribution by Illumina Inc Licensed to Promega Corporation under U S Patent Nos 5 338 671 and 5 587 287 and their corresponding foreign patents Promega Read Before Using this Product This Product and its use and disposition is subject to the following terms and conditions If Purchaser does not agree to these terms and conditions then Purchaser is not authorized by Illumina to use this Product and Purchaser must not use this Product 1 Definitions Application Specific IP means Illumina owned or controlled intellectual property rights that pertain to this Product and use thereof only with regard to specific field s or specific application s Application Specific IP excludes all Illumina owned or controlled intellectual property that cover aspects or features of this Product or use thereof that are common to this Product in all possible applications and all poss
45. ns Section 9 d below Illumina shall i defend indemnify and hold harmless Purchaser against any third party claim or action alleging that this Product when used for research use purposes in accordance with these terms and conditions and in accordance with this Product s Documentation and Specifications infringes the valid and enforceable intellectual property rights of a third party and ii pay all settlements entered into and all final judgments and costs including reasonable attorneys fees awarded against Purchaser in connection with such infringement claim If this Product or any part thereof becomes or in Illumina s opinion may become the subject of an infringement claim Illumina shall have the right at its option to A procure for Purchaser the right to continue using this Product B modify or replace this Product with a substantially equivalent non infringing substitute or C require the return of this Product and terminate the rights license and any other permissions provided to Purchaser with respect this Product and refund to Purchaser the depreciated value as shown in Purchaser s official records of the returned Product at the time of such return provided that no refund will be given for used up or expired Consumables This Section states the entire liability of Ilumina for any infringement of third party intellectual property rights Exclusions to Illumina Indemnification Obligations Illumina has no obligation to defen
46. or Oligo Pools FPA and FPB The FPA and FPB libraries for each sample must be run together on the same flow cell for the MiSeq software to analyze the results for each sample L NOTE Control libraries generated from ACD1 with ACP1 must be pooled and run separately from those libraries prepared with FPA and FPB as they require a longer MiSeq run of 151 cycles Add 10 ul of IN NaOH to 140 ul EBT buffer Vortex the solution Transfer 5 ul of each 4 nM library to be sequenced from the LNP plate to its own tube in a PCR 8 tube strip E NOTE After use the sealed LNP plate can be stored at 25 C to 15 C for up to 7 days Add 15 ul of the NaOH EBT solution to each 5 ul of library and incubate for 5 minutes at room temperature Label 1 microcentrifuge tube PAL Pooled Amplicon Library Add 10 ul of each of the 8 library NaOH EBT solutions into the PAL tube Pipette to mix Make sure that the pooled libraries are mixed well Label 1 microcentrifuge tube DAL Diluted Amplicon Library In the DAL tube mix 792 ul of HT1 with 8 ul of 20 pM PhiX library Using the same tip pipette up and down 3 5 times to rinse the tip and ensure complete transfer Add 8 ul from the PAL tube to the DAL tube containing HT1 and PhiX Using the same tip pipette up and down 3 5 times to rinse the tip and ensure complete transfer Vortex the DAL tube at top speed L NOTE You can make and save additional DAL from the remaining 72 ul of unused PAL
47. ore use Material 20000848 Part 15042911 v01 Index Sequences Use the following sequences for entry on your MiSeq system sample sheet 17 Index PCR Primer Index Sequence A701 ATCACGAC A702 ACAGTGGT A703 CAGATCCA A704 ACAAACGG A705 ACCCAGCA A706 AACCCCTC A707 CCCAACCT A708 CACCACAC A709 GAAACCCA A710 TGTGACCA A711 AGGGTCAA A712 AGGAGTGG A501 TGAACCTT A502 TGCTAAGT A503 TGTTCTCT A504 TAAGACAC A505 CTAATCGA A506 CTAGAACA A507 TAAGTICC A508 TAGACCTA TruSight Tumor 26 Reference Guide 39 seouenbes xepu 40 Material 20000848 Part 15042911 v01 Technical Assistance For technical assistance contact Illumina Technical Support Table 1 Illumina General Contact Information Website www illumina com Email techsupport illumina com Table2 Ilumina Customer Support Telephone Numbers Region Contact Number Region Contact Number North America 1 800 809 4566 Italy 800 874909 Australia 1 800 775 688 Netherlands 0800 0223859 Austria 0800 296575 New Zealand 0800 451 650 Belgium 0800 81102 Norway 800 16836 Denmark 80882346 Spain 900 812168 Finland 0800 918363 Sweden 020790181 France 0800 911850 Switzerland 0800 563118 Germany 0800 180 8994 United Kingdom 0800 917 0041 Ireland 1 800 812949 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 to supp
48. ortillumina com select a product then select Documentation amp Literature TruSight Tumor 26 Reference Guide 9UB SISSY JEOIUYDS E EE a E Ee GATAACAGTAACACACT TC TGTTAACCT T TIGTTGATCCACTGATTCAACGTACCG TATCAAT TGAGAC TAAATAT IAACGTACCAT TAAGAGCTACCGTCTTCTGTTAACCT TAAGAT TACTI GATCCAC TGATTCAACGTACCGI GACT GATT GAACGACCAAGAT TACT IGATGCACT GAT I CAAGG TACUGTAACGAACGTAT CAAT GAGAC EE GTTAACCTTAAGAT TACTTGATCCACTGATTCAACGTACCGTAACGS GAATGATAACAGTAACACACTTCTGTTAACCTTAAGAT TACTTGATCCACTGAT TCAACGTACCGTAAAGATTAC T TGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGAC T DEREN ACH BURA OPC AG Ts e GATAACAGTAACACACT TCTGTTAACCTTAAGAT TACTTGATCCACTGAT TCAACGTACCGTAACGAACG TATCAAT TGAGAC TAAATATTAACG TACCAT TAAGAGCTACCGTCT TC TGT TAACCT TAAGATTACT T CTGATT ee EE as EE El ee Kee E TAE LASTI EO ET SAU RA LO TRA UAR NARRA IET CTGTTAACCTT AGAT BL ATA TTC ATE eS EH Roe L TT eU AACGACG CTAAATA CATTAAGAGCTAC TTACTTGATCCACTGATTCAACGTACCGTAACGAACQG TATCAAT TGAGACTAAATAT TAACG TACCAT TAAGAGCTACCGTGCAAC GAAAAG AACAGTAACAC GATAACAGTAACAGACTTCYGL IAACG AGA TACT GATOCACTOA GAACGIAGOG TRAO GANGO IAI CAAT I GAGAG AAATAT TAACG TAGGAHARGAGG GGG TON TOTT TACT TANGATTACT TGATCUACT GAT ICAA ACCA MSS TAC CAS vss AACCTTAAGATTACTTGATCCACT IVRE RAS T AACGAACGTATCAAT TGAGACTAAATATTAACG TACCAT TAAGAGCTACCGTGCAACGACGAACT TCTGT T A AAGAT TACTTGA GCTACCGTGCAACGAAAATAACC lTAAGATTACT TGATCCACTGAT TCAACGTACTTCTGTTAACCTTAAGATTACTTGATCCACTGAT TCAACGTACCG TAACGAACGTATCAATTGAGAC TAAGCTACCGTG
49. r agrees not to nor authorize any third party to engage in any of the following activities i disassemble reverse engineer reverse compile or reverse assemble the Product ii separate extract or isolate components of this Product or subject this Product or components thereof to any analysis not expressly authorized in this Product s Documentation iii gain access to or attempt to determine the methods of operation of this Product or iv transfer to a third party or grant a sublicense to any Software or any third party software Purchaser further agrees that the contents of and methods of operation of this Product are proprietary to Illumina and this Product contains or embodies trade secrets of Illumina The conditions and restrictions found in these terms and conditions are bargained for conditions of sale and therefore control the sale of and use of this Product by Purchaser 5 Limited Liability TO THE EXTENT PERMITTED BY LAW IN NO EVENT SHALL ILLUMINA OR ITS SUPPLIERS BE LIABLE TO PURCHASER OR ANY THIRD PARTY FOR COSTS OF PROCUREMENT OF SUBSTITUTE PRODUCTS OR SERVICES LOST PROFITS DATA OR BUSINESS OR FOR ANY INDIRECT SPECIAL INCIDENTAL EXEMPLARY CONSEQUENTIAL OR PUNITIVE DAMAGES OF ANY KIND ARISING OUT OF OR IN CONNECTION WITH WITHOUT LIMITATION THE SALE OF THIS PRODUCT ITS USE ILLUMINA S PERFORMANCE HEREUNDER OR ANY OF THESE TERMS AND CONDITIONS HOWEVER ARISING OR CAUSED AND ON ANY THEORY OF LIABILITY WHETHER IN CONTRACT
50. r tap on the bench to mix and then repeat this step Incubate at room temperature without shaking for 2 minutes Place the plate on the magnetic stand for 2 minutes Transfer 40 ul of the supernatant from the CLP plate to the SGP plate Centrifuge the SGP at 1 000 x g for 1 minute NOTE Store the SGP plate at room temperature during the library quantification and normalization steps After library normalization store the SGP plate at 25 C to 15 C until ready to quantify and normalize any remaining samples TruSight Tumor 26 Reference Guide 2 5 dn ueal9 YOd Protocol Library Quantification To achieve the highest quality of data on the Illumina MiSeq sequencing platform it is important to create optimum cluster densities This step requires accurate quantification of DNA libraries Illumina recommends quantifying libraries generated from FFPE samples using the Agilent Technologies Bioanalyzer 2100 Procedure 20 1 Load 1 ul of the resuspended library on an Agilent Technologies 2100 Bioanalyzer using the Agilent DNA 1000 Refer to the Agilent DNA Kit Guide Part Number G2938 90014 and Agilent DNA 1000 Kit Quick Start Guide Part Number G2938 90015 for complete instructions on using the Agilent Technologies 2100 Bioanalyzer Check the size and purity of the sample The expected final product is a band at 300 330 bp as in Figure 5 If generating libraries using ACP1 the expected final product is present at 350 380
51. repare the IAP Indexed Amplification Plate as described in the following section TruSight Tumor 26 Reference Guide 1 9 soBIJO punog jo uoneB6r 1 uoisue1Xx3 Protocol PCR Amplification In this step the extension ligation products are amplified using primers These primers add index sequences for sample multiplexing i5 and i7 as well as common adapters required for cluster generation P5 and P7 Consumables PMM2 PCR Master Mix 2 i5 primers A501 A508 i7 primers A701 A712 TDP1 TruSeq DNA Polymerase 1 0 05 N NaOH freshly prepared from 10 N NaOH 96 well skirted PCR plate Microseal B adhesive film Troughs Preparation 20 1 Prepare the following consumables Item Storage Instructions PMM2 25 C to 15 C Thaw at room temperature Vortex to mix and then briefly centrifuge Thaw at room temperature Vortex each tube to Index primers 25 C to 15 C mix and then briefly centrifuge i5 and i7 To prepare fresh 0 05 N NaOH add 20 ul of 10 N NaOH to 3 98 ml of sterile water Arrange the index primers in the TruSeq Index Plate Fixture as follows Index 1 i7 adapters A701 A712 in columns 1 12 Index 2 i5 adapters A501 A508 in rows A H Collect all liquid in the bottoms of the tubes by holding them in place in the rack and tapping it against the bench d NOTE If fewer than 48 samples 96 reactions are being prepared or alternate index combinations are being employed the Index Plate
52. request Illumina will attempt to pass through such indemnity if any to Purchaser TruSight Tumor 26 Reference Guide V Revision History Document Date Material 20000848 September Document 15042911 2015 v01 Part 15042911 Rev A May 2013 TruSight Tumor 26 Reference Guide Description of Change Updated to new library prep style Rebranded to TruSight Tumor 26 Corrected Consumables list in Library Denaturing and Pooling section Removed 1X TE and replaced with EBT to match protocol Corrected Box 2 and Box 3 in TruSight Tumor 26 Kit Updated Consumables list Added Deparaffinization Solution OLAamp DNA FFPE Tissue Kit and KAPA SYBR FAST qPCR Master Mix 2X Universal Removed 100x TE Buffer Corrected PhiX control kit name to PhiX Control v3 and corrected the catalog number Initial release Material 20000848 Part 15042911 v01 Table of Contents Revision History Lo ooooocccccccccccocccccccccccccccccncccncnccnnc cnn rrr rrr vi Table of Contents 200000 cee cece cece cece cece cece cece ce cece noraa narenn viii Chapter 1 Overview 1 Introduction 2 How Does the Assay Work 3 DNA Input Recommendations 22 22 e cece eee ccceee cece cecccceeee 4 Additional Resources 5 Es An 7 INrOdUCHON 8 Tips and Techniques aaea aaaea araar naonana 9 TruSight Tumor 26 Workflow 10 Qualification of DNA Extracted from FFPE Samples
53. t to 40 C can adversely affect hybridization TruSight Tumor 26 Reference Guide 1 5 004 OBIJO jo uonezipugAH Protocol Remove Unbound Oligos This process removes unbound oligos from genomic DNA using a filter capable of size selection Two wash steps using SW1 ensure complete removal of unbound oligos A third wash step using UB1 removes residual SW1 and prepares samples for the extension ligation step Consumables ELM4 Extension Ligation Mix 4 SW1 Stringent Wash 1 UB1 Universal Buffer 1 Filter plate with lid keep spare filter plates as general lab supplies Adapter collar reusable Midi plate Troughs Y y 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 Y WARNING This set of reagents contains mercaptoethanol Perform the following procedure in a hood or well ventilated area Preparation 16 1 Prepare the following consumables Item Storage Instructions ELM3 25 C to 15 C Thaw at room temperature for 20 minutes sw1 2 C to 8 C Set aside at room temperatur
54. that it confirms is covered by this warranty Repaired or replaced Consumables come with a 30 day warranty Hardware may be repaired or replaced with functionally equivalent reconditioned or new Hardware or components if only a component of Hardware is non conforming If the Hardware is replaced in its entirety the warranty period for the replacement is 90 days from the date of shipment or the remaining period on the original Hardware warranty whichever is shorter If only a component is being repaired or replaced the warranty period for such component is 90 days from the date of shipment or the remaining period on the original Hardware warranty whichever ends later The preceding states Purchaser s sole remedy and Illumina s sole obligations under the warranty provided hereunder Third Party Goods and Warranty Illumina has no warranty obligations with respect to any goods originating from a third party and supplied to Purchaser hereunder Third party goods are those that are labeled or branded with a third party s name The warranty for third party goods if any is provided by the original manufacturer Upon written request Illumina will attempt to pass through any such warranty to Purchaser Indemnification a Infringement Indemnification by Illumina Subject to these terms and conditions including without limitation the Exclusions to Illumina s Indemnification Obligations Section 9 b below the Conditions to Indemnification Obligatio
55. tional terms found in the Software s end user license agreement Specifications means Illumina s written specifications for this Product in effect on the date that the Product ships from Illumina 2 Research Use Only Rights Subject to these terms and conditions and unless otherwise agreed upon in writing by an officer of Illumina Purchaser is granted only a non exclusive non transferable personal non sublicensable right under Illumina s Core IP in existence on the date that this Product ships from Illumina solely to use this Product in Purchaser s facility for Purchaser s internal research purposes which includes research services provided to third parties and solely in accordance with this Product s Documentation but specifically excluding any use that a would require rights or a license from Illumina to Application Specific IP b is a re use of a previously used Consumable c is the disassembling reverse engineering reverse compiling or reverse assembling of this Product d is the separation extraction or isolation of components of this Product or other unauthorized analysis of this Product e gains access to or determines the methods of operation of this Product f is the use of non Illumina reagent consumables with Illumina s Hard ware does not apply if the Specifications or Documentation state otherwise or g is the transfer to a third party of or sub licensing of Software or any third party software AII Material 20
56. ughs About Reagents Using ACPD1 and ACP1 enables Illumina Technical Support to troubleshoot in the event you need assistance Illumina technical support recommends including control samples in your assay periodically to establish baselines and monitor overall performance If you are using controls use ACDI instead of gDNA and ACP1 instead of FPA and FPB ACP1 is specific for Homo sapiens and does not work with DNA from other species Preparation 14 1 Prepare the following consumables Item Storage Instructions Optional ACD1 25 C to 15 C Thaw at room temperature for up to 30 minutes Optional ACP1 25 C to 15 C Thaw at room temperature for up to 30 minutes FPA 25 C to 15 C Thaw at room temperature for up to 30 minutes FPB 25 C to 15 C Thaw at room temperature for up to 30 minutes OHS3 25 C to 15 C Thaw at room temperature If precipitate is observed incubate at 37 C for 10 minutes and vortex for 1 minute Repeat as needed until precipitate is no longer visible Material 20000848 Part 15042911 v01 Procedure 1 oN O OF Item Storage Instructions Genomic DNA 25 C to 15 C Thaw at room temperature for up to 30 minutes Place thawed tubes on ice Set a 96 well heat block to 95 C Label a new 96 well PCR plate HYP_Plate_ID Dilute 10 ul of genomic DNA extracted from FFPE samples Use the following table to determine the fold dilution required for each calculated delta Ca
57. warranty for the Hardware unless the upgrade was TruSight Tumor 26 Reference Guide 9 conducted by Illumina at Illumina s facilities in which case the upgraded Hardware shipped to Purchaser comes with a Base Hardware Warranty Exclusions from Warranty Coverage The foregoing warranties do not apply to the extent a non conformance is due to i abuse misuse neglect negligence accident improper storage or use contrary to the Documentation or Specifications ii improper handling installation maintenance or repair other than if performed by Illumina s personnel iii unauthorized alterations iv Force Majeure events or v use with a third party s good not provided by Illumina unless the Products Documentation or Specifications expressly state such third party s good is for use with the Product Procedure for Warranty Coverage In order to be eligible for repair or replacement under this warranty Purchaser must i promptly contact Illumina s support department to report the non conformance ii cooperate with Illumina in confirming or diagnosing the non conformance and iii return this Product transportation charges prepaid to Illumina following Hlumina s instructions or if agreed by Illumina and Purchaser grant Illumina s authorized repair personnel access to this Product in order to confirm the non conformance and make repairs Sole Remedy under Warranty Illumina will at its option repair or replace non conforming Product
Download Pdf Manuals
Related Search