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TruSight Tumor Sample Preparation Guide - Support

1. 4 Chapter 2 TruSight Tumor Protocol 1 5 Introduction 6 TruSight Tumor Sample Preparation Workflow 7 Qualification of DNA Extracted from FFPE Samples 8 Hybridization of Oligo Pool 12 Removal of Unbound Oligos oranana 15 Extension Ligation of Bound Oligos 20 PCR Amplification 21 Verify Library Preparation Optional 2202 ccc cece cece eee eeeceeeee 25 PCR Clean Up 26 Library Quantification _ 29 Library Normalization 30 Library Denaturing and Pooling 31 Appendix A Supporting Information ooo 35 Introduction 36 Acronyms 37 How Does the TruSight Tumor Sample Preparation Assay Work 39 Tr
2. User Part 15042911 Rev A Preparation 1 Remove the TruSight Tumor Sample Preparation Oligo Pools FPA FPB and genomic DNA from 15 to 25 C storage and thaw at room temperature for up to 30 minutes Place thawed tubes on ice If running Illumina provided controls thaw ACD1 and or ACP1 as well 2 Set a 96 well heat block to 95 C 3 Remove OHS3 from 15 to 25 C storage and thaw at room temperature If precipitate is observed incubate at 37 C for 10 minutes and vortex 1 minute Repeat as needed until precipitate is no longer visible NOTE Using the provided controls 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 NOTE j The control ACPI is specific for Homo sapiens and will not work with DNA from other species Procedure 1 Label a new 96 well PCR plate HYP Plate ID 2 Dilute 10 ul of genomic DNA extracted from FFPE samples Use the table below to determine the fold dilution required for each calculated delta Cq Delta Cq p Hs 5405 DES 1 5 to 4 1 5 0 5 Dilution 16x 8x Ax 2x No dilution NOTE If preparing libraries from the control DNA in parallel with FFPE DNA samples dilute 5 ul of ACDI with 45 ul of TE Buffer Add 10 ul to each of two control wells for FPA and FPB and or two control wells for ACP1 a TruSight Tum
3. 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 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
4. 1 2 to 8 C Pre Amp E 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 Please contact your account manager for more information 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 Dispose of containers and any unused contents in accordance with the governmental safety standards for your region For more information see the MSDS for this kit at http www illumina com msds 42 Part 15042911 Rev A Box 2 TruSight Tumor Oligo Set Pre Amplification Acronym Reagent Name Storage Temperature Area QCP Quality Control Primers 15 to 25 C FPA FPB 157 to 25 C OCT 15 to 25 C Box 3 Post Amplification Acronym Reagent Name Storage Temperature Area HT1 Hybridization Buffer 15 to 25 C Post Amp EBT Elution Buffer with Tris Post Amp TruSight Tumor Sample Preparation Index Kit Box 1 Pre Amplification Reagent Name Storage Temperature Area i5 Index Primers A501 to A508 8 tubes 15 to 25 C Pre Amp i7 Index Primers A701 to A712 12 tubes 15 to 25 C Pre Amp Box 2 Pre Amplification Reagent Name Storage Temperature Area i5 Index Tube Caps White Room temperature Pre Amp i7 Index Tube Caps Orange Pre A
5. Information 48 Daily Cleaning of Pre PCR Area A daily cleaning of the pre PCR area using a 0 5 Sodium Hypochlorite 10 Bleach solution helps to eliminate PCR product that has entered the pre PCR area Identify pre PCR areas that pose the highest risk of contamination and clean these areas with a 0 5 Sodium Hypochlorite 10 Bleach solution before beginning any pre PCR processes High risk areas might include but are not limited to the following items Bench tops Door handles Refrigerator freezer door handles Computer mouse Keyboards Daily Cleaning of Post PCR Area Reducing the amount of PCR product in the post PCR area helps reduce the risk of contamination in the pre PCR area Daily cleaning of the post PCR area using a 0 5 Sodium Hypochlorite 10 Bleach solution helps achieve this Identify post PCR areas that pose the highest risk of contamination and clean these areas with a 0 5 Sodium Hypochlorite 10 Bleach solution daily High risk areas might include but are not limited to the following items Thermal cyclers Bench space used to process amplified DNA Door handles Refrigerator freezer door handles Computer mouse Keyboards Weekly Cleaning of All Lab Areas Once a week perform a thorough cleaning of the pre PCR and post PCR areas using 0 5 Sodium Hypochlorite 10 Bleach Clean all bench tops and laboratory surfaces Clean all instruments that are not cleaned daily Thoroughly mop lab floors Make
6. Support page Go to the TruSight Tumor Sample Preparation support page and click Downloads Prepare Libraries and Sample Sheet Prepare your libraries using the protocol detailed in this user guide In parallel you must prepare a sample sheet which will be used by the MiSeq to identify each sample and its corresponding index To prepare your sample sheet use the Illumina Experiment Manager a wizard based application which allows for the recording of your sample ID workflow indices and other parameters applicable to your 96 well plate The Illumina Experiment Manager can be run on any Windows platform You can download the Illumina Experiment Manager from the Illumina website at http www illumina com Go to the TruSight Tumor Sample Preparation support page and click Downloads Sequence Libraries on MiSeq TruSight Tumor Sample Preparation libraries must be sequenced on a MiSeq sequencing system using a paired end sequencing run For more details on using the MiSeq instrument or setting up your run please see the MiSeq System User Guide Parts 15042911 Rev A Analyze Data with Illumina Supported Software Analyze Data with MiSeq Reporter MiSeq Reporter processes the base calls generated by the MiSeq sequencing system It is an on instrument software which is built in to the instrument s processes MiSeq Reporter produces information such as alignment and structural variants For TruSight Tumor libraries the Amplicon DS workflow
7. as general lab supplies After the overnight incubation confirm the heat block has cooled to 40 C NOTE If the heat block fails to cool to 40 C overnight library preparation will need to be repeated Remove the HYP plate from the heat block and centrifuge at 1 000 xg at 20 C for 1 minute to collect condensation Using a multichannel pipette set to 60 ul transfer the entire volume of each sample onto the center of the corresponding pre washed wells of the FPU plate Change tips after each column to avoid cross contamination Cover the FPU plate with the filter plate lid and centrifuge the FPU at 2 400 xg at 20 C for 5 minutes Wash the FPU plate as follows a Using a multichannel pipette add 50 ul of SW1 to each sample well b Cover the FPU plate with the filter plate lid and centrifuge the FPU at 2 400 xg for 5 minutes Repeat the wash as described in the previous step Discard all the flow through containing formamide waste and unbound oligos collected up to this point in an appropriate hazardous waste container then reassemble the FPU The same MIDI plate can be re used for the rest of the pre amplification process Part 15042911 Rev A 8 Using a multichannel pipette add 45 ul of UBI to each sample well NOTE t Make sure that all liquid has drained after centrifugation Repeat centrifugation if necessary Any residual wash buffer may inhibit subsequent enzymatic reactions 9 Cover the FPU plate with the filter plate li
8. for 14 18 hours NOTE Moving the plate from the 95 C heat block to another pre heated block set to 40 C will adversely affect hybridization Part 15042911 Rev A Removal of 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 WARNING y 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 Dispose of containers and any unused contents in accordance with the governmental safety standards for your region For more information see the MSDS for this kit at http www illumina com msds WARNING y This set of reagents contains f mercaptoethanol The following procedure may be performed in a hood or well ventilated area if desired Estimated Time Total duration 20 minutes Hands on 20 minutes Consumables Item Quantity Storage ELMS thawed in preparation 1 tube 15 to 25 C for Extension Ligation SW1 Stringent Wash 1 1 tube DOE UB1 Universal Buffer 1 1 tube 2 to 8 C Filter plate with lid 1 plate Adapter collar reusable 1 plate MIDI plate 1 plate Troughs As needed TruSight Tumor Sample Preparation Guide Supplied By Ilumina Illumina Ilumi
9. for post amplification steps Q Instruments BioShake iQ High Speed Thermal mixer Part 1808 0506 or 45 jueuudinb43 Supporting Information 46 Thermal Cycler The following table lists the recommended settings for selected thermal cycler models Ilumina recommends that you validate any thermal cyclers not listed below if your lab has not yet performed the TruSight Tumor Sample Preparation 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 Eppendorf Mastercycler Gradient S Heated Plate Pro S Simulated Tube 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 the protocol should be verified prior to use Part 15042911 Rev A Prevent PCR Product Contamination The PCR process is commonly used in the laboratory to amplify specific DNA sequences Unless proper laboratory hygiene is used PCR products can contaminate reagents instrumentation and genomic DNA samples causing inaccurate and unreliable results PCR product contamination can shut down lab processes and significantly delay normal operations Make sure that the lab is set up appropriately to reduce the risk of PCR product contamination Physically Separate Pre PCR and Post PCR Areas Physically separate laboratory
10. sure that personnel responsible for weekly cleaning are properly trained on prevention of PCR product contamination Parts 15042911 Rev A Items Fallen to the Floor The floor is contaminated with PCR product transferred on the shoes of individuals coming from the post PCR area therefore anything falling to the floor must be treated as contaminated Disposable items that have fallen to the floor such as empty tubes pipette tips gloves lab coat hangers must be discarded Non disposable items that have fallen to the floor such as a pipette or an important sample container must be immediately and thoroughly cleaned with a 0 5 Sodium Hypochlorite 10 Bleach solution to remove PCR product contamination Clean any lab surface that has come in contact with the contaminated item Individuals handling anything that has fallen to the floor disposable or non disposable must discard their lab gloves and put on a new pair TruSight Tumor Sample Preparation Guide 4 9 UOI EUIWE U09 19NPp01d4 HOd 1u9 9Jg Supporting Information MiSeq Sample Sheet Preparation Create your Sample Sheet for MiSeq sequencing using the Illumina Experiment Manager Ilumina recommends the Illumina Experiment Manager to prepare your Sample Plate and Sample Sheet Select MiSeq as your instrument then select Targeted Resequencing and Amplicon DS as the workflow Ensure you select the Homo Sapiens genome folder 4 NOTE The assay control prepared with ACD1 ACP1
11. 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 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 request Illumina will attempt to pass through such indemnity if any to Purchaser TruSight Tumor Sample Preparation Guide Vl Table of Contents Table of Contents vii Chapter 1 Overview 1 Introduction Lu Q u temone noU E E e naani nba ture Ld eae 2 DNA Input Recommendations 3 Additional Resources
12. 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 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
13. to yield the ACq values for each sample TruSight Tumor Sample Preparation Guide 1 1 s duueS 3444 WO1 D919841X3 VNG Jo uoneoyieno TruSight Tumor Protocol Hybridization of Oligo Pool 12 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 reproductive toxin Personal injury can occur through inhalation ingestion skin contact and eye contact Dispose of containers and any unused contents in accordance with the governmental safety standards for your region For more information see the MSDS for this kit at http www illumina com msds E NOTE Illumina does not support the use of gDNA samples giving a delta Cq value of greater than 4 Estimated Time Total duration 14 18 hours overnight Hands on 15 minutes Consumables Item TruSight Tumor Sample Preparation Oligo Pools FPA FPB OHS3 Oligo Hybridization for Sequencing 3 Genomic DNA See DNA Input Recommendations on page 3 96 well skirted PCR plate Optional Adhesive aluminum foil seal if a heat sealer is not available Troughs Quantity 1 tube per pool 1 tube As needed 1 plate 2 seals As needed Storage 15 to 25 C 15 to 25 C 15 to 25 C Supplied By Ilumina Illumina User User User
14. tubes 2 tubes screwcap recommended PCR eight tube strip 1 2 5 L Ice bucket il Preparation 1 Set a heat block with a microcentrifuge tube insert to 96 C 2 Inan ice bucket prepare an ice bath Place thawed HT1 in bath to chill TruSight Tumor Sample Preparation Guide Supplied By Ilumina Illumina U ser ser ser ser ser ser Bui oog pue Buunjeuegq eqq 31 TruSight Tumor Protocol 3 Prepare a fresh dilution of 0 1 N NaOH by adding 100 pl stock 1 N NaOH to a microcentrifuge tube containing 900 ul laboratory grade water q CAUTION y Using freshly diluted NaOH is essential in order to completely denature samples for 4 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 Prepare PhiX Control Library 1 In a microcentrifuge tube add 2 ul of stock 10 nM PhiX library to 8 ul 1X EBT buffer to yield 10 ul of 2 nM PhiX library Add 10 pl 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 xg 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 pre chilled HT1 to the 20 pl denatured PhiX library to make 20 pM Phix library Dp NOTE The denatured 20 pM
15. 16 17 18 19 20 Changing tips is not required if you use care to avoid cross contamination Seal the CLP plate with Microseal B and shake on a microplate shaker at 1 800 rpm for 5 minutes After shaking if any samples are not resuspended gently pipette up and down or lightly tap the plate on the bench to mix then repeat this step Incubate at room temperature without shaking for 2 minutes Place the plate on the magnetic stand for 2 minutes Label a new 96 well PCR plate SGP Storage Plate Carefully transfer 40 ul of the supernatant from the CLP plate to the SGP plate Change tips between samples Seal the SGP plate with Microseal B and then centrifuge at 1 000 xg for 1 minute SAFE STOPPING POINT After PCR Clean Up the SGP plate may be kept at room temperature during the upcoming library quantification and normalization steps Once quantified samples have been normalized the SGP plate may be stored at 20 C until ready to quantify and normalize any remaining samples Part 15042911 Rev A Library Quantification In order to achieve the highest quality of data on the Illumina MiSeq sequencing platform it is important to create optimum cluster densities This requires accurate quantitation of DNA libraries Ilumina recommends quantifying libraries generated from FFPE samples using the Agilent Technologies Bioanalyzer 2100 Quality Control 1 Load 1 ul of the re suspended library on an Agilent Technologies 2100 B
16. AAAGAT TAC T TGAT AAGAGCTACCGTAAC AGAATGATAACAGTAACACACT TCTGT TAACCT TAAGAT TACT TGATCCACTGAT TCAACG TACCG TAACGAAC GTATCAATTGAGACTA CACACTTCTGTTAAAA ACG TACCAT TAAGAGCTACCGTGCAACAGTAACACACTTCTGT TAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAC IAAAGAATGATAACAT TA AGAATGATAACAG TAACACACT TCTGT TAACCTTAAGAT TACTTGATCCACTGAT TCAACGTACCG TAACGAACGTAT AATTGAGAC T CTGATTCAACGTAAAA CAACGTACCGTAACGAACGTATCAT TAAGAT TACTTGATCCAC TGATTCAACGTACCGTAACGAACG TATCAAT TGAGACTAAATAT TAA CTGTTAACCTTAAGAA BALIACTIGATCGAG GAT ICAACG TAAGATIAG HIGATCCAGT GATT CAACGIACCG AACGAAGGIAICAMLTGAGCTIG IGT AAG HAGCAACGACGAAAAC SGACGAAAAGAATGATAACAGTAACACACTTCTGT TAACCT TAAGAT TACTTGATCCACTGATTCAACGTACCGTAAAGAT TACTTGATC AAGAGC TACCGTAAC FOR RESEARCH USE ONLY ILLUMINA PROPRIETARY Catalog FC 130 9003DOC Part 15042911 Rev A May 2013 This document and its contents are proprietary to Illumina Inc and its affiliates Illumina and are intended solely for the contractual 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 docume
17. AACGTACCGTAACGAACGTATCAATTGAGAC TAAGC TACCGTGCAACGACGAAAAGAATGATAAAT T GAC AAAACAATOATAACACTAACACACTIOTOTIRACCITTAAGATTACTIGATOGAOTGATTCAACOTACCOTAMAGATTACITGATGCACTGA TO AACOTACCOTAACOAACGTATORA IGAGACTAAATATTAACO ACCATTAAGAGCTACCGTAAC AGAATGATAACAGTAACACACTTCTGT TAACCT TAAGAT TACT TGATCCACTGAT TCAACG TACCG TAACGAACGTATCAAT TGAGAC TAAATAT TAACGTACCAT TAAGAGCTACCGTGCAACGACGAAAAGAATGATAACAGTAACACACTTCTGTTAAAA ACGTACCAT IAAGAGCTACCGTGOCAACAGTAACACACT T TT IMAGALIAGI TEATG ACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGAC TAAATAT TAACGTACCAT TAAGAGCTACCGTGC GGAERAMAGAAT GATAACATTA AGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACC E E T TGATCCACTGATTCAACGTAAAA RTT ACT WERT SEM qe D NUO ae EE PEE CARET ROI ae ann GATAA De Ae e SHE at PAAGAA 3AT TACTTGATCCACTGAT T PAAGATTACTTGA AA ae AAT TGAGACTAGCAACGACGAA AA x Ca c AACGTACCGTAACGAACG TATCAAT Tt ITTCAACG COTA AAC Et RPLY PAA UA SR GANSA AA RA AK O AD TATUM ARTA TAA LIAAGATT AAAATATT CGACGAAAAGAATGATAA A OAOA TAN BAG LILO GG GT TAACCTTAAGAT TACT TGATCCACT GAT ICAACGTACCG IAACGAACGTATCAAT IGAGACTAAATAT TAACG TACCAT TAAGAGCTACCGTCTTCTGTTAACCTTAAGAT TAC T TGATCCAC TGAT ICAACGTAAAA VE TAC TIGATCCACT CATTCAACG TIAAGATIAC TH GATCCACTCATICAARCTACCOTRACGARCOTAT CAN GAGCTIOTOTTAACOTTAAGATIRCTICATCCACTOATI LANGO ACCGTAACGAACGTATCAATTGAGACTAGCAACGA CGAAAA6 SGTATCAATTGAGACTAAATATTAACGTACCATTAAGAGTCTGTTAACCTTAAGAT TACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATATTAACGTACCATTAAGAGCTACCGT
18. ACG NCCAT TAAGAGC TACCGTGCAACTTAACCTTAAGAT TACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT T GAGACTAAATAT TAACG TACCAT TAAGAGCTACCGTGCAACGACGAACTTCTGT TAACCT TAAGAT TACT TGAT 3CIACCGTGCAACGAAAATAACCTTAAGATTACT TGATCCACTGAT TCAACGTACTTCTGTTAACCTTAAGAT TACTTGATCCACTGATTCAACGTACCG TAACGAACG TATCAAT TGAGACTAAGCTACCGTGCAACGACGAAAAGAAT GAT ASAS Ne a Ue RATE T MAE GNG PAGG PAGGANA AAKALA AGA TAE Tn 3ATAACAGTAACACACT TCTGTTAACCT T M TCAATTGAGACT m v PN O a CGACGAAAAGAATGA bored Ip CCAT TAAGAGC TACCGTGCAACAGTAACACACT TCTGTTAACCTTAAGAT TACT TGATCCACTGAT TCAACGTACCG TAACGAACGTAT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTGCAACGACGAAAAGAAT GATAA ATACA AACACACTICTO AAC PAAGAT TACTTGATQCACTOATICAA CO TACCG TAACOAACGIAT CARET GAGACTAAATATIAACOTACCATTAAGAGCIASCOTCH CTO TANGCAAGATIAC LT GATCOACTGATICAACG GTACCGT O eee ATCAAITG O Ta EU a TTAAGAGCTACCGTGCAACGACGAAAAGAATGATAACAGTAACACACTTCTGTTAACCT TA ZTTGATCCACTGATTCAACGTIAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACG TATCAAT TRGAGCTTCTGT TAACCT T A A RATA TA TCAATTGAGACTAGCAACGACG AAAAGAATGAT AAGAGTAACACACTICIGLIAACGT AAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCA STOM Den iTACCGTAACGAACGTATCAAT TGAGAC TAAATATTAACGTACCAT TAAGAGCTACC 3aATAACAG TAACACACT TCTGT TAACCT TAAGAT TACT TGATCCACT GAT TCAACGTACCGTAACGAACGTATCAATT GAGACTAAATAT TAACG ANKAGAGC TACCGTCTTCTGTTAACCTTAAGATTACTTGA EAR AT GRABE OPUS NC E TCAT TAAGAT TACTTGATCCACTGAT TCAACGTACCGTAACGAACG TAT CAAT TGAGACTAAATAT TAACG TACCAT TAAGAGC TACCGTGCAACGACGAAAAGAATGATAAC
19. AG TAACACACT TCTGT TAACC T T IAAAAGAATGATAACAGTAACACACT TCT GT TAACC T TAAGAT TA CAGATCCACTOATTCAACCTACCOTAANCATTACT TGATCCACT GATT CAAROTACCGTAACCAAC ETAT CAAM GAGAGTAAATATTAACGIACCATIANGAGCTACE CCAT TAAGAGCTACCGTGCAACAGTAACACACT TCTGTTAACCTTAAGATTACT TGATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGT GCAACGACGAAAAGAAT GATAA 3ATAACAGTAACACACTTCTGTTAACCT TAAGATTACTTGATCCACTGATTCAACGTACCG TAACGAACGTAT AAT TGAGACTAAATAT TAACGTACCAT TAAGAGC TACCG TCTTCTGTTAACCT TAAGAT TACTTGATCCACTGAT TCAACG 2TT GATCCACTGATTCAACGT TAAGAT TACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGCT TCTGT TAACCT TAAGAT TACT TGATCCACTGAT TCAACG TACCGTAACGAACGTAT CAAT TGAGAC TAGCAACGACQ ERA ARA ETAT SAL PAGAGTOTETHA NAH PASALI NOT I GAT ETE GST EA LA CARE SAGA ERAT DR DRE TAM AA OLA SAMA CO ARO GTACCGTAACGAACGTATCATTAAGATTACTTGATCCAGCTGATTCAACGTACCGTAACGAACGTATCAATTGAGA FA AASA ACCGTGCAACGACGAAAAGAATGATAACAGTAACACACTTCTGT TAACCT Td gt TTGATCCACTGATTCAACGT TAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGCTTCTGTTAACCT TAAGAT TACT TGATCCACTGAT TCAACGTACCGTAACGAACGTA TGARIGAGACTAGCAACGACG IAAAAGAATGATAACAGTAACACAC T TCTGT TAACCT TAAGAT TACTT GATCCACTGAT T CAACGTACCGTAAAGAT TACTTGATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACC 3ATAACAGTAACACACT TCTGTTAACCT TAAGAT TA A ATTACTT ELI OS ACUNA ZACTGATTCAACGTACCAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTCTTCTGT TAACCT TAAGAT TACT TGATCCACTG
20. ATTCAACGTACCG TAACGA AAAAGANTGATAACAGTAAGAGAGTIGTGY IAAGC TAAGATIACT TGATCGACTGAT CACO AC CGI MAGA TACT IAT LAG GA ICAAGGTACCGTAACGAACGIATCAAI TGAGAC TAAATATTAAUGTACCATTAAGAGG TAGO cereis e DA ERS GTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACG TATCAA E ia appaia BA ACUNA n n l C7 gq ARR a GATTACTTGATCCACTGATTCAACG TGAGACTAAATAT TAACGTTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATA TALAGA NE GCTTCTGT TAACCTTAAGATTACT TGATCCACTGATTCAACGTACCGTA ATCAATTGAGA CTAAAY ATTAACGTACTTAACCTTAAGATTACTTGATCCACTGATTCAACG TACCGTAACGAACGTCT TC TGT TAACCT TAAGAT T KETIGMCCACIGATTCAACGT ACCGTAACGAACGTATCAAT TGAGACTAACGACGA CAN UE etr ELS MM e AT NG e E DET E NE 3ATAACAGTAACACACTT AACCTIAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATT GAGAC OUEST GTACCATTAAGAGCTACCGTCTTCTGTTAACCTTAAGAT TACT TGATCCACTGAT TCAACG CCA I TANGAGCTACCGTECAACTIAACCTT AAGA HAGTIGATCCACIGATTICAACCTACECTARCOARCOTA TCAATTGA GA CTAAATATTAACG TACCAT TAAGAGCTACCGTGCAACGACGAACTICTGTT AACOTIANGATTAGTIGAT TACOS GCAACGAAAATAACG TAAGATIACT GAICCACTGAT ICAACGTAGLICTGT IAACC I TAAGAT TACT TGATCCAGT GAT GAAGGTACUGIAACGAACG TAI GAAT GAGAG TAGO AGGGTGGAAGG ACGAAAAGAATGAT IAAAAGAATGATAACAGTAACACAC TTCTGT TAACCT TAAGAT TACTTGATCCACTGATTCAACGTACCGTAAAGATTAC T TGATCCACTGATTCAACG TACCGTAACGAACG TA TTGAGACTAAA CGTACCAT TAAGAGCTACC 3ATAACAGTAACACACT TCTGTTAACCTTAAGAT TACT T DA CCAC CTGATTCAACGTACCGTAACGAACG TAY SANTI GAGACIAAATALTANCGT ACCATTAAGAGC TACCGTOGAA AOGA CEAMAAGAAT GA ADAC TA CACACTI
21. C NON AD C SI C Ne e a e Ng paa poe e aAa e a MEE E aara AA 3ATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTA COCA SAG MAT GAGA T GGTAGYATIPAGAGOTANSOTGTTETE TANAGA AAA APTA EART SAT CHANG GTACCGTAACGAACGTATCATTAAGATTACTTGATCCACTGATTCAACGTACCGT RACCAACGIA TCAATTGAGACTAAATAT TAACGTACCATTAAGAGC TACCGTGCAACGACGAAAAGAATGATAACAGTAACACACT TCTGT TAACCT T STEAL OGAGT GALT CAACGTTAAGATTACTIGATCGACT GATT GAAGG TACOGTAACOAACGTALGAATTGAGCTIC TS HAAGGTTAAGATIACTTGATCCACTGALT CAAGUTACCO TAAO GAACOTATCAATTOAGAG TAGOAACGACU 7AAAAGAATGATAACAGTAACACACTTCTGT TAACCT TAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATAT TAACGTACCAT TAAGAGCTACC Ilumina San Diego California U S A 1 800 809 ILMN 4566 1 858 202 4566 outside North America techsupport illumina com www illumina com
22. DAL tube 1 2 times to mix Incubate immediately in the ice water bath for 5 minutes then transfer contents to the template position in the MiSeq reagent cartridge 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 Proceed to library sequencing as instructed in the MiSeq System User Guide Part 15042911 Rev A Supporting Information Introduction 222225225225 2b U2h2 et a ede deeds a bb Ud den e ERE 36 ACTONYMS tocado et tot aa oe AA 37 How Does the TruSight Tumor Sample Preparation Assay Work 39 TruSight Tumor Sample Preparation Process Overview 40 TruSight Tumor Sample Preparation Kit Contents 42 User Supplied Consumables 22 2 se esses e ellen llis 44 EQUINA 45 Prevent PCR Product Contamination MiSeq Sample Sheet Preparation q Y pm ossi ss zin ai a Pe ee Fa aa asi PU Ba ff Sere es as 1 XTGEGG CA rt regagrcercH t peo asar o GG AA lt TruSight Tumor Sample Preparation Guide 3 5 v xipueddaw Supporting Information Introduction The protocols described in this guide assume that you are familiar with the contents of this appendix have confirmed your kit contents and have obtained all of the requisi
23. GCAACGAAAAGAAT GATAACAG TAACAAC AAGATTAC GTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACC GCAACGACGAAAAGAATGAT TGTTAACCTTAAGAA CAACGTACCGTAACGAACGTATCAT TAAGA TTGATCCACTGATTCAACGTACC CGTATCAAI A AAACGTACCAT TAAGAGCTACCGT TT Y 3ATTAC TCCACT TTAAGATTACTTGATCCACTGATTCAACG TACCGTAAG CGTATCAATTGAGCTICTGTT TTGAGACTAGCAACGACG 2 GATAACAGTAACACAC T TCTGT T AA TTGATCCACTGATTCAACGTACCGTAAAGA Ti GAGCTACCGTAAC A TGATAACAG TAACA E GTTAACCT TAAGAT TACT TGT TGATCCACTGATTCAACGTACCGTATCAAT TGAGAC ATTAAC GATCCACTGATTCAACG GT CAYCCACTOATICAACOTA ATTA TCCACTGATTC ACCGT CAATTGA TA ITAACGAACAT T CA e LITO IAA ACTTGATOCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGAT TCAAGGTA ATTAAGA meen AGAATGATAACAGTAACACACTTCTGT TAACCT TAAGAT TACTT CT CAACG TAACGAAC CAA T GA iTTAA ITTACTTGATCCACTGATT i TAACGAACGTATCAATT ATTCAACGTACCGTAACGTA CGTATCAAT TGAGACTAAATAT TAACGTACTTAACC IT TACTTGATCCACTGATTCAACGTACCGTAACGAACGTCTTCTGTTAA GAGAG T TAACGTACCAT TAAGAGCTACAACCTTAAGAI GATCCAC ORIS A S COGTAACGAACG TATCAAT TGAGAC TAAATAT TAAC AACAGTAACACACTCOA AGAATGATAACAGTAACACACT TCTGT T GG TR CR ARO ACCGTAACGAACGTATCAATTGAGACT ARAIATIAADGIACCATAAGAGSTAGCGICHICTGIIARGGT AAGATIAC TT GATCCACT GAT TG AACGT NASHA NAG AGO IA CUA Ke LANGAN GATTACTTGATCCAC IG AAO PAGG Sea ACE A CRAT S ie TTACTTGATCCITA AAGAGCTACCGTGCAACGAAAATAACCTTAAGATTACTTGATCCACTGATTCAACGTACTTCTGTTAACCTTAAGATTAGTTGATGCAC GAATGATAAAT T CGAAAAGAATGATAACAG TAACACAC IS GTTAACCTIAAGAT TACTTGATCCACTGATTCAACGTACCG T
24. 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 Hardware 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 All 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 thi
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26. PhiX library can be stored up to three weeks at 15 to 25 C as single use aliquots After three weeks cluster numbers tend to decrease Prepare Samples for Sequencing 32 Ae U Ne If the LNP plate was stored frozen thaw at room temperature Centrifuge the LNP plate at 1 000 xg at 20 C for 1 minute to collect condensation Determine the samples to be pooled for sequencing If the LNP plate was stored frozen using a P200 multichannel pipette set to 40 ul mix each library to be sequenced by pipetting up and down 3 5 times Change tips between samples Part 15042911 Rev A Library Denaturing and Pooling When using the TruSight Tumor assay Illumina recommends sequencing four tumor samples 8 libraries total per run when using V2 chemistry This process is outlined in the following steps If sequencing a different number of samples adjust accordingly oo N O A HS NOTE Each sample is represented by two libraries which are generated from the TruSight Tumor Oligo Pools FPA and FPB It is critical that the FPA and FPB libraries for each sample be run together on the same flowcell in order for the MiSeq software to properly analyze the results for each sample f NOTE Control libraries generated from ACD1 with ACP1 must be pooled and run separately from those prepared with FPA and FPB as they will 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 e
27. Sample Preparation Guide 2 5 euondo uonejgedoejg eq AJU A PCR Clean Up components Estimated Time Total duration 50 minutes Hands on 20 minutes Consumables Item TruSight Tumor Protocol EBT Elution Buffer with Tris AMPure XP beads 80 Ethanol freshly prepared 96 well MIDI plates Microseal B adhesive film Troughs Preparation 1 Bring the AMPure XP beads to room temperature 2 Prepare fresh 80 ethanol from absolute ethanol i NOTE Quantity 1 tube 440 ul per 8 samples 3 3 ml per 8 samples 2 As needed As needed Storage Room temperature 2 ik BAC Room temperature This process uses AMPure XP beads to purify the PCR products from the other reaction Supplied By Ilumina User Always prepare fresh 8096 ethanol for wash steps Ethanol can absorb water from the air impacting your results Procedure 1 Centrifuge the IAP plate at 1 000 xg at 20 C for 1 minute to collect condensation 26 Part 15042911 Rev A Oo 0 N Mm 11 12 13 14 Label a new MIDI plate CLP_Plate_ID Clean up Plate Invert AMPure XP beads 10 times Vortex vigorously and then invert again 10 times f 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 Using a multichannel pipette set to 60 ul transfer 55 ul PCR product from the IAP plate to the CLP plate Chan
28. ach 4 nM library to be sequenced from the LNP plate to its own tube in an eight tube PCR strip tube i joue the sealed LNP plate may be stored at 15 to 25 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 pl of each library NaOH EBT solution there should be 8 into the PAL tube Mix the pooled libraries well by pipetting up and down 10 times 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 10 Mix DAL by vortexing the tube at top speed TruSight Tumor Sample Preparation Guide 3 3 Buljood pue Buunjeuegq eqq TruSight Tumor Protocol 34 11 12 13 14 NOTE 4 If you would like to make and save additional DAL from the remaining 72 ul of unused PAL for future runs it may be stored at 15 to 25 C for up to 3 days Longer storage may lead to sub optimal cluster densities Centrifuge the DAL tube at 1 000 xg at 20 C for 1 minute to collect contents Incubate the DAL tube in a heat block at 96 C for 2 minutes After the incubation invert the
29. become more critical to the success of these assays The TruSight Tumor Sample Preparation 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 require a largely intact DNA template that can be denatured The formalin fixation and paraffin embedding of tissues impacts this requirement by fragmenting cross linking and otherwise damaging DNA through a variety of chemical modifications Assessing the extent of this damage and adjusting the DNA extraction procedure to compensate for it as much as possible is essential to improving the success of the TruSight Tumor Sample Preparation assay with FFPE DNA DNA Extraction Recommendations Ilumina recommends the Qiagen Supplementary Protocol Purification of genomic DNA from FFPE tissue using the QIAamp DNA FFPE Tissue Kit and Deparaffinization Solution for the purposes of extracting 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 Deparaffinize with 320 ul of Qiagen Deparaffinization Solution Digest with proteinase K in a thermomixer overnight at 1 000 rpm Decrease elution volume to 30 ul to maximize DNA concentration TruSight Tumor Sample Preparation Guide 3 suonepueuuulooeH 1ndu YNG Overv
30. bp range or 350 380 bp for ACP1 libraries The expected concentration range is 4 300 nM Record the values 30 Storage Supplied By Room Ilumina temperature User User 2 Label a MIDI plate LNP plate Library Normalization Plate and dilute 4 ul of all samples 520 nM to 4 nM with the EBT buffer e g if a sample is 254 nM add 4 ul of library to 250 ul of EBT buffer to give 4 nM 3 For any samples lt 20 nM dilute as needed to ensure that the volume of the final 4 nM working stock is at least 20 ul SAFE STOPPING POINT If you do not plan to proceed to Library Denaturing and Pooling on page 31 and q subsequent sequencing on the MiSeq after completion of Library Normalization store the sealed LNP plate at 15 to 25 C for up to 7 days Part 15042911 Rev A Library Denaturing and Pooling In preparation for cluster generation and sequencing equal volumes of normalized library are denatured then are combined diluted in hybridization buffer and heat denatured prior to sequencing on the MiSeq PhiX is used as an internal control for sequencing Estimated Time Total duration 10 minutes Hands on 10 minutes Required Equipment Heat block with microcentrifuge tube insert Consumables Item Quantity Storage HT1 Hybridization buffer 1 tube 15 to 25 C 2 ul 10 nM PhiX Library 2 ul 15 to 25 C Stock 1 0 N NaOH diluted 15 to 25 C to 0 1 N NaOH Laboratory grade water 1X TE Buffer Microcentrifuge
31. by Promega 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 For Research Use Only not for any clinical or therapeutic use in humans or animals This E A Part 15042911 Rev A 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 possible 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 I
32. cifications 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 Product s 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 Illumina 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 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 whicheve
33. columns from the FPU plate to the IAP plate in a similar manner Tips must be changed after each column to avoid index and sample cross contamination d After all the samples have been transferred the waste collection MIDI plate of the FPU can be discarded The metal adapter collar should be put away for future use 7 Cover the IAP plate with Microseal B and seal with a rubber roller 8 Centrifuge at 1 000 xg at room temperature for 1 minute TruSight Tumor Protocol 9 Transfer the IAP plate to the post amplification area 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 t NOTE On the PCR machine set the reaction volume to 60 ul and the temperature ramp speed to maximum SAFESTOPPING POINT If you do not plan to immediately proceed to PCR Clean Up following the l completion of PCR the plate can remain on the thermal cycler overnight or store it at 2 to 8 C up to 48 hours 2 4 Part 15042911 Rev A Verify Library Preparation Optional After PCR combine 5 ul of amplified product with 15 ul of DEPC DI H20 and run on a 4 TBE agarose gel along with 100 bp ladder to confirm the presence of the 300 330 bp library product If generating libraries with ACP1 the product should be present at 350 380 bp Alternatively the products can be run on a bioanalyzer TruSight Tumor
34. d and centrifuge the FPU at 2 400 xg for 5 minutes TruSight Tumor Sample Preparation Guide 1 9 soBIJO punoqun Jo enouay TruSight Tumor Protocol 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 This results in the formation of products containing the targeted regions of interest flanked by sequences required for amplification Estimated Time Total duration 50 minutes Hands on 5 minutes Consumables Item Quantity Storage Supplied By ELMS Extension Ligation Mix 1 tube 15 to 25 C Illumina 3 Adhesive aluminum foil seal 1 seal User Troughs As needed User Procedure 20 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 Changing tips between columns is not required if you use care to avoid cross contamination 2 Seal the FPU plate with adhesive aluminum foil and then cover with the lid to secure the foil during incubation 3 Incubate the entire FPU assembly in the pre heated 37 C incubator for 45 minutes Ensure waste is removed from bottom of the plate 4 While the FPU plate is incubating prepare the IAP Indexed Amplification Plate as described in the following section Part 15042911 Rev A PCR Am
35. d 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 Illumina for any infringement of third party intellectual property rights Exclusions to Illumina Indemnification Obligations Illumina has no obligation to defend indemnify or hold harmless Purchaser for any Illumina Infringement Claim to the extent such infringement arises from i the use of Part 15042911 Rev A 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 condit
36. ding to the quality of the extracted DNA Estimated Time Total duration 3 hours Hands on 60 minutes Consumables Item Quantity QCT Quality Control 1 tube Template QCP Quality Control Primer 1 tube Genomic DNA As needed See DNA Input Recommendations on page 3 KAPA SYBR FAST Master Mix 1 plate Universal 2x Other qPCR mixes can be used but should be tested Nuclease free water As needed 48 or 96 well plate 1 plate Preparation Storage 15 to 25 C 15 to 25 C 15 to 25 C 15 to 25 C Supplied By Ilumina Illumina User User User User 1 Remove the OCT QCP KAPA SYBR FAST Master Mix Universal 2x and genomic DNA from 15 to 25 C storage and thaw at room temperature for up to 30 minutes Part 15042911 Rev A Procedure 8 Place thawed tubes on ice If using OCT for the first time aliquot 5 ul of OCT into different PCR strip tubes for long term storage to avoid freeze thawing Add 5 ul of OCT to 495 ul of Nuclease free water in a microfuge tube Vortex the dilution to thoroughly mix the sample Add 1 ul of QCP to 9 ul of Nuclease free water in a microfuge tube NOTE j Make a larger dilution if qualifying more than one genomic DNA sample Vortex the dilution to thoroughly mix the sample Add 1 5 ul of Qiagen extracted genomic DNA to 148 5 ul of Nuclease free water in microfuge tubes to make a 100 fold dilution Vortex the dilutions to thoroughly
37. ge tips between samples Seal the CLP plate with Microseal B Shake the CLP plate on a microplate shaker 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 Using a multichannel pipette set to 100 ul and with the CLP plate on the magnetic stand carefully remove and discard the supernatant Change tips between samples f NOTE Delays during this step may lead to bead clumping following removal of the supernatant Proceed immediately to next step as soon as all supernatant is removed With the CLP plate on the magnetic stand wash the beads with freshly prepared 80 ethanol as follows a Add 200 ul of freshly prepared 80 ethanol to each sample well using a multichannel pipette Avoid disturbing the beads b Incubate the plate on the magnetic stand for 30 seconds or until the supernatant appears clear c Carefully remove and discard the supernatant Repeat the 80 ethanol wash described in the previous step Use a P20 multichannel pipette to remove excess ethanol Aspirate all remaining ethanol using a fine tip pipette Do not disturb or touch the beads 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 TruSight Tumor Sample Preparation Guide P T dn ueal9 HOd TruSight Tumor Protocol 28 15
38. iew Additional Resources The following resources are available for TruSight Tumor Sample Preparation protocol guidance and sample tracking Access these and other resources on the Illumina website at support illumina com sequencing kits ilmn Then select TruSight Tumor Sample Preparation Support Resource Best Practices TruSight Tumor Sample Preparation Experienced User Card part 15038313 Ilumina Experiment Manager EM Description Provides best practices specific to this protocol Review this before starting sample preparation Topics include General Advice on Sample Handling e Ensuring Consistency Handling Magnetic Beads e Handling Reagents e Avoiding Cross Contamination e Washing with 8076 Ethanol During PCR Clean Up Click Best Practices on the TruSight Tumor Sample Preparation Support page Provides protocol instructions but with less detail than what is provided in this user guide New or less experienced users are strongly advised to follow this user guide and not the EUC Click Documentation amp Literature on the TruSight Tumor Sample Preparation Support page Enables you to create and edit appropriate sample sheets for Ilumina sequencers and analysis software and record parameters for your sample plate To download the software click Downloads on the TruSight Tumor Sample Preparation Support page To download the documentation click Documentation amp Literature on the TruSight Tumo
39. illumina TruSight Tumor oample Preparation Guide D AA CARTA tose es RASA eg UY AGAAT GATAACAGTAACACACT TCTGTT TACTTGT TGATCCACTGATTCAACG TACCGTATCAAT TGAGACTAAATATTAACGTACCAT TAAGAGCTACCGTCTTCTGTTAACCTTAAGAT TACTT GATCCAC TGATICAACGTACCGTAAAAA GAGA TGA CAACOTACCAAGATIACTIGATCC ACT GA TCAACOTACCON AACGAACGTAT CAT GAGACTARATATAACGTAGCALPAAGAGCIACCGTCTTCIGTIAACCTIANGATIACTTGATGCAC GANI CAAUG TAGS TAACGAACATE SJGACGAAAAGAATGATAACAG TAACACACTTCTGT TAACC T TAAGAT TACT TGATCCACTGAT TCAACG TACCGTAAAGATTAC T TGATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TGAGAC TAAATAT TAACGTACCAT TAAGAGCTACCGTAAC AGAATGATAACAG TAACACACTTCTGT TAACCTTAAGAT TACTTGATCCACTGAT TCAACGTACCG TAACGAACG TATCAAT TGAGAC TAAATATTAACGTACCAT TAAGAGCTACCGTCT TC TGT TAACCT TAAGATTACTTGAT CCACTGAT TCAACG TAAAA AT CAAT T7 GAGAC TAAATAT TAACGT TG TTAACCTTAAGATTACT TGATCCACTGAT TCAACG TACCG TAACGAACGTATCAAT TGAGACTAAATAT TAACG TACCAT TAAGAGCT TC TG T TAAC E AEN GALEAE TEN AA AA AA AACGTATCAATTGAGACTAAATAT TAACGTAC T TAACCT TAAGAT TACT TGATCCACTGAT I CAACG TACCG TAACGAACG TCTTCTGT TAACCT TAAGAT TACT T GATCCACTGAT TCAACGTACCGTAACGAACG TAT CAAT TGAGAC TAACGACGAAACG AGAAT GATMCAGTAADACACTTGTG LARGO IMAGA TAC IGATGCAGIGAT TGAACGTACOG TAAGGAAGGTATCANT AGAD TAAATAT TAAGGTACGA TANDA RU TAG GIU ICT Aa PANA BIEN AC AACCTTAAGAT TACTTGA CTG CCGTAA CGTATCAAT TG CTAAATATTAACGTACC ACCGTIG GAACTTCTGTTAACCTTAAGAT TA NIS PACER AACGAAAATAACCTI NAGKITA AG GONG TAI BAKAT ACTTCTGTIAACCTTAAGATTACTTGATCCACTGAT TC
40. ioanalyzer using the Agilent DNA 1000 Refer to 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 2 Check the size and purity of the sample The final product should be a band at 300 330 bp as in Figure 4 If generating libraries using ACP1 the final product will be present at 350 380 bp This concentration should correlate with the relative library intensities previously observed on the agarose gel see Verify Library Preparation Optional on page 25 Figure 4 Representative DNA Sample Prep Library Size Distribution 300 330 bp 15 100 200 300 500 700 bp NOTE If gt 10 of the library product is present in the 150 250 bp range we recommend repeating the PCR Clean Up Procedure using 40 ul of product and 32 ul of AMPure XP beads TruSight Tumor Sample Preparation Guide p 9 uoneouynuengo eqq TruSight Tumor Protocol Library Normalization This process normalizes the quantity of each library to ensure more equal library representation in your pooled sample Estimated Time 30 minutes Consumables Item Quantity EBT Elution Buffer with Tris As needed MIDI plate As needed Microseal B adhesive film As needed Procedure 1 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
41. ion in PDF is available for download from the Illumina website Go to www illumina com support select a product then click Documentation amp Literature TruSight Tumor Sample Preparation Guide 5JUB SISSY EOIUYDE AAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATATTAACGTACCATTAAGAGCTACC 3ATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGTTGATCCACTGATTCAACGTACCGTATCAAT TGAGAC TAAATAT TAACGTACCAT TAAGAGCTACCGTCTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGT PAGI GAT HANGO a Ns AN IAAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGAT TACTT GATCCACTGAT TCAACGTACCGTAAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACC 3ATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATATTAACGTACCATTAAGAGCTACCGTCTTCTGTTAACCT TAAGATTACTTGATCCACTGATTCAACG EAGER IPA TATA GULO TIPANG TI PAGA AGIT I BATAC AG IBANG OTA EAER TEAR BAG TCO IA a ny Lan CAGE TEREA ATCAAT TGAGAC TAAATAT TAACGTACT TAACCT TAAGAT TACT TGATCCACTGATT CAACGTACCGTAACGAACGTCTTCTGTTAACCT TAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAACGACGA 3ACTAAATATTAACGTACCAT TAAGAGCTACAACCT TAAGAT TACT TGATCCACTGATTCAACGTACCGTAACGAACG TAT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAGC TACCGTGCAACGACGAAAAGAAT GATAACAGTAACACA 3ATAACAGTAACACACTTCTGTTAACCT TAAGATTACT TGATCCACTGATTCAACGTACCG TAACGAACG TATCAAT T GAGACTAAATAT TAACG TACCAT TAAGAGCTACCG TCT TCTGT TAACCTTAAGAT TACT TGATCCACT GAT TCA
42. ions 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 e 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 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 d 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
43. is chapter describes the TruSight Tumor protocol Review Best Practices before proceeding See Additional Resources on page 4 for information on how to access TruSight Tumor 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 sequencers and analysis software See Additional Resources on page 4 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 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 libraries as they require longer cycles 6 Part 15042911 Rev A TruSight Tumor Sample Preparation Workflow The following diagram illustrates the workflow using the TruSight Tumor Sample Preparation Kit Safe stopping points are marked between steps Figure 1 TruSight Tumor Sample Preparation Workflow Qualification of DNA Extracted from FFPE Samples Hands On 60 min Incubation 120 min PCR Clean Up Hands O
44. is provided as a plug in for MiSeq Reporter This workflow produces aligned reads in the BAM format and outputs variants in vcf files These files merge the variant information obtained from sequencing of both strands For more information about this software please see the MiSeq System User Guide MiSeq Reporter Amplicon DS Workflow Reference Guide or MiSeq Reporter s online help http www illumina com help miseq_reporter default htm TruSight Tumor Sample Preparation Guide A SSo20Jg UO jeseda 1g ajdwes sown 1ybisna Supporting Information TruSight Tumor Sample Preparation Kit Contents The TruSight Tumor Sample Preparation Kit contains the following components and is shipped on dry ice unless specified otherwise below As soon as you receive your kit store the kit components at the specified temperatures and in designated pre amplification and post amplification areas TruSight Tumor Sample Preparation Kit Catalog FC 130 2001 TG Catalog TG 130 2001 Box 1 Pre Amplification Acronym Reagent Name Storage Temperature Area ACD1 Amplicon Control DNA 1 15 to 25 C Pre Amp ACP1 Amplicon Control Oligo Pool 1 15 to 25 C Pre Amp OHS3 Oligo Hybridization 15 to 25 C Pre Amp for Sequencing Reagent 3 ELM3 Extension Ligation Mix 3 15 to 25 C Pre Amp PMM2 PCR Master Mix 2 15 to 25 C Pre Amp TDP1 TruSeq DNA Polymerase 1 15 to 25 C Pre Amp SW1 Stringent Wash 1 2 to 8 C Pre Amp UB1 Universal Buffer
45. itioned correctly according to Figure 3 Collect all liquid in the bottoms of the tubes by holding them in place in the rack and tapping it against the bench 1 NOTE If less than 48 samples 96 reactions are being prepared or alternate index combinations are being employed the Index Plate Fixture does not need to be used and the indices can be added to the appropriate wells of the IAP plate manually Figure 3 TruSeq Index Plate Fixture TruSight Tumor Protocol A i5 primers white caps B i7 primers orange caps C IAP plate 5 Label a new 96 well PCR plate IAP Indexed Amplification Plate 2 2 Part 15042911 Rev A 10 Procedure 1 Using a multichannel pipette add 9 ul of i5 primers clear solution to each column of the IAP plate Tips must be changed after each row to avoid index cross contamination To avoid index cross contamination discard the original white caps and apply new white caps Using a multi channel pipette add 9 ul of i7 primers yellow solution to each row of the IAP plate Tips must be changed after each row to avoid index cross contamination To avoid index cross contamination discard the original orange caps and apply new orange caps For 96 reactions add 60 ul of TDP1 to 2 58 ml of PMM2 If preparing fewer reactions use the following calculation of reactions 0 625 ul TDP 26 875 ul PMM2 Do not pipette volumes of less than 5 ul of TDP1 Invert the PMM2 TDP1 PCR master mix 20 times
46. mix the samples Determine the plate layout of the qPCR reaction For 10 samples Illumina recommends this plate layout 1 2 3 4 5 6 A OCT Sample 1 Sample 3 Sample 5 Sample 7 Sample 9 B OCT 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 Ilumina recommends using nuclease free water Prepare the SYBR master mix reaction as follows The master mix contains extra volume TruSight Tumor Sample Preparation Guide 9 s duueS 3444 WO1 p3198 1IX3 YNG Jo uoneoyieno TruSight Tumor Protocol 10 11 12 13 14 15 16 10 Table 1 SYBR Master Mix Reactions Consumable ul per ul per ul per well plate plate 48 wells 96 wells KAPA SYBR FAST 5 0 ul 275 ul 550 ul Master Mix Universal 2x QCP 1 0 pl 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 Take care to pipette accurately into the wells as small variations will affect the assay Add 2 ul of the OCT dilution the sample dilutions or nuclease free water to each well of the plate Take care to pipette accurately into the wells as small variations will affect the assay Seal the plate
47. mp Additional Required Components Consumable Catalog Storage Temperature Area TruSeq Custom Amplicon Filter Plate FC 130 1006 Room temperature Pre Amp TruSeq Index Plate Fixture and Collar FC 130 1007 Room temperature Pre Amp Kit Reusable TruSight Tumor Sample Preparation Guide 4 3 s u3 U0 py uolpued luq ajduwes Joun 1ybisna Supporting Information User Supplied Consumables Quantity As needed As needed As needed 3 3 As needed 3 As needed 2 40 ml As needed 2 As needed As needed As needed As needed As needed 44 Consumable PhiX control kit 10 N NaOH prepare from tablets or use a standard solution 100x TE Buffer Sigma Aldrich Part T9285 Dilute to 1x TE Buffer 96 well skirted PCR plates 0 2 ml polypropylene 96 well storage plates 0 8 ml MIDI plates Agencourt AMPure XP 60 ml kit Adhesive aluminum foil seal Conical tubes 15 ml Eppendorf microcentrifuge tubes screw top recommended Ethanol 200 proof for molecular biology Microseal B adhesive seals PCR Eight Tube Strips Solution basin PVC non sterile trough Agarose gel 2 or 4 DNA 1000 Kit for Bioanalyzer DNA molecular weight markers Ice bucket Supplier Illumina Part 15034071 General lab supplier General lab supplier Bio Rad Part MSP 9601 Fisher Scientific Part AB 0859 Fisher Scientific Part AB 0765 Beckman Coulter Part A63881 A63880 Beckman Coulter Pa
48. n 20 min Processing 30 min Reagents Reagents EBT QcT QCP AMPure XP beads EtOH Output Output Plate SGP Plate Hybridization of Oligo Pool anes on Ik Library Quantification Incubation 2 18 hours Reagents FPA FPB OHS3 Output HYP Plate Library Normalization Hands On 30 min Processing 50 min Reagents EBT Removal of Unbound Oligos Output Hands On 20 min LNP Plate Reagents ELM3 CU SW1 UB1 1 Output FPU Plate Library Denaturing and Pooling Hands On 10 min Reagents HT1 Extension Ligation of O Bound Oligos TE Buffer Hands On 5 min Output Incubation 45 min PAL amp DAL tubes Reagents ELM3 Qutput CU FPU Plate 1j B Preamp PCR Amplification B Postamp Hands On 30 min TY Cold Storage Cycle Time 105 min Option Reagents Fill in the lab tracking PMM2 form as you perform TDP1 the assay i5 primers i7 primers NaOH Output IAP Plate TruSight Tumor Sample Preparation Guide MO P4JOM Uorneaedaag ejduues sown 1ybisna TruSight Tumor Protocol Qualification of DNA Extracted from FFPE Samples During this step a gPCR reaction will be performed to determine 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 can be calculated for each sample and used to predict its performance in the TruSight Tumor Sample Preparation assay The exact amount of FFPE DNA input will vary accor
49. na Illumina Ilumina User User 15 soBIJO punoqun jo enouay TruSight Tumor Protocol Preparation 1 16 Remove ELM3 from 15 to 25 C storage and thaw at room temperature ELMS is used in the Extension Ligation step and takes approximately 20 minutes to thaw Remove SW1 and UB1 from 2 to 8 C storage and set aside at room temperature Part 15042911 Rev A 3 Assemble the filter plate assembly unit FPU in the following order from top to bottom NOTE 4 For instructions on viewing a video demonstration of this step see page 8 Figure 2 Filter Plate Unit Assembly A Lid B Filter plate C Adapter collar D MIDI plate 4 Apply the FPU barcode plate sticker to the filter plate 5 Pre wash the FPU plate membrane as follows a Using a multichannel pipette add 50 ul of SW1 to each well TruSight Tumor Sample Preparation Guide 1 y soBIJO punoqun Jo enouay TruSight Tumor Protocol Procedure 18 1 4 NOTE Pre wash only the wells to be used in the current assay You may use fresh unused wells of a previously opened filter plate but wells that have been used in a previous assay should never be re used b Cover the FPU plate with the filter plate lid and keep it covered during each centrifugation step c Centrifuge the FPU at 2 400 xg at 20 C for 5 minutes Pre heat incubator not heat block to 37 C f NOTE Illumina strongly recommends keeping spare filter plates FC 130 1006 on hand
50. nt 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 OR ANY USE OF SUCH PRODUCT S OUTSIDE THE SCOPE OF THE EXPRESS WRITTEN LICENSES OR PERMISSIONS GRANTED BY ILLUMINA IN CONNECTION WITH CUSTOMER S ACQUISITION OF SUCH PRODUCT S FOR RESEARCH USE ONLY 2013 Illumina Inc All rights reserved Illumina IlluminaDx BaseSpace BeadArray BeadXpress cBot CSPro DASL DesignStudio Eco GAIIx Genetic Energy Genome Analyzer GenomeStudio GoldenGate HiScan HiSeq Infinium iSelect MiSeq Nextera NuPCR SeqMonitor Solexa TruSeq TruSight VeraCode the pumpkin orange color and the Genetic Energy streaming bases design are trademarks or registered trademarks of Ilumina Inc All other brands and names contained herein are the property of their respective owners Patent pending for methods performed by components in this kit product includes GoTaq Hot Start Polymerase manufactured
51. o a single tube and sequenced on the MiSeq System Custom Custom Nadaanan Probe Region of interest Probe2 a lens Custom Custom P7 N Index 1 Index 2 P5 P7 Index 1 Index 2 P5 Hybridization of custom oligonucleotide probes Extension and ligation Addition of indices and sequencing adapters by PCR Final amplicon ready for sequencing with MiSeq 00W gt TruSight Tumor Sample Preparation Guide 3 9 uoneJjedoJjg ajdwes Joun 1yBIsn1 ay ssoq MOH Supporting Information TruSight Tumor Sample Preparation Process Overview 40 The TruSight Tumor Sample Preparation process overview can be summarized into the following steps Place Your Order Place your order directly through your Mylllumina account Go to the TruSight Tumor Sample Preparation product page on the Illumina website A Mylllumina account is required When placing your order make sure you also order MiSeq reagents and additional components needed to perform the TruSight Tumor Sample Preparation See the TruSight Tumor Sample Preparation Kit Contents on page 42 in this guide for information on additional components such as filter plates Download Your Manifest Files Two manifest files are required for TruSight Tumor Sample Preparation sequencing on the MiSeq They provide the list of regions targeted by the assay which is used by the MiSeq for alignment and analysis The manifest files can be downloaded from the TruSight Tumor Sample Preparation
52. or Sample Preparation Guide 1 3 004 OBIIO JO UO eZIPLQAH TruSight Tumor Protocol Add 10 ul of each sample as prepared in step 2 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 oligo pool 1 FPA to all sample containing wells on the left half of the HYP plate Then add 5 ul of oligo pool 2 FPB to all sample containing wells on the right half of the HYP plate Change tips after each column to avoid contamination f NOTE If preparing libraries from ACD1 add 5 ul of FPA to one control well and 5 ul of FPB to the second control well If preparing libraries using ACP1 add 5 ul of ACP1 to two additional control wells of ACD1 Using a multichannel pipette add 35 ul of OHS3 to each sample in the HYP plate Gently pipette up and down 3 5 times to mix Change tips after each column to avoid contamination 1 NOTE Ensure any crystals or precipitate in OHS3 have dissolved after following procedure described in Preparation step 3 Seal the HYP plate with a heat sealer lumina recommends the Agilent PlateLoc Thermal Microplate Sealer If a heat sealer is not available use an aluminum foil seal Centrifuge at 1 000 xg at 20 C for 1 minute Place the HYP plate in the pre heated block at 95 C and incubate for 1 minute Change the temperature of the same heat block to 40 C and incubate
53. plification In this step the extension ligation products are amplified using primers that add index sequences for sample multiplexing i5 and i7 as well as common adapters required for cluster generation P5 and P7 Estimated Time Total duration 100 minutes depending on thermocycler Hands on 30 minutes Consumables Item Quantity Storage Supplied By PMM2 PCR Master Mix 2 1 tube 15 to 25 C Illumina i5 primers A5XX 1 tube per primer 15 to 25 C Illumina i7 primers A7XX 1 tube per primer 15 to 25 C Illumina TDP1 1 tube 152t01 25 E Illumina TruSeq DNA Polymerase 1 Microseal B adhesive film 1 User 0 05 N NaOH freshly prepared Asneeded User from 10 N NaOH 96 well skirted PCR plate 1 plate User Troughs As needed User Preparation 1 Prepare fresh 0 05 N NaOH by adding 20 ul of 10 N NaOH to 3 98 ml of sterile water 2 Remove PMM2 and the index primers i5 and i7 from 15 to 25 C storage and thaw on a bench at room temperature Vortex each tube to mix and briefly centrifuge the tubes in a microcentrifuge TruSight Tumor Sample Preparation Guide 2 1 uoneoyiduv HOd 3 Arrange i5 primer tubes white caps clear solution vertically in the Index Plate Fixture aligning tubes A501 through A508 with rows A through H 4 Arrange i7 primer tubes orange caps yellow solution horizontally in the Index Plate Fixture aligning tubes A701 through A712 with columns 1 through 12 Confirm i5 and i7 primer tubes are pos
54. r Sample Preparation Support page Part 15042911 Rev A Trusight Tumor Protocol Introduction u l lll ded ckotie see bud bl Le ded cade cerrada atra pad 6 TruSight Tumor Sample Preparation Workflow 7 Qualification of DNA Extracted from FFPE Samples 8 Hybridization of Oligo Pool 12 Removal of Unbound Oligos 15 Extension Ligation of Bound Oligos 20 PGR AMplificatiON L l u u u sts ecco epee yh ac cen rire co nA eod dne dee labeled sues 21 Verify Library Preparation Optional UU 25 PCR Clean Up 26 Library Quanlification ll u u aces Apa kad oda soa Magalak Jam LATA plasa hd NG 29 Library Normalization u uuu O lu saya nn RR RR RR RII is uE uQ aaa 30 Library Denaturing and Pooling 31 omit Ma Pai E asawa EE E 3 A M T TI A o ff tome mr a a pst AR ME d Secas 4 Ak Ta nail E E TruSight Tumor Sample Preparation Guide 5 c Je1deuo TruSight Tumor Protocol Introduction Th
55. r 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 Obligations 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 an
56. re supplied for 48 samples and indexes provided enable sample indexing of 4 samples per sequencing run TruSight Tumor leverages 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 High Accuracy Low Frequency Variant Detection Highly accurate somatic variant analysis at limit of detection below 5 allele frequency across 175 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 Part 15042911 Rev A DNA Input Recommendations Formalin fixed paraffin embedded FFPE human tissues are a valuable source of material for molecular analysis and clinical studies A number of processes and protocols now exist for the extraction and purification of nucleic acids from FFPE samples however as the assays used to evaluate DNA and RNA have evolved from simple monoplex PCR to higher plexity products the quality and amount of nucleic acid extracted from FFPE material has
57. rt 538619 General lab supplier General lab supplier General lab supplier Bio Rad Part MSB 1001 General lab supplier Labcor Part 730 001 General Lab Supplier Agilent 5067 1504 for 300 samples General Lab Supplier General Lab Supplier Part 15042911 Rev A Equipment Pre PCR Equipment Supplier 37 Incubator Forced Air Oven VWR International or comparable Heat Block 96 well Scigene Hybex Microsample Incubator for PCR plate Tabletop Note This model is recommended for this assay Passive cooling as opposed to active cooling performed in a PCR thermocycler is recommended for maximum target enrichment specificity and uniformity Centrifuge General lab supplier Plate centrifuge that attains designated speeds of protocol NOTE Use a dedicated set of pipettes pipette tips vortexer and centrifuge for pre amplification steps Post PCR Equipment Supplier Magnetic Stand 96 Invitrogen DynaMag 96 Side Skirted Post PCR Plate Shaker Q Instruments BioShake XP High Speed mixer Part 1808 0505 Tabletop Centrifuge General lab supplier Plate centrifuge that attains designated speeds of protocol Gel Electrophoresis General lab supplier Supplies and Apparatus Bioanalyzer System Agilent Technologies Heat Block for 1 5 ml General lab supplier centrifuge tubes TruSight Tumor Sample Preparation Guide NOTE Use a dedicated set of pipettes pipette tips vortexer heat block and centrifuge
58. s Product including without limitation any rights from third parties or rights to Application Specific IP Illumina 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 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 Part 15042911 Rev A 4 Unauthorized Uses Purchaser agrees a to use each Consumable only one time and b to use only Illumina consumables reagents with Illumina Hardware The limitations in a b do not apply if the Documentation or Specifications for this Product state otherwise Purchaser 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
59. should be given the Sample ID and Sample Name Control in your Sample Plate and Sample Sheet files i7 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 i5 Index PCR Primer Index Sequence A501 TGAACCTT 5 O Part 15042911 Rev A i5 Index PCR Primer A502 A503 A504 A505 A506 A507 A508 TruSight Tumor Sample Preparation Guide Index Sequence TGCTAAGT TGTTCTCT TAAGACAC CTAATCGA CTAGAACA TAAGTICC TAGACCTA 51 uoneJdedojg 1eeus a dwes basin o2 Part 15042911 Rev A Technical Assistance For technical assistance contact Illumina Technical Support Table 3 Illumina General Contact Information Illumina Website www illumina com Email techsupport illumina com Table4 Illumina Customer Support Telephone Numbers Region Contact Number Region Contact Number North America 1 800 809 4566 Italy 800 874909 Austria 0800 296575 Netherlands 0800 0223859 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 MSDSs Material safety data sheets MSDSs are available on the Illumina website at www illumina com msds Product Documentation Product documentat
60. space where pre PCR processes are performed DNA extraction quantification and normalization from the laboratory space where PCR products are made and processed post PCR processes Never use the same sink to wash pre PCR and post PCR troughs Never share the same water purification system for pre PCR and post PCR processes Store all supplies used in the protocols in the pre PCR area and transfer to the post PCR area as needed Use Dedicated Equipment and Supplies Dedicate separate full sets of equipment and supplies pipettes centrifuges oven heat block etc to pre PCR and post PCR lab processes and never share between processes Dedicate separate storage areas freezers and refrigerators to pre PCR and post PCR consumables Because the pre and post amplification reagents are shipped together it is important to unpack the reagents in the pre PCR lab area and then move the post amplification reagents to the proper post PCR storage area Pre PCR and Post PCR Lab Procedures To prevent PCR product contamination it is important to establish lab procedures and follow best practices Illumina recommends daily and weekly cleaning of lab areas using 0 5 Sodium Hypochlorite 10 Bleach CAUTION To prevent sample or reagent degradation make sure that all vapors from the cleaning solution have fully dissipated before beginning any processes TruSight Tumor Sample Preparation Guide 47 uoneulue1uo 19NPp01d4 HOd 1u9 9Jg Supporting
61. te equipment and consumables 3 6 Part 15042911 Rev A Acronyms Table2 TruSight Tumor Sample Preparation Acronyms Acronym ACD1 ACEN CLP DAL EBT ELM3 FPA FPB FPU HT1 HYP IAP LNP OHS3 PAL PMM2 QCP Definition Amplicon Control DNA 1 Amplicon Control Oligo Pool 1 CLean up Plate Diluted Amplicon Library Elution Buffer with Tris Extension Ligation Mix 3 TruSight Tumor Oligo Pool A TruSight Tumor Oligo Pool B Filter Plate Unit Hybridization Buffer HYbridization Plate Indexed Amplification Plate Library Normalization Plate Oligo Hybridization for Sequencing Reagent 3 Pooled Amplicon Library PCR Master Mix 2 Quality Control Primers Quality Control Template TruSight Tumor Sample Preparation Guide 3 e SUWUAUOIOV Supporting Information Acronym 38 Definition StoraGe Plate Stringent Wash 1 TruSeq DNA Polymerase 1 Universal Buffer 1 Part 15042911 Rev A How Does the TruSight Tumor Sample Preparation Assay Work For each amplicon two 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 followed by extension and ligation to form DNA templates consisting of the regions of interest flanked by universal primer sequences Using indexed primers supplied with the kit DNA templates are then amplified by PCR The library products are then pooled int
62. to mix well You will add this mix to the IAP plate in the next section f NOTE Do not vortex When the 45 minute extension ligation reaction is complete remove the FPU from the incubator Remove the aluminum foil seal and replace with the filter plate lid Removing the aluminum foil seal before centrifugation is recommended to ensure the reaction supernatant will drain into the waste plate effectively Centrifuge the FPU at 2 400 xg for 2 minutes Using a multichannel pipette add 25 ul of 0 05 N NaOH to each sample well on the FPU plate Ensure the NaOH is fully dispersed across each membrane by gently pipetting up and down if necessary Incubate the FPU plate at room temperature for 5 minutes While the FPU plate is incubating 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 Change tips between samples Transfer samples eluted from the FPU plate to the IAP plate as follows a Seta multichannel P20 pipette to 20 ul TruSight Tumor Sample Preparation Guide 2 2 uoneoyiduv HOd b Pipette the contents in the first column of the FPU plate up and down 5 6 times then transfer 20 ul from the FPU plate to the corresponding column of the IAP plate Gently pipette up and down 5 6 times to thoroughly combine the DNA with the PCR master mix NOTE Slightly tilt the FPU plate to ensure complete aspiration and to avoid air bubbles c Transfer the remaining
63. uSight Tumor Sample Preparation Process Overview 40 TruSight Tumor Sample Preparation Kit Contents 42 User Supplied Consumables 44 Equipment 45 Prevent PCR Product Contamination MiSeq Sample Sheet Preparation Technical Assistance TruSight Tumor Sample Preparation Guide Vl Part 15042911 Rev A Overview got o AA AA IA 2 DNA Input Recommendations 3 AdditionaliRESOUNCES uuu seco uu u uuu nasa yaa nas isis its 4 a hi K Ka ape Dan Y NG PATA Y y 5 N NN 33 Z MEE K EI a aawat rt B NU NG aaa y e Tree lt tt rar aga n AATGCGGCA rey rogas TC6T OH pao id d un ocres pa Fi o ar md o kA Z yt aS TruSight Tumor Sample Preparation Guide 1 LJe3deuo Overview Introduction TruSight Tumor Sample Preparation takes a deeper view of variation in solid tumors including lung colon melanoma gastric and ovarian This enables clinical researchers to look beyond point mutations within hot spots in single genes for a more comprehensive view of somatic variation The TruSight Tumor content set provides amplicon based library preparation reagents DNA QC sample indexes and oligos targeting identified regions of interest Sufficient reagents a
64. 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 Hardware 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 TruSight Tumor Sample Preparation Guide IV 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 warranty for the Hardware unless the upgrade was 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 Spe
65. with the appropriate seal depending on the instrument that you are using taking care to avoid cross contamination and to avoid smudging the surface of the lids Place the plate on an adapter if needed and centrifuge the plate to 250 xg for 1 minute Ensure the seal is free of any liquid or dust place the plate on the gPCR machine in the correct orientation then close the lid and run the following thermal profile Procedure Temperature Time Hot Start 50 C 2 minutes 95 C 10 minutes x40 95 C 30 seconds 60 C 30 seconds 72 C 30 seconds Confirm that the instrument captures images after the 72 C step Part 15042911 Rev A 17 18 19 20 NOTE The Cq threshold should be set to a value that avoids inaccurate measurements due to background 100 RFU on the BioRad 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 QCT amplification Ensure that there is good amplification for the QCT and remove outliers from a triplicate group that are gt 0 5 Cq different from the rest of the group J NOTE Four or more outliers per plate indicate technical errors Replicates exhibiting abnormal amplification curves should be excluded see Illumina sequencing white paper Generating Sequencing Libraries Using DNA from FFPE Samples for more details Subtract the average Cq for the QCT from the average Cq for each sample

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