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TruSight DNA Amplicon Sequencing Panel - Support

1. Beads 1 LNW1 Library Normalization Wash 1 LNS2 Library Normalization Storage buffer 2 0 1 N NaOH less than one week old 96 well skirted PCR plate 15 ml conical tube Microseal B adhesive film 26 Quantity 1 tube 1 tube 2 tubes 1 tube 3 ml per 96 samples 1 plate 1 tube As needed Storage 25 C to 15 C LAU AC 2 C to 8 C Room temperature Supplied By Illumina Illumina Ilumina Ilumina User User User User Part 15054779 Rev B Y y WARNING A component in 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 applicable local governmental safety standards For more information see the SDS for this kit at support illumina com sds ilmn Y WARNING This set of reagents contains mercaptoethanol Perform the following procedure in a hood or well ventilated area if desired Preparation 1 Prepare fresh 0 1N NaOH 2 Remove LNAI from 25 C to 15 C storage and bring to room temperature Use a 20 C to 25 C water bath as needed dy NOTE LNA1 might form visible precipitates or crystals Before use vortex vigorously and then hold the tube in front of a light and visually inspect to make sure that all precipitate has dissolved 3 Remove LNB1 and LNW
2. 10 minutes Significantly incomplete drainage of SW1 compromises target enrichment specificity Repeat the wash as follows a Using a multichannel pipette add 45 ul of SW1 to each sample well Take care to avoid cross contamination or change tips between columns b Cover the FPU plate with the filter plate lid and centrifuge to 2 400 x g for 5 minutes y NOTE If the SW1 does not drain completely after 5 minutes the plate can be centrifuged again for up to 10 minutes Significantly incomplete drainage of SW1 compromises target enrichment specificity 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 reused for the rest of the pre amplification process Using a multichannel pipette add 45 ul of UB1 to each sample well Take care to avoid cross contamination or change tips between columns Cover the FPU plate with the filter plate lid and centrifuge the FPU at 2 400 x g for 5 minutes NOTE i If the UB1 does not drain completely after 5 minutes the plate can be centrifuged again for up to 10 minutes Part 15054779 Rev B Extension Ligation of Bound Oligos This process connects the hybridized upstream and downstream oligos A DNA polymerase extends from the upstream oligo through the targeted region followed by ligation to the 5 end of the downstream oligo using a DNA ligase The ex
3. 5 ul of each library to be sequenced from the SGP plate column by column to a PCR 8 tube strip Change tips after each column to avoid cross contamination Seal SGP with Microseal B and set aside 4 NOTE After use store the sealed SGP plate at 25 C to 15 C Combine and transfer the contents of the PCR 8 tube strip into the PAL tube Mix PAL well Apply the DAL Diluted Amplicon Library barcode sticker to a fresh Eppendorf tube Create DAL by combining the volumes of HT1 and PAL indicated in Table 2 based on your MiSeq Reagent Kit version Upon transferring PAL using the same tip pipette up and down 3 5 time to rinse the tip and ensure complete transfer E NOTE e Volumes for diluting PAL with HT1 were established using recommended equipment e g plate shaker calibrated for shaking speed Typical laboratory conditions e g 20 C to 25 C were strictly followed during the normalization procedure If cluster density is too high or too low adjust the dilution ratio to better suit the equipment temperature and handling in your laboratory after validation Table 2 Pooling Dilution Volume of HT1 Volume of PAL MiSeq v2 594 ul 6 ul MiSeq v3 580 ul 20 ul Mix DAL by vortexing the tube at top speed r NOTE i If you would like to save the remaining PAL for future use store the PAL tube at 25 C to 15 C Make sure that the diluted library DAL is freshly prepared and used immediately for MiSeq loading Storage of the DAL result
4. DNA Amplicon Sequencing Panel Library Prep Kit Contents 40 User Supplied Consumables 22200000 43 Edlprelibs e pci eo rui A o 44 MiSeq Sample Sheet Preparation 0 0000022 c cece cece eee cece e cece ee ees 46 Illumina Amplicon Viewer 2222222 200022 cece cece ccceeeeccccecccceeeeeeees 48 Technical ASSISTANCE teta a austen 49 TruSight DNA Amplicon Sequencing Panel Library Preparation Guide IX Part 15054779 Rev B Overview LJe1deuo IAUOGUCTION AA lul ex c np ete Isic DULL LE ID ee eet IA LEE UE 2 DNA Input Recommendations 2 2 2 2 2 22 e cece cece cece cc cece ceceeeeeceeeeeceeeeeeeeees 3 AdditionalResoUl ces t A O i last ais 4 Unis Ga My i Mina AA py a e zoe es gt na n 7 ji s uL ar Tarna ar pa S TGCGGCATEA rGGASTOSTC OO T 4 A p OCR di 5 AU vore A e S tow Se TruSight DNA Amplicon Sequencing Panel Library Preparation Guide 1 Overview Introduction The TruSight DNA Amplicon Sequencing Panel Library Prep Kit uses the proven TruSeq Custom Amplicon TSCA assay and allows you to sequence targeted regions of the genome The targeted regions span upwards of 600 kb with up to 1 536 amplicons in a single multiplex reaction This highly targeted approach enables a wide range of applications for discovering validating and screening genetic variants in a rapid and efficient manner TruSight
5. DNA Amplicon Sequencing Panel enables a high level of multiplexing by generating up to 1 536 amplicons within a single reaction and integrated indexes support sequencing up to 96 samples per MiSeq run The TruSight DNA Amplicon Sequencing Panels leverages the long paired end read capability speed and high data quality of the MiSeq System Excellent Multiplexing Capability Amplify up to 1 536 amplicons in a single reaction and sequence up to 96 samples in a single MiSeq run Revolutionary Assay with Fast and Simple Workflow Generate up to 1 536 amplicons across 96 samples within a single plate with less than 3 hours hands on time Automated Data Analysis Perform variant calling and analysis across all samples using simple on instrument automated analysis software Complete Amplicon Sequencing Solution for MiSeq Get the convenience of a fully integrated DNA to data solution from assay sequencing and automated data analysis to offline software for reviewing results Part 15054779 Rev B DNA Input Recommendations Supported Type of DNA Amplicon Size Input Up to 15 ul High quality 150 175 250 50 ng recommended genomic DNA 425 bp Input DNA Quantitation Illumina recommends quantifying the starting genomic material Quantify the starting genomic material using a fluorescence based quantification method such as PicoGreen rather than a UV spectrometer based method Fluorescence based methods which employ a double stra
6. 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 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 A adhesive seals Microseal B adhesive seals PCR 8 tube strips Solution basin PVC non sterile trough Agarose gel 276 for 250 bp and 425 bp amplicons or 476 for 150 bp 175 bp and 250 bp amplicons DNA 1000 Kit for Bioanalyzer DNA molecular weight markers Ice bucket TruSight DNA Amplicon Sequencing Panel Library Preparation Guide Supplier General lab supplier Bio Rad Part MSP 9601 Fisher Scientific Part AB 0859 Fisher Scientific Part 4 AB 0765 Beckman Coulter Part A63881 A63880 Beckman Coulter Part 538619 General lab supplier General lab supplier General lab supplier Bio Rad Part MSA 5001 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 43 so qeuunsuo pelddns 1esn Supporting Information Equipment Pre PCR Equipment 37 incubator Heat block 96 well Tabletop centrifuge d NOTE Supplier Forced Air Oven VWR International or comparable Scigene Hybex Microsample Incubator for PCR
7. and not the EUC Enables you to create and edit appropriate sample sheets for Illumina sequencers and analysis software and record parameters for your sample plate Use the TruSeq Amplicon IEM workflow when creating sample sheets for the TruSight DNA Amplicon Sequencing Panel Part 15054779 Rev B Protocol Introduction cnc cnc ns 6 TruSight DNA Amplicon Sequencing Panel Library Prep Kit Workflow 7 Hybridizatioriof Oligo POOL zit ssa Cossio a dead tl 8 Removal of Unbound Oligos ooo 11 Extension Ligation of Bound Oligos 2 2 0 ccc ceccccccccccccccececccccceceeccccecceeeeeeees 15 PGR AMPI ANON e e o o Dee Le du REEL aa EAS 16 PCR Clean Up ess soon hec esencia e ot E CLE e Pe Ad 21 Library Normalization ecserin eee cece cece eee e cece RR RR cece cee ceeceeeeeeeeees 26 Library Pooling and MiSeq Sample Loading 200 00 c cece eee c eee eccecccceceeeees 31 a gt j IA E x SV kA A x d A A ty ini o coi yi A dl i Ed pos 1 ban gt a TruSight DNA Amplicon Sequencing Panel Library Preparation Guide 5 c Je1deuo Protocol Introduction This chapter describes the TruSight DNA Amplicon Sequencing Panel Library Prep Kit protocol Review Best Practices before proceeding See Additional Resources on page 4 for information on how to access TruSight DNA Amplicon Sequencing Panel Library Prep Kit Best Practices on the Illumina website Revie
8. been used in a previous assay After the pre wash step if there is a significant amount gt 15 ul well of residual buffer in multiple wells 210 wells plate switch to a fresh filter plate y NOTE Illumina strongly recommends keeping spare filter plates FC 130 1006 on hand as general lab supplies After the 80 minute incubation confirm that the heat block has cooled to 40 C While the HYP plate is still in the heat block reinforce the seal using a rubber roller or sealing wedge Remove the HYP plate from the heat block and centrifuge to 1 000 x g at 20 C for 1 minute to collect condensation Using a multichannel pipette set to 65 ul transfer the entire volume of each sample onto the center of the corresponding pre washed wells of the FPU plate Take care to avoid cross contamination or change tips between columns Cover the FPU plate with the filter plate lid and centrifuge the FPU at 2 400 x g at 20 C for 5 minutes Wash the FPU plate as follows a Using a multichannel pipette add 45 ul of SW1 to each sample well Changing tips between columns is not required if you use care to avoid cross contamination b Cover the FPU plate with the filter plate lid and centrifuge the FPU at 2 400 x g for 5 minutes TruSight DNA Amplicon Sequencing Panel Library Preparation Guide 1 3 soBIJO PUNOJUN Jo enouay Protocol t NOTE i If the SW1 does not drain completely after 5 minutes the plate can be centrifuged again for up to
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11. the reaction supernatant drains into the waste plate effectively Centrifuge the FPU at 2 400 x g for 5 minutes Using a multichannel pipette add 25 ul of 50 mM NaOH to each sample well on the FPU plate Ensuring that pipette tips come in contact with the membrane pipette the NaOH up and down 5 6 times Tips must be changed after each column 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 Part 15054779 Rev B a Seta multichannel P20 pipette to 20 ul b Using fine tips pipette the NaOH 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 combine the DNA with the PCR master mix d NOTE Slightly tilt the FPU plate to ensure complete aspiration and to avoid air bubbles c Transfer the remaining 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 Put the metal adapter collar away for future use If only a partial FPU plate is
12. used clearly mark which wells have been used Store the FPU plate and lid in a sealed plastic bag to avoid contamination of the filter membrane 7 Cover the IAP plate with Microseal A film and seal with a rubber roller 8 Centrifuge to 1 000 x g at 20 C for 1 minute 9 Transfer the IAP plate to the post amplification area TruSight DNA Amplicon Sequencing Panel Library Preparation Guide 1 9 uoneoyiduv HOd Protocol 10 Perform PCR on a thermal cycler using the following program and the recommended number X of PCR cycles The following table contains the number of amplicons in your TSO and the number of PCR cycles required NOTE The ACD1 ACP1 control can be processed using the same conditions as your TSO Table 1 50 99 ng Amplicon Size Number of PCR Cycles X 96 amplicons 33 97 384 amplicons 28 385 768 amplicons 27 769 1 536 amplicons 26 95 C for 3 minutes X cycles of 95 C for 30 seconds 66 C for 30 seconds 72 C for 60 seconds 72 C for 5 minutes Hold at 10 C SAFESTOPPINGPOINT 1 If you do not plan to proceed to PCR Clean Up on page 21 immediately the plate can remain on the thermal cycler overnight You can also store it at 2 C to 8 C up to two days If storing at 2 C to 8 C replace Microseal A with Microseal B 2 O Part 15054779 Rev B PCH Clean Up This process uses AMPure XP beads to purify the PCR products from the other reaction components Estimated Time Total duration 50 m
13. well Take care to avoid cross contamination or change tips between columns b Seal the LNP plate with a Microseal B adhesive seal c Shake the LNP plate on a microplate shaker at 1 800 rpm for 5 minutes Place the plate on the magnetic stand for 2 minutes or until the supernatant has cleared e Carefully remove and discard the supernatant in an appropriate hazardous waste container Remove the LNP plate from the magnetic stand and repeat the wash with LNWI as follows a Using a multichannel pipette add 45 ul of LNW1 to each well Take care to avoid cross contamination or change tips between columns b Seal the LNP plate with a Microseal B adhesive seal c Shake the LNP plate on a microplate shaker at 1 800 rpm for 5 minutes Place the plate on the magnetic stand for 2 minutes or until the supernatant has cleared e Carefully remove and discard the supernatant in an appropriate hazardous waste container f Use a P20 multichannel pipette to remove excess LNW1 Sv NOTE Using a P20 multichannel to remove residual LNW1 is important to avoid reagent carryover into the storage buffer and to reduce volume variability which would affect library normalization Remove the LNP plate from the magnetic stand and add 30 ul of 0 1 N NaOH less than a week old to each well to elute the sample Seal the LNP plate with a Microseal B adhesive seal Shake the LNP plate on a microplate shaker at 1 800 rpm for 5 minutes During the 5
14. 222 i 48 o iff rre spa 16 A sr TruSight DNA Amplicon Sequencing Panel Library Preparation Guide 3 5 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 of the requisite consumables and equipment 3 6 Part 15054779 Rev B How Does the TruSight DNA Amplicon Sequencing Panel Library Prep Kit Assay Work One pair of oligos is designed for each amplicon Hybridization of oligos to genomic DNA occurs in a 96 well plate 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 PCR amplifies DNA templates pools the templates into a single tube and sequences them on the MiSeq System Probe 1 Region of interest Probe 2 Wes V P7 Nindex 1 Index 2 PS D P7 Index 1 Index 2 P5 Hybridization of oligonucleotide probes Extension and ligation Addition of indexes and sequencing adapters by PCR Final amplicon ready for sequencing with MiSeq UOU gt TruSight DNA Amplicon Sequencing Panel Library Preparation Guide 37 Buiduanbas uooi duuy VNG 1uBignu ay Seog MOH Supporting Information Acronyms 38 Table 3 TruSeq Custom Amplicon Library Preparation Acronyms Acronym ACD1 ACP1 TSO CER DAL EBT ELM4 FPU HT1 HYP LNA1 L
15. 5 Apply the IAP Indexed Amplification Plate barcode plate sticker to a new 96 well PCR plate TruSight DNA Amplicon Sequencing Panel Library Preparation Guide 1 y Protocol Procedure 18 10 1 Using a multichannel pipette add 4 ul of i5 primers clear solution to each column of the IAP plate Changing tips between columns is not required To avoid index cross contamination discard the original white caps and apply new white caps provided in the kit Using a multichannel pipette add 4 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 provided in the kit Remove all the index primer tubes from the working area For 96 samples add 56 ul of TDP1 to 2 8 ml of PMM2 1 full tube For fewer than 96 samples calculate the volumes of TDP1 and PMM2 needed Invert the PMM2 TDP1 PCR master mix 20 times to mix well You will add this mix to the IAP plate in the next section Unused volume is already included in the calculation y NOTE Always add TDP1 to PMM2 before use Never store the combined PMM2 TDP1 master mix 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
16. 779 Rev B Library Pooling and MiSeq Sample Loading 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 Estimated Time Total duration 10 minutes Hands on 10 minutes Consumables Item Quantity Storage HT1 Hybridization Buffer 1 tube 25 C to 15 C MiSeq reagent cartridge 1 cartridge PIC iD SAC Eppendorf tubes screw cap 2 tubes recommended PCR 8 tube strip 1 2 5 LIce bucket 1 Preparation 1 Set a heat block suitable for 1 5 ml centrifuge tubes to 96 C Supplied By Illumina Illumina User User User 2 Remove a MiSeq reagent cartridge from 25 C to 15 C storage and thaw at room temperature 3 han ice bucket prepare an ice water bath by combining 3 parts ice and 1 part water Procedure 1 If the SGP plate was stored frozen thaw the SGP plate at room temperature 2 Centrifuge the SGP plate at 1 000 x g at 20 C for 1 minute to collect condensation TruSight DNA Amplicon Sequencing Panel Library Preparation Guide 31 Duipeo ajduwes basi pue Buljoog AJejgr1 Protocol 32 Apply the PAL Pooled Amplicon Library barcode sticker to a fresh Eppendorf tube If the SGP plate was stored frozen mix each library to be sequenced by pipetting up and down 3 5 times using a P200 multichannel pipette Change tips between samples Using a P20 multichannel pipette transfer
17. AACAGTAACACACT TCTGT T AAGATTACTTGATCCACTGATTCAACGTACCG TAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAI TAAGAGC TACCGTCT TCTGT T AAGAI T POLI GA SE SAL CAO ATACCGT AAC AAC TACA AA G TACITGATOGACIGATTOAA ATA CCGTAACGAACGTATCAATTGAGAC TAAATAT TAACGTACCAT TAAGAGCTACCGT GCAACGA CORARACAN AGAT TAACAGTAACACACTTCTGTTAACCT T GAL COACTOATTOAACQITAAGAHACTIGATCCACTGATIGAACGTACCGTAACGAACG TAI CAATTGAGC TTC TG HACC TAAG TTACI IGATCCACT GAT CAAGOTACCCTAACGAACG TAY CARTTGAGACTAGGAACGACG IAAAAGAATGATAACAGTAACACACT TCTGT TAACCT TAAGAT TACTTGATCCACTGAT TCAACGTACCGTAAAGAT TACT TGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT T GAGACTAAATAT TAACGTACCAT TAAGAGCTACC m lumina 5200 Illumina Way San Diego California 92122 U S A 1 800 809 ILMN 4566 1 858 202 4566 outside North America techsup port illumina com www illumina com
18. AAGATIAC TT GATCCACT GAT IG AACGT A EAS en ne TM T GAT TACTTGATCCAC WB E ee RR A SALES RE VTACTTGATCCTTA BE AE AA AC GATTACTTGATCCACTGATTCAACGTACTTCTGTIAACCTTAAGATTACTTGATOCAC GAATGATAAAT T GGAAAAGAATGATAACAG TAACACAC IS GTTAACCTIAAGAT TACTTGATCCACTGATTCAACGTACCG TAAAGAT TAG T TGAT AAGAGCTACCGTAAC AGAATGATAACAGTAACACACT TCTGT TAACCT TAAGAT TACT TGATCCACTGAT TCAACG TACCGTAACGAAC GTATCAATTGAGACTA CACACTTCTGTTAAAA ACGTACCAT TAAGAGCTACCGTGCAACAGTAACACACT TCTGTTAACCT TAAGAT TACT TGATCCACTGAT TCAACGTACCGTAAC AAGAATGATAACAT TA AGAATGATAACAG TAACACACT TCTGT TAACCTTAAGAT TACTTGAT CCACT GAT TCAACGTACCG TAACGAACGTAT AATTGAGAC T CTGATTCAACGTAAAA CAACGTACCGTAACGAACGTATCAT TAAGAT TACTTGATCCAC TGATTCAACGTACCGTAACGAACG TATCAAT TGAGACTAAATAT TAA TCTGTTAACCTTAAGAA BALIAGT IGATCGAG GAL ICAACG TAAGATIAG LIGATCCAGT GALI CAACGTACCG AACGAAGG TATOAALTGAGCT TO IGT AAG HAGCAACGACGAAAAC SGACGAAAAGAATGATAACAG TAACACACT TCTGT TAACCT TAAGAT TACTTGATCCACTGAT TCAACGTACCGTAAAGAT TACTTGAT C TTAAGAGCTACCGTAAC FOR RESEARCH USE ONLY ILLUMINA PROPRIETARY Catalog FC 130 9007DOC Part 15054779 Rev B October 2014 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 d
19. ATCAAT T GAGAC TAAAT AAA OTACCATTAACACOTACO CCAT TAAGAGCTACCGTGCAACAGTAACACACT TCTGTTAACCT TAAGATTACT TGATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTGCAACGACGAAAAGAAT GATAA 3ATAACAGTAACACACTTCTGTTAACCT TAAGATTACTTGATCCACTGATTCAACGTACCG TAACGAACGTAT AAT TGAGACTAAATAT TAACGTACCAT TAAGAGC TACCG TCTTCTGTTAACCT TAAGAT TACTTGATCCACTGAT TCAACG ZI TGATCCACTGATTCAACGTTAAGAT TACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGCT TCTGT TAACCTTAAGAT TACT TGATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT T GAGAC TAGCAACGACQ EAM ETATE EN AT A RAI AT ALTA D A ALT C A A Ve ROV VAN nf ie aL aTACCGTAACGAACGTATCATTAAGATTACTTGATCCAC TGATTCAACGTACCGTAACGAACG TATCAAT TGAGA e eS RA pl Fess CGACGAAAAGAATGATAACAGTAACACACTTCTGT TAACCT T ZTTGATCCACTGATTCAACGTIAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACG ATCAAT TRGAGCTTCTGT TAACCT TAAGAT TACT TGATCCACTGAI TCAACGTACCGTAACGAACGTAT C ANT GA ACTAS AA BAG IAAAAGAATGATAACAGTAACACAC T TCTGT TAACCT TAAGAT TACTT GATCCACTGAT TCAACGTACCGTAAAGAT TACT TGATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACC 3ATAACAGTAACACACT TCTGTTAACCT TAAGAT TACT T ACE ADD MEE UA Ee e AAATATTAACGTACCAT TAAGAGCTACCGTCTTCTGTTAACCT TAAGAT TAI SIUE UMS CIE ZACTGATTCAACGTACCAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TRAGACTAAATAT TAACGTACCAT TAAGAGC TACCGT CTTC TGT TAACCT TAAGAT TACT T GATCCAC TGATTCAACGTACCGTAACGA ANAAGAATGATAACAGTAACACAC TI CTO TTAACCTTAAGATAGTIGATCCACTOATT CAACG TACOS IAAAGATIAC TT GATOCACTOAT
20. CATTAAGAGCTACC 3aATAACAGTAACACACT TCTGTTAACCT TAAGAT TACT TGT TGATCCACTGATTCAACGTACCG TATCAAT TGAGAC TAAATAT TAACGTACCAT TAAGAGCTACCGTCTTCTGT TAACCTTAAGATTACTTGATCCACTGAT TCAACGTACCGT ATA TEMO REG ADIAT SET TR TOIT e AA EA AA AT TS UNI IAAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGAT TACTT GATCCACTGAT TCAACGTACCGTAAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACC 3ATAACAGTAACACACT lCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACG TATCAAT TGAGACTAAATATTAACGTACCAT TAAGAGCTACCGTCTTCTGTTAACCT TAAGATTACTTGATCCACTGAT TCAACG TGAGACTAAATAT TAACGTTGTTAACCTTAAGATTACTTGATCCACTGATTCAACG TACCGTAACGAACG TATCAAT TGAGACTAAATATTAACGTACCAT TAAGAGCT TCTGT TAACCT TAAGAT TACTTGATCCACTGAT TCAACG TACCG TA ATCAAT TGAGAC TAAATAT TAACGTACT TAACCT TAAGAT TACT TGATCCACTGATTCAACGTACCGTAACGAACG CT TCTGTTAACCTTAAGAT TACTT GATCCACTGATTCAACGTACCGTAACGAACG TATCAAT T GAGAC TAACGACGA 3AC TAAATAT TAACGTACCAT TAAGAGCTACAACCT TAAGAT TACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGC TACCGTGCAACGACGAAAAGAAT GATAACAG TAACACA RUINA SEI ARTES MUSS A TO EE US elles He IO STU ER ETE eS EUN e AE CAOS TO COTA ae Ar CCATTAAGAGC TACCGTGCAACTTAACCTTAAGAT TACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT T GAGACTAAATAT TAACG TACCAT TAAGAGCTACCGTGCAACGACGAACT T CTGT TAACCT T e Ee EA 3CIACCGTGCAACGAAAATAACCTTAAGATTACT TGATCCACTGAT TCAACGTACTTCTGTTAACCTTAAGAT TACTTGATCCACTGATTCAACGTACCG TAACGAACG TATCAAT TGAGAC TAAGCTACCGTGCAACGACGAAAAGAAT GAT AAAAGAATGA
21. CCACTGA VACGTACCATTAAGAGCTA KA RASCI TCCACT GAT TCAACGTA CGAACGTATCAAT TGAGA AACGTACCAI TAAGAGC T CAACGACG TG AT TACTTGATCCT TA AAGAGCTACCG C TRAGATIAG PTGATCOACTOATICARCGN ACTTCTGTIAACCTIAAGAT TACTT GATCCACTGATTCAACGTA TAACGAACGTATCAATTGAGACTAAGCTACCGTGCAAC TT G GA A ENCGANAAGAAT GATAACAGTAACACACTYGTGTAACG TIAAGAT TACTTGATOCACTGAT TOAA GTACCO TAAAGALIAG LIGATCOACTGATIGAACGTAGCG TAACGAAGG TATCAATT GAGAGTAAATATTAACGTACCATTAAGACGTACUGTAAL AGAATGATAACAGTAACACACT TCTGT TAACCT TAAGAT TACT TGATCCACTGAT TCAACG TACCG TAACGAACGTATCAAT TGAGAC TAAATAT TAACGTACCAT TAAGAGCTACCGT GCAACGACGAAAAGAAT GATAACAG TAACACACTTCTGT TAAAAL ACGTACCAT IAAGAGCTACCGTGCAACAGTAACACACT T TT TATO CT ACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGAC TAAATAT TAACG TACCAT TAAGAGCTACCGTGC OSVETU GATAACATTAY AGAAT GATAACAGTAACACACTTCTGTTAACCT TAAGATTACTTGAT CCACTGATTICAACGTACC ERE E I TGATCCACTGATTCAACGTAAAA SANTAS TOATOGACTON TOAACS TAAGATTAGT IGATOGAC GAT CAACGTACCG TRA GGAACGTATORATTOAGOII ae EE eee Oe SES ar GATAA RON TT TTAAGAA CT UCM ee SIL CON M EA AAA ACTIC AOL OTIRAG AEH TARA IR eae ACA EA TACTA CACI N TON AGB Rao AGACITA AAA PRACA e ACTGATT AACETACOCTAACGAACGTALCAT TAAQATTACTIGATOCACIOATTORAGOTAC CTAACGAACCTATOAATIGAGAC TAAATAT TAACGTACCATTAAGAGCTACC C AATGATAA AGTAACACACI TOTGTTAA PT AAGAN Y TGATAACAGTAACACACTTCTGTTAACCT TAA GATCCACTGATTCAACG GAI TCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATAT TAACGTACCAT TAAGAGCTACCG TAAC NUM Eel ng e E i AAGAT TAC
22. I from 2 C to 8 C storage and bring to room temperature Use a 20 C to 25 C water bath as needed 4 Vigorously vortex LNB1 for at least 1 minute with intermittent inversion until the beads are well resuspended Make sure that there is no pellet found at the bottom of the tube when the tube is inverted TruSight DNA Amplicon Sequencing Panel Library Preparation Guide P T uonezi euJoN AJeJqr Protocol Procedure 28 1 For 96 samples add 4 4 ml of LNAT to a fresh 15 ml conical tube NOTE i If you do not plan to use full tubes for 96 samples a P1000 set to 1000 ulis required to resuspend the beads completely in step 2 Mix only the required amounts of LNA1 and LNBI for the current experiment Never use a P200 pipette to handle LNB1 Store the remaining LNA1 and LNB1 separately at their respective recommended temperatures To preserve stability never freeze LNB1 beads or mix with LNA1 if not used immediately Use a P1000 pipette set to 1000 ul to resuspend LNB1 thoroughly by pipetting up and down 15 20 times until the bead pellet at the bottom is resuspended NOTE It is critical to resuspend the LNB1 bead pellet at the bottom of the tube The use of a P1000 ensures that the beads are homogeneously resuspended and that there is no bead mass at the bottom of the tube Resuspension is essential for achieving consistent cluster density on the flow cell Immediately after LNB1 is thoroughly resuspended use a P1000 pipette to
23. LIABILITY OR OTHERWISE SHALL IN NO EVENT EXCEED THE AMOUNT PAID TO ILLUMINA FOR THIS PRODUCT 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 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 desig
24. LNP Plate HYP Plate Removal of Unbound Oligos Hands On 20 min Reagents LNA1 swi LNB1 UBI LNW1 LNS1 Output FPU Plate BE Output SGP Plate Extension Ligation of Library Normalization Hands On 30 min Processing 50 min Reagents Library Pooling for MiSeq Bound Oligos S Sequencing Hands On 5 min E Incubation 45 min Hands On 10 min Reagents Reagents ELM3 HTI Output Output FPU Plate PAL amp DAL tubes PCR Amplification Hands On 30 min Cycle Time 85 105 min Reagents PMM2 TDP1 i5 primers 17 primers Nal Output IAP Plate Pre Amp PostAmp YF Cold Storage Option TruSight DNA Amplicon Sequencing Panel Library Preparation Guide eL Aeq jaueg Bulouenbss uooljduy VNG 1UBISNI Protocol Hybridization of Oligo Pool During this step an oligo containing upstream and downstream oligos specific to your targeted regions of interest is hybridized to your genomic DNA samples Y y WARNING A component in 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 applicable local governmental safety standards For more information see the SDS for this kit at support illumina com sds ilmn Estimated Time Total duration 1 hour 35 minutes Hands on 15 minutes Consumables Item Quantity Storage
25. NB1 ENP LNS2 LNW1 OHS2 PAL Definition Amplicon Control DNA 1 Amplicon Control Oligo Pool 1 TruSight Oligos Clean up Plate Diluted Amplicon Library Elution Buffer with Tris Extension Ligation Mix 4 Filter Plate Unit Hybridization Buffer Hybridization Plate Indexed Amplification Plate Library Normalization Additives 1 Library Normalization Beads 1 Library Normalization Plate Library Normalization Storage Buffer 2 Library Normalization Wash 1 Oligo Hybridization for Sequencing Reagent 2 Pooled Amplicon Library Part 15054779 Rev B Acronym Definition PCR Master Mix 2 StoraGe Plate Stringent Wash 1 TruSeq DNA Polymerase 1 Universal Buffer 1 TruSight DNA Amplicon Sequencing Panel Library Preparation Guide 39 SUJ UOJOY Supporting Information TruSight DNA Amplicon Sequencing Panel Library Prep Kit Contents The TruSight DNA Amplicon Sequencing Panel Library Prep Kit contains the following components and is shipped on dry ice unless specified otherwise 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 DNA Amplicon Sequencing Panel Library Prep Kit v1 5 Catalog FC 130 1010 Box 1 Pre Amplification Acronym Reagent Name Storage Temperature Area ACD1 Amplicon Control DNA 1 25 C to 15 C Pre Amp ACP1 Amplicon Control Oligo Pool 1 25 C to 15 C Pre Amp OHS2 Oli
26. PMN2 PCR Master Mix 2 1 tube 25 C to 15 C Illumina i5 primers A5XX 1 tube per primer 25 C to 15 C Ilumina 17 primers A7XX 1 tube per primer 25 C to 15 C Illumina TDP1 1 tube 25 C to 15 C Illumina TruSeq DNA Polymerase 1 Microseal A adhesive film 1 User 50 mM NaOH less than one 3 5 ml for 96 samples User week old prepared from 10 N NaOH 96 well skirted PCR plate 1 plate User Troughs As needed User Preparation 16 1 Prepare fresh 50 mM NaOH 2 Remove PMM2 and the index primers i5 and i7 from 25 C to 15 C storage and thaw on a bench at room temperature Allow approximately 20 minutes to thaw PMM2 and the index primers Part 15054779 Rev B 3 After the index primers are thawed vortex each tube to mix and briefly centrifuge the tubes in a microcentrifuge Use 1 7 ml Eppendorf tubes as adapters for the O microcentrifuge E NOTE 5 For low throughput runs with low numbers of index combinations the Index Plate Fixture is not needed The indexes can be added to the appropriate wells of the IAP plate o manually zh 4 Arrange the primers in a rack i e the TruSeq Index Plate Fixture using the following d arrangements ct a Arrange i5 primer tubes white caps clear solution vertically aligned with rows A O through H 2 b Arrange i7 primer tubes orange caps yellow solution horizontally aligned with columns 1 through 12 A i5 primers white caps B i7 primers orange caps C IAP plate
27. SeqMonitor 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 Part 15054779 Rev B 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 TruSight DNA Amplicon Sequencing Panel Library Preparation Guide 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
28. Supplied By TSO TruSight Oligos 1 tube 25 C to 15 C Ilumina OHS2 Oligo Hybridization 1 tube DIE io SAC Illumina for Sequencing 2 ACD1 1 tube 25 C to 15 C Illumina Genomic DNA As needed 25 C to 15 C User See DNA Input Recommendations on page 3 96 well skirted PCR plate 1 plate User Adhesive aluminum foil seal 2 seals User Troughs As needed User 8 Part 15054779 Rev B Preparation 1 2 Procedure Follow the DNA Input Recommendations on page 3 to quality quantitate DNA samples Remove the TSO OHS2 ACD1 and genomic DNA from 25 C to 15 C storage and thaw at room temperature 4 NOTE OHS2 might form visible precipitates or crystals Before use vortex vigorously and then hold the tube in front of a light and visually inspect to make sure that all precipitates have dissolved Set a 96 well heat block to 95 C Preheat an incubator to 37 C to prepare for the extension ligation step E NOTE f Using the provided controls enables Ilumina Technical Support to troubleshoot in the event you need assistance Illumina Technical Support recommends including control samples in your assay to establish baselines and monitor overall performance After a baseline is established ACD1 is not necessary in every library preparation e The control ACPI is not necessary for the TruSight DNA Amplicon Sequencing Panel although it is included Use TSO with ACD1 as a positive control Apply the HYP Hybridization Plate barc
29. T TGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT T GAGACT GA TAACAT TA AGAAT GATAACAG TAAC CTTGATCCACTGATICAACGTACCGTAAC TCAA GATCCACTGATTCAACG AC IGATCCACTGATT OAACGTIAAGATTAG TIGATCCACTOATTCAACG TACCOTRACG CGTATCAAT TGAGCT TCTGT TAACCTTAAGAT TACT TGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAGCAACGAC t 2 CTAAATATTAACGTACCAT TAAGAGTCTGT TAAC GAT TACTTGATCCACT GAT TCAACG TACCGTAACGAACG TAT CAAT TI CAGTAACAAC CAACGTACCGTAACGAACGTATCATTAAGA TTGATCCACTGATTCAACGTACC CGTATCAAT TGAGA AACGTACCAT TAAGAGCTACCGT CTI 3ATTAC TCCACT TTA NI CCACTGAIT CGTACCGTAAG CGTATCAATTGAGCTICTGTT TTGAGACTAGCAACGACG 2 GATAACAG TAACACAC T TCTGT T AT TAA GATCCACTGAT TCAACGTACCGTAAAGA Ti GAGCTACCGTAAC A TGATAACAG TAACA E TTAAGATTACTTGTTGATCCACTGATTCAACGTACCGTATCAAT TGA ATTAAC GATCCACTGATTCAACG GU SAY CCACTQATTCAACQTA TCCACTGATTC CCGT CAATTGAGACT ITAACGAACAT T AI GATAACAG TAACACAC T TCTGT TAACCTTAA ACTTGATOCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGAT TCAACGTA AT IAAGAGC T CEA AGAATGATAACAG IAACACACTTCTGT TAACCT TAAGAT TACT T TGATTCAACG TAACGAACGTATCAA ACTTGAI CCACTGAT I T GA TTACTTGATCCACTGATT TAAC CGTATCAA ATTCAACGTACCGTAACGTA CGTATCAATTGAGAC TAAATAT T ACTIAACC GAT TACT TGATCCACT GATT CAACGTACCGTAACGAACGTOTTCTGTTAACCT T GAGAG T TAACGTACCAT TAAGAGCTACAACCT TAAGA GATCCAC NA T NI COGTAACGAACG TATCAAT TGAGAC TAAATAT TAAC AACAGTAACACACTCOA AGAATGATAACAGTAACACACTTCTGT T EB HE ACER ARO AACCG TAACGAACGTATCAAT TGAGAC T ARAIATIAADGIACCATAAGAGS TACOS TOT CTOTIAACO
30. TAACAGTAACAGAGTTCTG TAACCTIAAGAT TAC TTGATCCACTGAT CAACGTACCGTAAAGAT TACTTGATCCACTOAT TCAAGG TACCGTAACGAACGTATCAATT GAGACTAAATAT TAACGTACCAT TAAGAGCTACC 3ATAACAGTAACACACT TCTGTTAACCT T iuo pM a la ATCAAT TGAGACT FE ot V AE aue CGACGAAAAGAATGA ME CCAT TAAGAGC TACCGTGCAACAGTAACACACT TCTGT TAACCTTAAGAT TACT TGATCCACTGAT TCAACGTACCG TAACGAACGTAT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTGCAACGACGAAAAGAAT GATAA SAACAGTAAGACAGT TCTGLIAACCTTAAGAT TACT GAT COAG GAT TCAACG ACC TAAGGAAGG TAT CAAT GAGAGAAATAT IAAGG ACCA AAGAGCTAGCGTCT IGGL TACO IMAGA TAC GAT COAG TGAT GARG GTACCGTAACGAACGTATCATTAAGATTACTTGATCCACTGATT CAACGTACCGTAACGAACGTATCAATTG WEGE EA AU re eee eee CCGTGCAACGACGAAAAGAATGATAACAG TAACACACT TCTGT TAACCT TA ZTTGATCCACTGATTCAACGTIAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACG TATCAAT TGAGCTTCTGT TAACCT TAAGAT TACT TI GRUT TGATTCAACGTACCGTAACGAACGTATCAAT TGAGACTAGCAACGACG IAAAAGAATGATAACAGTAACACAC T TOTGT TAACCT TAAGAT TACTTGATCCACTGAT TCAACGTACCGTAAAGAT TAC T TGATCCAC AT GTAACGAACGTATCAAT TGAGACTAAATATTAACGTACCAT TAAGAGC TACC aATAACAG TAACACA SARAI RIA CCTTAAGAT TACTTGATCCACTGATTCAACG TACCG TAACGAACG TATCAAT TGAGAC TAAATAT TAAC AN KAGASCIACOGTOLICTOTTAN CCTTAAGATTACTTGA PSA SOT Wer s TCAT TAAGAT TACTTGATCCACTGAT TCAACGTACCGTAACGAACG TAT CAAT TGAGACTAAATAT TAACG TACCAT TAAGAGC TACCGTGCAACGACGAAAAGAATGATAACAG TAACACACT TCTGTTAACC T T IAAAAGAATGATAACAGTAACACACT TCTGTTAACC T TAAGAT TACTTGAT CCACTGAT TCAACGTACCGTAAAGAT TACT IGATCCACTGAT TCAACGTACCG TAACGAACG T
31. TSAACG TACOGTAACGAACGTAT CART GAGAGTAAATATIAACGTACCATIAAGAGCTAGG Se e DATAS GTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACG TATCAA ET eda ata aie Se D US PLoS LESE METTE CALI ACTTGATCCACTGATTCAACG TGAGACTAAATAT TAACGTTGTTAACCTTAAGATTACTTGATCCACTGAT TCAACG TACCGTAACGAACG TATCAAT TGAGACTAAATA M em SUME GCTTCTGTTAACCT TAAGAT TACTTGATCCACTGAT TCAACGTACCGTA ATCAATTGAGA CTAANTALT AACGTACT TAACCTTAAGAI TACT TGATCCACTGATTCAACGTACCGTAACGAACG TCTTCTGT TAACCT TAAGAT T AC TI GATCCACIG TICAACG ACCGIAACGAACGTATCAATTGAGACTAACGACGA M n E Le 3ATAACAGTAACACACTTCTGTTAACCTTAAGATTACT TGATCCACTGATTCAACG TACCG TAACGAACG TATCAAT TGAGAC gee die TTAAGAGCTA TTCTGTTAACCTTAAGAT TACT TGATCCACTGAT TCAACG CCA I TAAGAGC TACCETGCAAC M CCIT AAGA HAGTIGATCCACIGATTCAACCTACECTARCOARCOTA TCAATTGA GA CTAAA ARRECA IRAGAGOTACC TOCAA AC DA OAA CTICTGTT AOC TANGAT TACTAQAT TACOS GUAACGAAAATAACG TAAGATIACT GAICCACTGAT GAACGTACT TGT TACO TANGA TACT TGATCCAGT GAIT GAACG ACUI AACGAACG TAI GAAT GAGA AAG ACSI GGAAGG ACGAAAAGAAT GAT AAAAGAATGA e TOTE OANGEAN GATTACTTGATCCACTGATTCAACGTACCG TAAAGAT TACT TI RAICA MCN ET e a VA OS NV TTGAGACTAAA CGTACCAT TAAGAGCTACC 3ATAACAGTAACACACT TCTGTTAACCT T AAT ATO CACTOA TCAAC GU ACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTGCAACGA CEAMAAGAA GA MAACAGTRACACAGHTCT OM CCATTAAGAGCTACEGT GUAACAGTAACACACTTCTGT TAACCT TAAGATACT GATCCAG GAT ICAACGTAGGS IAACGAACG IAT CAAT I GAGAGTAAATAT TAACGTACGAI TAAGAGCTACCG I GCAACGACGAAAGAAT SATAN 3aAT
32. ble 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 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 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 Products Documentation i
33. er version of RTA contact Illumina Technical Support for advice on how to proceed i7 Index PCR Primer Index Sequence A701 A702 A703 A704 A705 A706 A707 ATCACGAC ACAGTGGT CAGATCCA ACAAACGG ACCCAGCA AACCCCTC CCCAACCT Part 15054779 Rev B i7 Index PCR Primer A708 A709 A710 A711 A712 i5 Index PCR Primer A501 A502 A503 A504 A505 A506 A507 A508 Index Sequence CACCACAC GAAACCCA TGTGACCA AGGGTCAA AGGAGTGG Index Sequence TGAACCTT TGCTAAGT TGTICTCT TAAGACAC CTAATCGA uoneJdedojg 1eeus ejduues basi CTAGAACA TAAGTICC TAGACCTA TruSight DNA Amplicon Sequencing Panel Library Preparation Guide 47 Supporting Information lumina Amplicon Viewer 48 Upon completion of MiSeq sequencing your data are automatically analyzed with MiSeq Reporter and can be visualized using the Illumina Amplicon Viewer Amplicon Viewer has been designed and developed for off instrument visualization and analysis of TruSight DNA Amplicon Sequencing Panel data Amplicon Viewer allows you to view data including coverage Q score variant call score etc from multiple MiSeq amplicon runs simultaneously and interactively You can also export custom reports based on selected samples targets variants Amplicon Viewer requires MiSeq Reporter data as input You can download the Amplicon Viewer through your Mylllumina account For more information about this software see www illumina com help amplicon vi
34. es at a time See note on on page 6 Clarified that unused volume is already included in calculation when preparing fewer than 96 samples and calculating volumes of TDP1 and PMM2 See step 10 in the Preparation section for PCR Amplification 15054779 A May 2014 Initial Release TruSight DNA Amplicon Sequencing Panel Library Preparation Guide MI Part 15054779 Rev B Table of Contents Revision History 22222 iii vii Table of Contents 202222222 o ix Chapter 1 Overview 02 oo ccoccceeceeecececece ooo nooo 1 AA i ala o DV 2 DNA Input Recommendations 2222222202 3 Additional Resources 000000 4 Chapisr2 Protocol eer dea t e eres ua ah 5 Introduction CREER is REDEEM 6 TruSight DNA Amplicon Sequencing Panel Library Prep Kit Workflow 7 Hybridization of Oligo Pool 2 0 0 ee eee ooo 8 Removal of Unbound Oligos 2 22 22 eee ee 11 Extension Ligation of Bound Oligos 2 2 22 ono 15 PCR Amplification o 22 02 22222 ie 16 PCR Clean Up nn 21 Library Normalization 2 2 0 2 eee nn 26 Library Pooling and MiSeq Sample Loading 220 22eeeeeee eee eeee 31 Appendix A Supporting Information occ 35 Introdu ctioni AA 36 How Does the TruSight DNA Amplicon Sequencing Panel Library Prep Kit ASSAY WOM us os ssh doesn rt dota idas 37 AcronymsS SMMMNNNNNEENNNNNNNNNNNNNWWWWWWVWVVJJDse 9 u 38 TruSight
35. ewer default htm To view a video demonstration of how to use the Illumina Amplicon Viewer go to TruSight DNA Amplicon Sequencing Panel Library Prep Kit support page and click the Training tab Part 15054779 Rev B Technical Assistance For technical assistance contact Illumina Technical Support Table 4 Illumina General Contact Information Address 5200 Illumina Way San Diego CA 92122 USA Website www illumina com Email techsupport illumina com Table5 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 Safety Data Sheets Safety data sheets SDSs are available on the Illumina website at support illumina com sds html Product Documentation Product documentation in PDF is available for download from the Illumina website Go to supportillumina com select a product then click Documentation amp Literature TruSight DNA Amplicon Sequencing Panel Library Preparation Guide A 9 99UEJSISSY EOIUYDE IAAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATAT TAACGTAC
36. 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 additional 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 Research Use Only Rights Subject to these terms and conditions and unless otherwise agreed upon in writing
37. go Hybridization 25 C to 15 C Pre Amp for Sequencing Reagent 2 ELM4 Extension Ligation Mix 4 25 C to 15 C Pre Amp PMM2 PCR Master Mix 2 25 C to 15 C Pre Amp TDP1 TruSeq DNA Polymerase 1 25 C to 15 C Pre Amp sw1 Stringent Wash 1 2 C to 8 C Pre Amp UB1 Universal Buffer 1 2 C to 8 C Pre Amp Barcode plate stickers for HYP FPU IAP Room temperature Pre Amp Y y WARNING A component in 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 applicable local governmental safety standards For more information see the SDS for this kit at support illumina com sds ilmn A O Part 15054779 Rev B Box 2 Pre Amplification This box is shipped at room temperature As soon as you receive your kit remove LNB1 from box 2 and store at 2 C to 8 C in the post amplification area Keep the filter plate in the pre amplification area at room temperature Acronym Reagent Name Storage Temperature Area Filter plate with lid Room temperature Pre Amp LNB1 Library Normalization Beads 1 2 C to 8 C Post Amp Box 3 Post Amplification Acronym Reagent Name Storage Temperature Area HT1 Hybridization Buffer 25 C to 15 C Post Amp LNA1 Library Normalization Additives 1 25 C to 15 C Post Amp LNW1 Library Normalization Wash 1 2 C to 8 C Post A
38. hird 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 Illumina for any infringement of third party intellectual property rights b 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 TruSight DNA Amplicon Sequencing Panel Library Preparation Guide V 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 Specification
39. ii 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 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 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
40. illumina TruSight DNA Amplicon Sequencing Panel Guide A AAA TN SSTP O eN PA AI IE U S IRAE CA TATA TATA SNA AGAAT GATAACAG TAACACACTTCTGT V IACITGI TGATCCACTGATTCAACG TACCGTATCAAT I GAGACTAAATAT TAACGTACCAT TAAGAGC TACCGTCT TC TGT TAACC T TAAGAT TACTT GATCCAC TGATICAACGTACCGTAAAAA CATCCACICATCAACOTACCAAGA TACTIGATOCAC GAL IAM GG TACO AACGAACGTAT CAT GAGAC TARA AT AA COTACCATIAAGAGOTACOGIOTICT GTIAACCI TAAGAI TAGIT GATOCACIGAI TORACGTACC CIAACCAACA SGACGAAAAGAATGATAACAGTAACACACTTCTGTTAACCTIAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTIGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGAC TAAATAT TAACGTACCAT TAAGAGCTACCGTAAC AGAATGATAACAG TAACACACTTOCTGT TAACCTTAAGAT TACTTGATCCACTGAT TCAACGTACCG TAACGAACG TATCAAT TGAGAC TAAATATTAACGTACCAT TAAGAGCTACCGTCT TC TGT TAACCT TAAGATTACTTGAT CCACTGAT TCAACG TAAAZA ATCAAT TGAGAC TAAATAT TAACGT T GT TAACCTTAAGAT TACT TGAT CCACTGAT ICAACG TACCG TAACGAACGTATCAAT TGAGACTAAATATTAACG TACCAT TAAGAGCT TCTG T TAAC eS E A VES VS TEE TAS AACGTATCAATTGAGAC TAAATAT TAACGTAC T TAACCT TAAGAT TACT TGATCCACTGAT I CAACG TACCG TAACGAACG TCTTCTGT TAACCT TAAGAT TACT T GATCCACTGATTCAACGTACCGTAACGAACG TAT CAAT TGAGAC TAACGACGAAACG AGAAT OATAACACTRACACAO T ICTA TRACCI IMAGA TAC GAT GCAGIGAT TGAACGTACOG TAAGGAAGGTATCANT TGAGAGTAAATAT TAAGGTACGA AAGAGGTAGCGTCT STATI Aen ce aya NO Seas rea AGAATGATAACAGTAACACACT TCTGT TAACCT TAAGAT TACT TGATCCACT GAT TCAACGTACCG TAACGAACG TAT CAAT TGAGAC TAAATAT TAACGTACCATTAAGAGCTACCGTCTTCTG CCTTAAGATTACTTGAT
41. inutes Hands on 20 minutes Consumables Item EBT Elution Buffer with Tris AMPure XP beads Freshly prepared 8076 ethanol EtOH 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 4 NOTE Quantity 1 tube As needed 40 ml per 96 samples As needed As needed Storage Room temperature DXGILOISS Room temperature Supplied By Illumina User User User User User Always prepare fresh 8076 ethanol for wash steps Ethanol can absorb water from the air impacting your results Procedure 1 Centrifuge the IAP plate at 1 000 x g at 20 C for 1 minute to collect condensation TruSight DNA Amplicon Sequencing Panel Library Preparation Guide 21 dn ueal9 Hod Protocol Lg To confirm that the library successfully amplified run an aliquot of the samples on a 4 agarose 5 ul or on a Bioanalyzer 1 ul The expected PCR product sizes for 250 bp amplicons are 350 bp Figure 3 Agarose Gel Example Expected ACP1 ACD1 PCR product is shown A Expected PCR Product for 250 bp amplicons 350 bp B Primers Part 15054779 Rev B Figure 4 Bioanalyzer Example Expected ACP1 ACD1 PCR product is shown B ru Sample 3 904 804 704 604 504 404 304 207 107 ad 10 15 100 200 300 500 1500 bp A Marker B Expected PCR Product for 250 bp amplicon
42. isclosed 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 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 O 2014 Illumina Inc All rights reserved Illumina 24sure BaseSpace BeadArray BlueFish BlueFuse BlueGnome cBot CSPro CytoChip DesignStudio Epicentre GAIIx Genetic Energy Genome Analyzer GenomeStudio GoldenGate HiScan HiSeq HiSeq X Infinium iScan iSelect ForenSeq MiSeq MiSeqDx MiSeq FGx NeoPrep Nextera NextBio NextSeq Powered by Illumina
43. ler Pro S TruSight DNA Amplicon Sequencing Panel Library Preparation Guide Temp Mode Calculated Calculated Gradient S Simulated Tube Lid Temp Heated Constant at 100 C Heated Heated Vessel Type Polypropylene plates and tubes Plate Plate 45 jueuudinb43 Supporting Information MiSeq Sample Sheet Preparation Create your Sample Sheet for MiSeq sequencing according to the MiSeg Sample Sheet Quick Reference Guide Illumina recommends the Illumina Experiment Manager to prepare your Sample Plate and Sample Sheet Select MiSeq as your instrument then select Targeted Resequencing and TruSeq Amplicon as the workflow Alternatively you can use your Experienced User Card and Lab Tracking Form and the appropriate index sequences corresponding to the PCR primers used in your assay 46 NOTE Give the assay control prepared with ACDI the Sample ID and Sample Name TSCA Control in your Sample Plate and Sample Sheet files NOTE If you have cancer samples as your DNA input make sure that the Use Somatic Variant Caller box is checked under TruSeq Amplicon Workflow Specific Settings NOTE Illumina recommends choosing a read length that does not exceed the TSO amplicon size NOTE For MiSeq instruments running RTA 1 17 28 MCS2 2 or higher low plexity index combinations have not been shown to cause problems during runs or with demultiplexing If running low plexity indexes on an instrument with an earli
44. microplate shaker at 1 800 rpm for 2 minutes t NOTE i Make sure that all samples are resuspended If there are samples in which the beads are not resuspended gently pipette up and down to resuspend the beads and repeat the previous two steps Part 15054779 Rev B 18 19 20 21 22 Incubate at room temperature without shaking for 2 minutes Place the plate on the magnetic stand for 2 minutes or until the supernatant has cleared Apply the LNP Library Normalization Plate barcode plate sticker to a new MIDI plate Using a P20 multichannel pipette and fine tips carefully transfer 20 ul of the supernatant from the CLP plate to the LNP plate Change tips between samples to avoid cross contamination NOTE If any beads are aspirated into the tips dispense the beads back to the plate and let the plate rest on the magnet for 2 minutes Make sure that the supernatant is clear Seal the LNP plate with Microseal B and then centrifuge to 1 000 x g for 1 minute to ensure all the supernatant is at the bottom of the well TruSight DNA Amplicon Sequencing Panel Library Preparation Guide 2 5 dn ueal9 Hod Protocol Library Normalization This process normalizes the quantity of each library to ensure more equal library representation in your pooled sample Estimated Time Total duration 1 hour 20 minutes Hands on 30 minutes Consumables Item LNAI Library Normalization Additives 1 LNBI Library Normalization
45. minute elution apply the SGP Storage Plate barcode plate sticker to a new 96 well PCR plate 29 uonezi euJoN AJeJqr Protocol 30 15 16 17 18 19 Add 30 ul LNS2 to each well to be used in the SGP plate After the 5 minute elution make sure that all samples in the LNP plate are resuspended completely If the samples are not resuspended gently pipette up and down or lightly tap the plate on the bench to resuspend the beads Then shake for another 5 minutes Place the LNP plate on the magnetic stand for 2 minutes or until the liquid is clear Using a multichannel pipette set to 30 ul transfer the supernatant from the LNP plate to the SGP plate Change tips between samples to avoid cross contamination NOTE i If any beads are aspirated into the tips dispense the beads back to the plate and let the plate rest on the magnet for 2 minutes Make sure that the supernatant is clear Seal the SGP plate with Microseal B and then centrifuge to 1 000 x g for 1 minute r NOTE The final library pool consists of single stranded DNA which does not resolve well on an agarose gel or Bioanalyzer chip qPCR can be used for quality control if desired For more information please see the Sequencing Library qPCR Quantification Guide SAFESTOPPING POINT 1 If you do not plan to proceed to Library Pooling and MiSeq Sample Loading and subsequent sequencing on the MiSeg store the sealed SGP plate at 25 C to 15 C Part 15054
46. mp LNS2 Library Normalization Storage Buffer 2 Room temperature Post Amp EBT Elution Buffer with Tris Room temperature Post Amp Barcode plate stickers for CLP LNP SGP Room temperature Post Amp PAL DAL Box 4 TruSight DNA Amplicon Sequencing Panel Oligo Kit Pre Amplification Acronym Reagent Name Storage Temperature Area TSO TruSight Oligo Tube 25 C to 15 C Pre Amp TruSeq Custom Amplicon Library Preparation Index Kit Catalog FC 130 1003 Box 1 Pre Amplification Reagent Name Storage Temperature Area i5 Index Primers A501 to A508 8 tubes 25 C to 15 C Pre Amp i7 Index Primers A701 to A712 12 tubes 25 C to 15 C Pre Amp Box 2 Pre Amplification Reagent Name Storage Temperature Area 15 Index Tube Caps White Room temperature Pre Amp i7 Index Tube Caps Orange Room temperature Pre Amp TruSight DNA Amplicon Sequencing Panel Library Preparation Guide 41 Meid jeue g 6urouenbes uoolduy VNG 1UBiSNy Supporting Information 42 Additional Components Consumable TruSeq Index Plate Fixture Kit Required and reusable TruSeq Custom Amplicon Filter Plate Highly recommended TruSeq Index Plate Fixture and Collar Kit Required and reusable Catalog Storage Temperature Area Part 15054779 Rev B User Supplied Consumables Quantity As needed 3 3 As needed 3 As needed 2 40 ml 1 As needed 2 As needed As needed As needed As needed As needed Consumable 10
47. n 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 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 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 Part 15054779 Rev B Revision History Part Revision Date Description of Change 15054779 B November Clarified minimum batch size The TruSight DNA Amplicon 2014 Sequencing Panel Library Prep Kit does not provide enough reagents to process fewer than 8 sampl
48. nce 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 Product s Documentation or Specifications expressly state such third party s good is for use with the Product d 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 e 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
49. nded DNA dsDNA specific dye specifically and accurately quantitate dsDNA even in the presence of many common contaminants In contrast UV spectrometer methods based on 260 OD readings are prone to overestimating DNA concentrations due to the presence of RNA and other contaminants commonly found in gDNA preparations TruSight DNA Amplicon Sequencing Panel Library Preparation Guide suonepueuuulooeH ndul YNA Additional Resources Overview Resource Training Best Practices TruSight DNA Amplicon Sequencing Panel Library Prep Kit Experienced User Card part 15054777 Illumina Experiment Manager IEM The following resources are available for TruSight DNA Amplicon Sequencing Panel Library Prep Kit protocol guidance and sample tracking These resources use the TruSeq Custom Amplicon assay and can utilize the same support tools Description Illustrates elements of the TruSight DNA Amplicon Sequencing Panel Library Prep Kit process Viewing these videos is recommended for new and less experienced users before starting sample preparation Provides best practices specific to this protocol Review before starting sample preparation Topics include General Advice on Sample Handling Handling Magnetic Beads Handling Reagents Avoiding Cross Contamination Provides protocol instructions but with less detail than what is provided in this user guide New or less experienced users are advised to follow this user guide
50. ns 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 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 IV Part 15054779 Rev B 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 c Exclusions from Warranty Coverage The foregoing warranties do not apply to the extent a non conformance is due to i abuse misuse neglect neglige
51. ny beads are aspirated into the tips dispense the beads back to the plate and let the plate rest on the magnet for 2 minutes and then make sure that the supernatant is clear Make sure that the supernatant is clear With the CLP plate on the magnetic stand wash the beads with freshly prepared 80 ethanol as follows a Using a multichannel pipette add 200 ul of freshly prepared 80 ethanol to each sample well Take care to avoid cross contamination or change tips between columns You do not need to resuspend the beads currently b Incubate the plate on the magnetic stand for 30 seconds or until the supernatant appears clear c Carefully remove and discard the supernatant Use a P20 multichannel pipette set to 20 ul to remove excess ethanol With the CLP plate on the magnetic stand perform a second ethanol wash as follows a Using a multichannel pipette add 200 ul of freshly prepared 80 ethanol to each sample well b Incubate the plate on the magnetic stand for 30 seconds or until the supernatant appears clear c Carefully remove and discard the supernatant d Usea P20 multichannel pipette to remove excess ethanol Remove the CLP plate from the magnetic stand and allow the beads to air dry for 10 minutes Using a multichannel pipette add 30 ul of EBT to each well of the CLP plate Take care to avoid cross contamination or change tips between columns Seal the plate with a Microseal B adhesive seal Shake the CLP plate on a
52. ode plate sticker to a new 96 well PCR plate Add 5 ul of control DNA ACDI and 5 ul of TE or water to 1 well in the HYP plate for the assay control Add 10 ul of Genomic to each remaining well of the HYP plate to be used in the assay For more dilute samples that is lt 25 ng ul up to 15 ul of DNA can be used Example Setup for High Quality Genomic DNA Input Volume DNA Concentration 50 ng 10 ul 5ng ul 50 ng up to 15 ul 233 ng ul TruSight DNA Amplicon Sequencing Panel Library Preparation Guide 9 004 OBI O jo uogezipag AH Protocol 10 Using a multichannel pipette add 5 ul of TSO to each well containing DNA Change tips after each column to avoid cross contamination If the samples have uneven volumes or are not sitting at the bottom of each well do the following a Seal the HYP plate with adhesive aluminum foil and secure the seal with a rubber roller or sealing wedge b Centrifuge to 1 000 x g at 20 C for 1 minute Using a multichannel pipette add 35 ul of OHS2 to each sample in the HYP plate When dispensing gently pipette up and down 3 5 times to mix Change tips after each column to avoid cross contamination y NOTE Ensure any crystals or precipitate in OHS2 have dissolved r NOTE Do not mix TSO and OHS2 for storage If combined TSO becomes unstable even when stored frozen Seal the HYP plate with adhesive aluminum foil and secure the seal with a rubber roller or sealing wedge Centrifuge to 1 000 x g a
53. 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 Magnetic stand 96 Post PCR plate shaker Tabletop centrifuge Gel electrophoresis supplies and apparatus Bioanalyzer System Heat block for 1 5 ml centrifuge tubes i NOTE Supplier Invitrogen DynaMag 96 Side Skirted Q Instruments BioShake iQ high speed thermoshaker part 1808 0506 or Q Instruments BioShake XP high speed lab shaker part 1808 0505 General lab supplier plate centrifuge that attains designated speeds of protocol General lab supplier Agilent Technologies General lab supplier Use a dedicated set of pipettes pipette tips vortexer heat block and centrifuge during post amplification steps Thermal Cycler The following table lists the recommended settings for selected thermal cycler models If your lab has not yet performed the TruSeq Custom Amplicon Library Preparation protocol 44 Part 15054779 Rev B Illumina recommends that you validate any thermal cyclers not listed Thermal Cycler Bio Rad DNA Engine Tetrad 2 MJ Research DNA Engine Tetrad Eppendorf Mastercyc
54. s 350 bp C Marker NOTE Illumina recommends assessing library quality by gel electrophoresis or Bioanalyzer is highly recommended for TruSight TSO oligo pools which are being used for the first time It is not necessary to perform this assessment on every sample in the experiment Illumina requires that you also include the control reaction generated with ACD1 in this assessment to enable Illumina Technical Support to troubleshoot in the event you need assistance Apply the CLP Clean up Plate barcode plate sticker to a new MIDI plate Using a multichannel pipette add 45 ul of AMPure XP beads to each well of the CLP plate z NOTE The ACDI control can be processed using the same conditions as your TSO Using a multichannel pipette set to 60 ul transfer the entire PCR product from the IAP plate to the CLP plate Change tips between samples Seal the CLP plate with a Microseal B adhesive seal Shake the CLP plate on a microplate shaker at 1 800 rpm for 2 minutes Incubate at room temperature without shaking for 10 minutes TruSight DNA Amplicon Sequencing Panel Library Preparation Guide 2 3 dn ueal9 Hod Protocol 24 10 11 12 13 14 15 16 17 Place the plate on a magnetic stand for 2 minutes or until the supernatant has cleared With the CLP plate on the magnetic stand and a multichannel pipette set to 100 ul carefully remove and discard the supernatant Change tips between samples y NOTE If a
55. s 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 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 upo
56. s in a significant reduction of cluster density 10 Using a heat block incubate the DAL tube at 96 C for 2 minutes Part 15054779 Rev B 11 12 13 14 After the incubation invert DAL 1 2 times to mix and immediately place in the ice water bath Keep the DAL tube in the ice water bath for 5 minutes z NOTE Perform the heat denaturation step immediately before loading DAL into the MiSeg reagent cartridge to ensure efficient template loading on the MiSeg flow cell Load DAL into a thawed MiSeq reagent cartridge into the Load Samples reservoir Sequence your library as indicated in the MiSeq System User Guide NOTE Illumina recommends choosing a read length that does not exceed the TSO amplicon size TruSight DNA Amplicon Sequencing Panel Library Preparation Guide 3 3 Buipeo7 ajduwes basi pue Buijoo y AJejgr1 34 Part 15054779 Rev B Supporting Information INTOQUCHION 2 11 ude doce cn con aso IL desa leg ukDercc ceed la da ana 36 How Does the TruSight DNA Amplicon Sequencing Panel Library Prep Kit Assay Work 37 A A E 38 TruSight DNA Amplicon Sequencing Panel Library Prep Kit Contents 40 User Supplied Consumables sssssssssssssseses ese cece s s s eser sra 43 EGUIDITIGDE one eene ace Deere ti uet 44 MiSeq Sample Sheet Preparation 0 2 2 ccc ccc cece cece cece cece cece eeeeeeeeceeeceeeees 46 Illumina Amplicon Viewer 22222222222
57. t 20 C for 1 minute Place the HYP plate in the preheated block at 95 C and incubate for 1 minute While the plate remains on the preheated block set the temperature to 40 C and continue incubating for 80 minutes E NOTE i During incubation the heat block temperature gradually decreases from 95 C to 40 C This process typically takes 80 minutes This gradual cooling is critical for proper hybridization therefore PCR thermal cyclers with active cooling are not recommended for this process Part 15054779 Rev B 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 Y WARNING A component in 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 applicable local governmental safety standards For more information see the SDS for this kit at support illumina com sds ilmn Y WARNING This set of reagents contains mercaptoethanol Perform the following procedure in a hood or well ventilated area if desired Estimated Time Total duration 20 minutes Hands on 20 minutes Consumables Item Q
58. tension ligation 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 ELM4 Extension Ligation Mix 4 1 tube 25 C to 15 C Ilumina Adhesive aluminum foil seal 1 seal User Troughs As needed User Procedure 1 Using a multichannel pipette add 45 ul of ELMA to each sample well of the FPU plate The extension ligation reaction takes place on the filter plate membrane Take care to avoid cross contamination or change tips between columns 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 preheated 37 C incubator for 45 minutes 4 While the FPU plate is incubating prepare the IAP Indexed Amplification Plate as described in the following section TruSight DNA Amplicon Sequencing Panel Library Preparation Guide 1 5 soBIJO punog jo uoneBrT uorisue1x3 Protocol PCR Amplification In this step the extension ligation products are amplified using primers that add sample multiplexing index sequences i5 and i7 as well as common adapters required for cluster generation P5 and P7 Estimated Time Total duration 85 105 minutes depending on the number of PCR cycles used Hands on 30 minutes Consumables Item Quantity Storage Supplied By
59. transfer 800 ul of LNBI to the 15 ml conical tube containing LNA1 Mix well by inverting the tube 15 20 times The resulting LNA1 LNB1 bead mix is enough for 96 samples Pour the bead mix into a trough and use it immediately in the next step Using a multichannel pipette add 45 ul of the combined LNA1 LNB1 to each well of the LNP plate containing libraries Changing tips between columns is not required if you use care to avoid cross contamination Seal the LNP plate with a Microseal B adhesive seal Shake the LNP plate on a microplate shaker at 1 800 rpm for 30 minutes NOTE i The 30 minute incubation is critical for proper library normalization Incubations of greater or less than 30 minutes affect library representation and cluster density Place the plate on a magnetic stand for 2 minutes or until the supernatant has cleared With the LNP plate on the magnetic stand use a multichannel pipette set to 80 ul to remove the supernatant and then discard in an appropriate hazardous waste container Part 15054779 Rev B 10 11 12 13 14 TruSight DNA Amplicon Sequencing Panel Library Preparation Guide t NOTE e If any beads are inadvertently aspirated into the tips dispense the beads back to the plate and let the plate rest for 2 minutes or until the supernatant has cleared Remove the LNP plate from the magnetic stand and wash the beads with LNW1 as follows a Using a multichannel pipette add 45 ul of LNW1 to each sample
60. uantity Storage ELM4 thawed in preparation 1 tube 25 C to 15 C for Extension Ligation SW1 Stringent Wash 1 1 tube LIC UN sae UB1 Universal Buffer 1 1 tube 2 C to 8 C Filter plate with lid 1 plate Adapter collar reusable 1 plate MIDI plate 1 plate Troughs As needed TruSight DNA Amplicon Sequencing Panel Library Preparation Guide Supplied By Ilumina Illumina Ilumina Ilumina Ilumina User User 11 soBIJO PUNOJUN Jo enouay Preparation 1 Remove ELM4 from 25 C to 15 C storage and thaw at room temperature ELM4 is used in the Extension Ligation step and takes approximately 20 minutes to thaw 2 Remove SW1 and UBI from 2 C to 8 C storage and set aside at room temperature Protocol 3 Assemble the filter plate assembly unit FPU in the following order from top to bottom 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 1 2 Part 15054779 Rev B Procedure 1 Pre wash the FPU plate membrane as follows a Using a multichannel pipette add 45 ul of SW1 to each well 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 x g at 20 C for 5 minutes NOTE Pre wash only the wells to be used in the current assay You can use fresh unused wells of a previously opened filter plate but do not use wells that have
61. w Appendix A Supporting Information to confirm your kit contents and make sure that you have obtained all of the requisite equipment and consumables 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 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 NOTE The TruSight DNA Amplicon Sequencing Panel Library Prep Kit does not provide enough reagents to process fewer than 8 samples at a time If you are processing less than 96 samples only 6 freeze thaw cycles are supported When calculating smaller reagent amounts the unused volume is already calculated in the totals listed in the protocol Part 15054779 Rev B TruSight DNA Amplicon Sequencing Panel Library Prep Kit Workflow The following diagram illustrates the workflow using the TruSight DNA Amplicon Sequencing Panel Library Prep Kit Safe stopping points are marked between steps Figure 1 TruSight DNA Amplicon Sequencing Panel Library Prep Kit Workflow Hybridization of Oligo Pool Hands On 15 min Incubation 80 min PCRClean Up Hands On 20 min Processing 30 min Reagents Reagents Tso EBT OHST AMPure XP beads ACD1 EtOH ACPI Ge utpu Output

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