TruSeq Genotype N e. Reference Guide. For Research Use Only. Not for use in diagnostic procedures. Document # v00 July 2017

Transcription

1 TrSeq Genotype N e Reference Gide Docment # v00 Jly 2017 For Research Use Only. Not for se in diagnostic procedres. ILLUMINA PROPRIETARY

2 TrSeq Genotype Ne Reference Gide This docment and its contents are proprietary to Illmina, Inc. and its affiliates ("Illmina"), and are intended solely for the contractal se of its cstomer in connection with the se of the prodct(s) described herein and for no other prpose. This docment and its contents shall not be sed or distribted for any other prpose and/or otherwise commnicated, disclosed, or reprodced in any way whatsoever withot the prior written consent of Illmina. Illmina does not convey any license nder its patent, trademark, copyright, or common-law rights nor similar rights of any third parties by this docment. The instrctions in this docment mst be strictly and explicitly followed by qalified and properly trained personnel in order to ensre the proper and safe se of the prodct(s) described herein. All of the contents of this docment mst be flly read and nderstood prior to sing sch prodct(s). FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN MAY RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TO USERS OR OTHERS, AND DAMAGE TO OTHER PROPERTY. ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S) DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE) Illmina, Inc. All rights reserved. Illmina, MiniSeq, MiSeq, Nextera, NextSeq, TrSeq, and the streaming bases design are registered or pending trademarks of Illmina, Inc. and/or its affiliate(s) in the U.S. and/or other contries. All other names, logos, and other trademarks are the property of their respective owners. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. ii

3 Table of Contents Chapter 1 Overview 1 Introdction 1 DNA Inpt Recommendations 1 Additional Resorces 1 Chapter 2 Protocol 3 Introdction 3 Tips and Techniqes 3 Library Prep Workflow 5 Qantify and Dilte DNA 6 Hybridize Oligo Pool 7 Remove Unbond Oligos 9 Extend and Ligate Bond Oligos 10 Amplify Libraries 11 Clean Up Libraries 13 Normalize Libraries 16 Pool Libraries 18 Appendix A Spporting Information 20 Introdction 20 Acronyms 20 Kit Contents 21 Consmables and Eqipment 23 Technical Assistance 26 Docment # v00 For Research Use Only. Not for se in diagnostic procedres. iii

4 Chapter 1 Overview Introdction 1 DNA Inpt Recommendations 1 Additional Resorces 1 Introdction Use this protocol to prepare p to 384 niqely indexed libraries sing the Illmina TrSeq Genotype Ne kit for targeted genotyping by seqencing. The kit spports genotyping by seqencing p to 5000 targets, inclding single ncleotide polymorphisms (SNPs) and small indels. This targeted approach allows a wide range of applications, sch as parentage assays, prity testing, and moleclar breeding. The Genotype Ne kit offers: Reliable identification of SNPs and small indels. Streamlined 96-well based workflow with less than 3 hors of hands-on time. Bead-based prification, which allows an atomation-friendly workflow. Cstom panels of p to 5000 markers for targeted genotyping of any plant or animal species. Compatibility with the MiniSeq, MiSeq, and NextSeq systems for high-qality seqencing reslts. DNA Inpt Recommendations Qantify the inpt DNA and assess the DNA qality before beginning library prep. DNA Type Spported Amplicon Size Inpt Genomic DNA 150 bp, 175 bp 50 ng Inpt DNA Diltion Qantify and dilte the DNA to the inpt amont of 50 ng. Yo can dilte and store more than the reqired DNA for later se. Dilte DNA in RS1 and SS1 and store as described in Qantify and Dilte DNA on page 6. Inpt DNA Qantification Qantify the starting genomic material sing a florescence-based qantification method, sch as a Qbit dsdna Assay Kit or PicoGreen. Do not se a UV-spectrometer-based method. Florescence-based methods employ a dye specific to doble-stranded DNA (dsdna). They specifically and accrately qantify dsdna, even when many common contaminants are present. UV spectrometer methods based on 260 OD readings can overestimate DNA concentrations de to the presence of RNA and other contaminants common to gdna preparations. Additional Resorces Visit the TrSeq Genotype Ne kit spport page on the Illmina website for docmentation, software downloads, training resorces, and information abot compatible Illmina prodcts. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 1

5 TrSeq Genotype Ne Reference Gide The following docmentation is available for download from the Illmina website. Resorce Cstom Protocol Selector TrSeq Genotype Ne Checklist (docment # ) TrSeq Genotype Ne Consmables and Eqipment List (docment # ) Description spport.illmina.com/cstom-protocol-selector.html A wizard for generating cstomized end-to-end docmentation that is tailored to the library prep method, rn parameters, and analysis method sed for the seqencing rn. Provides a checklist of steps for the experienced ser. Provides an interactive checklist of ser-provided consmables and eqipment. For information on the seqencing reagents provided with the TrSeq Genotype Ne kit, see the system gide for yor instrment. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 2

6 Chapter 2 Protocol Introdction 3 Tips and Techniqes 3 Library Prep Workflow 5 Qantify and Dilte DNA 6 Hybridize Oligo Pool 7 Remove Unbond Oligos 9 Extend and Ligate Bond Oligos 10 Amplify Libraries 11 Clean Up Libraries 13 Normalize Libraries 16 Pool Libraries 18 Introdction This section describes the TrSeq Genotype Ne protocol. Before proceeding, confirm the kit contents and make sre that yo have the reqired consmables and eqipment. This protocol reqires different magnetic stands for pre-pcr and post-pcr procedres. Althogh the kit incldes Amplicon Control Oligo Pool 3 (ACP3), se of a control is not spported so this reagent is not sed. Follow the protocol in the order described sing the specified parameters. Prepare p to 384 samples in batches of 96 samples. Prepare for Pooling If yo plan to pool libraries, record information abot yor samples before beginning library prep. For more information, see the TrSeq Genotype Ne spport page. Review Nextera Low Plex Pooling Gidelines (Pb. No ) when preparing libraries for Illmina seqencing systems that reqire balanced index combinations. Tips and Techniqes Unless a safe stopping point is specified in the protocol, proceed immediately to the next step. Avoiding Cross-Contamination When adding or transferring samples, change tips between each sample. When adding adapters or primers, change tips between each row and each colmn. Remove nsed index adapter tbes from the working area. Sealing the Plate Always seal the 96-well plate before the following steps in the protocol: Shaking steps Vortexing steps Centrifge steps Thermal cycling steps Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 3

7 TrSeq Genotype Ne Reference Gide Apply the adhesive seal to cover the plate, and seal with a rbber roller. Microseal 'B' adhesive seals are effective at -40 C to 110 C, and sitable for skirted or semiskirted PCR plates. Use Microseal 'B' for shaking, centrifging, and long-term storage. Microseal 'A' adhesive film is sed for thermal cycling steps to prevent evaporation. Plate Transfers When transferring volmes between plates, transfer the specified volme from each well of a plate to the corresponding well of the other plate. Centrifgation Centrifge at any step in the procedre to consolidate liqid or beads in the bottom of the well, and to prevent sample loss. To pellet beads, centrifge at 280 g for 1 minte. Handling Beads Pipette bead sspensions slowly. Before, allow the beads to come to room temperatre. Immediately before se, vortex the beads ntil they are well dispersed. The color of the liqid mst appear homogeneos. If beads are aspirated into pipette tips, dispense back to the plate on the magnetic stand, and wait ntil the liqid is clear (~2 mintes). When washing beads: Use the appropriate magnetic stand for the plate. Dispense liqid so that beads on the side of the wells are wetted. Keep the plate on the magnetic stand ntil the instrctions specify to remove it. Do not agitate the plate while it is on the magnetic stand. Do not distrb the bead pellet. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 4

8 TrSeq Genotype Ne Reference Gide Library Prep Workflow The following figre illstrates the TrSeq Genotype Ne library prep workflow. Figre 1 TrSeq Genotype Ne Workflow Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 5

9 TrSeq Genotype Ne Reference Gide Qantify and Dilte DNA This step qantifies and diltes inpt DNA to the appropriate concentration in the reqired dilent for sbseqent steps. Consmables RS1 (Resspension Soltion 1) SS1 (Sample Stabilization Soltion 1) Genomic DNA Microcentrifge tbes Abot Reagents For later se, yo can dilte and store more DNA than is reqired for the crrent protocol. Dilte DNA in RS1 and SS1 as described in the procedre. Prepare aliqots of dilted DNA in microcentrifge tbes and store at -25 C to -15 C for p to 4 weeks. Store thawed dilted DNA at 2 C to 8 C for p to 2 weeks. Avoid repeatedly freezing and thawing dilted DNA. Preparation 1 Prepare the following consmables. Reagent Storage Instrctions DNA 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. Flick to mix, and then centrifge briefly. Do not vortex. RS1 15 C to 30 C If stored at 2 C to 8 C, let stand for 30 mintes to bring to room temperatre. Vortex to mix, and then centrifge briefly. SS1 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. Flick to mix, and then centrifge briefly. Procedre 1 Qantify DNA sing a florometric method, sch as Qbit or PicoGreen. 2 Dilte DNA to 25 ng/µl in RS1. 3 Reqantify the dilted DNA sing the same florometric qantification method. 4 In a microcentrifge tbe, frther dilte the 25 ng/µl DNA in RS1 to a total volme of 4 µl with a total DNA inpt of 50 ng. 5 Add 1 µl SS1 to the 4 µl of 50 ng DNA. These volmes reslt in 5 µl of 50 ng inpt DNA. Table 1 Example for 50 ng DNA Inpt Reagent Stock Concentration Volme DNA 25 ng/µl 2 µl RS µl SS1 5x 1 µl Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 6

10 TrSeq Genotype Ne Reference Gide Hybridize Oligo Pool This step hybridizes a cstom oligo pool that contains pstream and downstream oligos specific to yor targeted regions of interest. Perform replicates to increase confidence in somatic variant calls. Consmables ELB (Extension-Ligation Bffer) ELE (Extension-Ligation Enzyme) CAT (TrSeq Genotype Ne Oligos) OHS2 (Oligo Hybridization for Seqencing 2) RS1 (Resspension Soltion 1) HYP (Hybridization Plate) barcode label Dilted DNA 96-well PCR plate Microseal 'B' adhesive film Microcentrifge tbes RNase/DNase-free 8-tbe strips and caps Prepare for a sbseqent step: SPB (Sample Prification Beads) SW1 (Stringent Wash 1) WARNING This set of reagents contains potentially hazardos chemicals. Personal injry can occr throgh inhalation, ingestion, skin contact, and eye contact. Wear protective eqipment, inclding eye protection, gloves, and laboratory coat appropriate for risk of exposre. Handle sed reagents as chemical waste and discard in accordance with applicable regional, national, and local laws and reglations. For additional environmental, health, and safety information, see the SDS at spport.illmina.com/sds.html. Abot Reagents CAT Yo can dilte and store more than the reqired amont of CAT for later se. Dilte CAT in RS1 as described in the procedre. Use a mltichannel pipette to dispense dilted CAT from a PCR 8-tbe strip that contains 70 µl in each tbe. Prepare aliqots of dilted CAT and store at -25 C to -15 C for p to 12 months. OHS2 Aspirate and dispense slowly de to the viscosity of the reagent. Before each se, vortex thoroghly and then centrifge briefly. Make sre that all precipitates have dissolved. When mixing, mix thoroghly. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 7

11 TrSeq Genotype Ne Reference Gide Preparation 1 Prepare the following consmables. Reagent Storage Instrctions DNA 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. Flick to mix, and then centrifge briefly. Do not vortex. CAT -25 C to -15 C Thaw at room temperatre for 30 mintes. Vortex to mix, and then centrifge briefly. OHS2 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. Vortex thoroghly to mix, and then centrifge briefly. RS1 15 C to 30 C If stored at 2 C to 8 C, let stand for 30 mintes to bring to room temperatre. Vortex to mix, and then centrifge briefly. SPB 2 C to 8 C Let stand to bring to room temperatre in preparation for the procedre to remove nbond oligos. Do not exceed 25 C. SW1 2 C to 8 C Let stand to bring to room temperatre in preparation for the procedre to remove nbond oligos. ELB -25 C to -15 C Thaw at room temperatre for 20 mintes, and then place on ice. ELE -25 C to -15 C Place on ice. 2 Save the following HYB program for 25 µl reaction volme on a Bio-Rad thermal cycler: Choose the preheat lid option and set to 100 C. Step 1: 95 C for 3 mintes. Step 2: From 90 C, decrease by 0.5 C, hold for 30 seconds, ramp at 0.1 C per second. Step 3: Go to step 2 for 59x. Step 4: From 60 C, decrease by 0.5 C, hold for 1 minte, ramp at 0.1 C per second. Step 5: Go to step 4 for 19x. Step 6: From 50 C, decrease by 1 C, hold for 2 mintes, ramp at 0.1 C per second. Step 7: Go to step 6 for 9x. Step 8: From 40 C, hold for 10 mintes, ramp at 0.1 C per second. 3 Label a new 96-well PCR plate HYP. Procedre 1 Dilte 2.5 µl CAT with 2.5 µl RS1 per sample well in a microcentrifge tbe. 2 Plse vortex to mix, and then centrifge briefly. 3 Add 5 µl RS1 to one well as a no template control. 4 Add 5 µl dilted DNA to the remaining wells. 5 Add 5 µl dilted CAT to all wells. 6 Add 15 µl OHS2 to each well. Using a P20 pipette, pipette slowly to mix. 7 If bbbles form, centrifge the plate at 100 g for 20 seconds. 8 Place on the preprogrammed thermal cycler and rn the HYB program. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 8

12 TrSeq Genotype Ne Reference Gide 9 For 96 samples, combine ELE and ELB as follows. Scale accordingly for p to 384 samples. a b Transfer 137 µl ELE to the contents of the ELB tbe. Flick and invert to mix. Do not vortex. NOTE Prepare the fll amont of ELE/ELB mixtre for the kit. If necessary, store aliqots at -25 C to -15 C for p to 3 months. Do not allow more than six freeze-thaw cycles. 10 Place the ELB/ELE mixtre on ice for se dring the procedre to extend and ligate bond oligos. Remove Unbond Oligos This step ses SPB to remove nbond oligos from gdna. Three wash steps with SW1 and one wash step with 60% ethanol ensre the complete removal of nbond oligos. Consmables and Eqipment SW1 (Stringent Wash 1) SPB (Sample Prification Beads) Freshly prepared 60% ethanol (EtOH) Magnetic Stand DynaMag-96 Side Skirted Magnet (se with 96-well fll-skirted PCR plates) DynaMag-96 Side Magnet (se with Eppendorf 96-well twin.tec PCR plates) WARNING This set of reagents contains potentially hazardos chemicals. Personal injry can occr throgh inhalation, ingestion, skin contact, and eye contact. Wear protective eqipment, inclding eye protection, gloves, and laboratory coat appropriate for risk of exposre. Use SW1 in a hood. Handle sed reagents as chemical waste and discard in accordance with applicable regional, national, and local laws and reglations. For additional environmental, health, and safety information, see the SDS at spport.illmina.com/sds.html. Abot Reagents Rinse SW1 over any beads on the side of the well when pipetting. SPB Make sre that beads are at room temperatre. Vortex SPB vigorosly before each se. When mixing, mix thoroghly. Preparation 1 Transfer 3 ml SPB to a tbe for pre-pcr se. 2 Transfer 6 ml SPB to a tbe for post-pcr se. 3 Prepare 200 µl per well of fresh 60% ethanol from 100% ethanol. Procedre 1 Add 25 µl SPB to each well of the HYB plate. Pipette slowly to mix. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 9

13 TrSeq Genotype Ne Reference Gide 2 Incbate at room temperatre for 5 mintes. 3 Place on the magnetic stand and wait ntil the liqid is clear (~2 mintes). 4 Remove and discard all spernatant from each well. 5 Keeping the plate on the magnetic stand, wash beads three times as follows. a b c Add 80 µl SW1 to each well. Incbate at room temperatre for 30 seconds. Remove and discard all spernatant from each well. 6 Use a 20 µl pipette to remove residal SW1 from each well. 7 Add 80 µl of 60% EtOH to each well. 8 Incbate at room temperatre for 30 seconds. 9 Remove and discard all spernatant from each well. 10 Use a 20 µl pipette to remove residal EtOH from each well. 11 Air-dry for p to 5 mintes. Proceed immediately to the next step. Extend and Ligate Bond Oligos This step connects the hybridized pstream and downstream oligos. A DNA polymerase extends from the pstream oligo throgh the targeted region, followed by ligation to the 5 end of the downstream oligo sing a DNA ligase. The reslt is the formation of prodcts containing the targeted regions of interest flanked by the seqences reqired for amplification. Consmables EDP (Enhanced DNA Polymerase) ELB/ELE mixtre EMM (Enhanced Master Mix) Microseal 'B' adhesive seal Microcentrifge tbe (1) Prepare for a sbseqent step: Index 1 (i7) adapters (N7XX) Index 2 (i5) adapters (S5XX) Abot Reagents Do not allow more than six freeze-thaw cycles of EMM. ELB/ELE mixtre Invert and flick to mix, and then centrifge briefly. Do not vortex. Prepare the fll amont of the mixtre. If needed, store aliqots at -25 C to -15 C for p to 3 months. Do not allow more than six freeze-thaw cycles. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 10

14 TrSeq Genotype Ne Reference Gide Preparation 1 Save the following EXT_LIG program for a 22 µl reaction volme on a thermal cycler: Choose the preheat lid option and set to 100 C 37 C for 45 mintes 70 C for 20 mintes Hold at 4 C 2 Prepare the following consmables. Reagent Storage Instrctions ELB/ELE mixtre -25 C to -15 C If frozen, thaw at room temperatre and then place on ice. Invert and flick to mix, and then centrifge briefly. Do not vortex. EDP -25 C to -15 C Place on ice. Flick to mix, and then centrifge briefly. EMM -25 C to -15 C Thaw at room temperatre for 20 mintes. Vortex to mix. Index adapters (i7 and i5) Procedre -25 C to -15 C Only prepare adapters being sed. Thaw at room temperatre for 20 mintes in preparation for the procedre to amplify libraries. Invert each tbe to mix. Centrifge briefly. 1 Remove the plate from the magnetic stand. 2 Using a P100 or P200 pipette, add 22 µl ELB/ELE mixtre to each well. 3 Using a pipette set to 20 µl or a P20 pipette, pipette to mix. Make sre that no beads remain in the pipette. 4 If bbbles form, centrifge at 100 g for 20 seconds. 5 Place on the thermal cycler and rn the EXT_LIG program. 6 Combine EDP and EMM in a microcentrifge tbe as follows. Nmber of Samples EDP EMM µl 21 µl µl 2006 µl Volmes inclde an additional 10%. 7 Pipette the EDP/EMM mixtre to mix, and then centrifge briefly. 8 Place the EDP/EMM mixtre on ice for se dring the procedre to amplify libraries. Amplify Libraries This step amplifies the extension-ligation prodcts and adds Index 1 (i7) adapters, Index 2 (i5) adapters, and seqences reqired for clster formation. Consmables EDP/EMM mixtre Index 1 adapters (N7XX) Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 11

15 TrSeq Genotype Ne Reference Gide Index 2 adapters (S5XX) Microseal 'B' adhesive seal Preparation 1 Save the following PCR program on a thermal cycler sing the appropriate nmber of PCR cycles, which are listed in the following table. 95 C for 3 mintes X cycles of: 98 C for 20 seconds 68 C for 20 seconds 72 C for 30 seconds 72 C for 1 minte Hold at 10 C Amplicon Plexity Nmber of PCR Cycles (X)¹ < 96 amplicons amplicons amplicons amplicons amplicons² 23 ¹ To achieve desired library yield and specificity, optimize the PCR cycle nmber for yor oligo pool. ² Alignment specificity might be redced for high plexity panels designed with larger amplicon sizes. NOTE PCR cycles are based on 50 ng inpt DNA and the nmber of amplicons in the CAT. 2 If sing an iceless cooler, eqilibrate the temperatre to 2 C to 8 C. Procedre 1 Arrange the Index 1 (i7) adapters in colmns 1 12 of the TrSeq Index Plate Fixtre. 2 Arrange the Index 2 (i5) adapters in rows A H of the TrSeq Index Plate Fixtre. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 12

16 TrSeq Genotype Ne Reference Gide Figre 2 TrSeq Index Plate Fixtre A B C D Rows A H: Index 2(i5) adapters(white caps) Colmns 1 12: Index 1(i7) adapters(orange caps) TrSeq Index Plate Fixtre HYP plate 3 Place the HYP plate containing beads on a TrSeq Index Plate Fixtre. 4 Add 4 µl of each Index 1 (i7) adapter down each colmn. Replace the cap on each i7 adapter tbe with a new orange cap. 5 Add 4 µl of each Index 2 (i5) adapter across each row. Replace the cap on each i5 adapter tbe with a new white cap. 6 Place the plate on ice or iceless cooler. 7 Add 20 µl EDP/EMM mixtre to each well. Pipette to mix. 8 Centrifge at 280 g for 1 minte. 9 Place the plate on ice or iceless cooler. 10 Immediately transfer to the post-pcr area. 11 Place on the preprogrammed thermal cycler and rn the PCR program for the appropriate nmber of cycles. The beads remain in the wells dring PCR. SAFE STOPPING POINT If yo are stopping, seal the plate and store at 2 C to 8 C for p to 2 days. Alternatively, leave on the thermal cycler overnight. Clean Up Libraries This step ses SPB (Sample Prification Beads) to prify the PCR prodcts from other reaction components. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 13

17 TrSeq Genotype Ne Reference Gide Consmables and Eqipment RSB (Resspension Bffer) SPB (Sample Prification Beads) Barcode labels CLP (Cleanp Plate) LNP (Library Normalization Plate) 96-well midi plates (2) Microseal 'B' adhesive seals Freshly prepared 80% ethanol (EtOH) Magnetic stand-96 (se with midi 96-well storage plates) Abot Reagents Vortex SPB vigorosly before se. Preparation 1 Prepare the following consmables. Reagent Storage Instrctions SPB 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. Do not exceed 25 C. RSB 15 C to 30 C If frozen, thaw at room temperatre for 20 mintes. Vortex to mix. 2 Prepare 400 µl fresh 80% ethanol per well from 100% ethanol. 3 Label a new midi plate CLP. 4 Label a new midi plate LNP. Procedre 1 Centrifge the HYP plate at 280 g for 1 minte. 2 Add 36 µl SPB to each well of the CLP plate. 3 Place the HYP plate on a magnetic stand and wait ntil the liqid is clear (~1 minte). 4 Transfer 45 µl clear spernatant from each well of the HYP plate to the corresponding well of the CLP plate. Transfer as few beads as possible. 5 Shake the plate at 1800 rpm for 2 mintes. 6 Incbate at room temperatre for 5 mintes. 7 Centrifge at 280 g for 1 minte. 8 Place on a magnetic stand and wait ntil the liqid is clear (~2 mintes). 9 Remove and discard all spernatant from each well. 10 Wash two times as follows. a Add 200 µl freshly prepared 80% EtOH to each sample well. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 14

18 TrSeq Genotype Ne Reference Gide b c Incbate on the magnetic stand for 30 seconds. Remove and discard all spernatant from each well. 11 Using a 20 µl pipette, remove residal EtOH from each well. 12 Remove from the magnetic stand and air-dry for p to 5 mintes. 13 Add 25 µl RSB to each well. 14 Shake the plate at 1800 rpm for 2 mintes. 15 If beads are not resspended, pipette to mix and then shake at 1800 rpm for 2 mintes. 16 Incbate at room temperatre for 2 mintes. 17 Centrifge at 280 g for 1 minte. 18 Place on a magnetic stand and wait ntil the liqid is clear (~2 mintes). 19 Transfer 20 µl prified library from each well of the CLP plate to the corresponding well of the LNP plate. 20 From the remaining liqid in the CLP plate, rn an aliqot of the samples and control sing any of the following methods: 5 µl on a 4% agarose gel. For p to six samples, 1 µl on an Agilent Bioanalyzer sing a DNA 1000 chip. For p to 96 samples, 1 µl on an Agilent 2200 TapeStation sing the D1000 ScreenTape assay. For p to 288 samples, 2 µl on an Advanced Analytical Fragment Analyzer sing the Standard Sensitivity NGS Fragment Analysis Kit. Table 2 Expected PCR Prodct Sizes Amplicon Size PCR Prodct Size 150 bp ~280 bp 175 bp ~310 bp Figre 3 Bioanalyzer Trace Example A B C Lower Marker Expected PCR Prodct for 175 bp Amplicons(~320 bp) Upper Marker Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 15

19 TrSeq Genotype Ne Reference Gide Figre 4 Fragment Analyzer Example A B C Lower Marker Expected PCR Prodct for 175 bp Amplicons(~320 bp) Upper Marker SAFE STOPPING POINT If yo are stopping, seal the plate and store at -25 C to -15 C for p to 6 months. Normalize Libraries This step normalizes the qantity of each library for balanced representation in pooled libraries. Only samples containing DNA reqire processing throgh the sbseqent steps. Consmables and Eqipment LNA1 (Library Normalization Additives 1) LNB1 (Library Normalization Beads 1) LNW1 (Library Normalization Wash 1) LNS2 (Library Normalization Storage bffer 2) SGP (Storage Plate) barcode label 0.1 N NaOH (freshly prepared) 96-well PCR plate, skirted 15 ml conical tbe Microseal 'B' adhesive seals Magnetic stand-96 (se with midi 96-well storage plates) Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 16

20 TrSeq Genotype Ne Reference Gide WARNING This set of reagents contains potentially hazardos chemicals. Personal injry can occr throgh inhalation, ingestion, skin contact, and eye contact. Wear protective eqipment, inclding eye protection, gloves, and laboratory coat appropriate for risk of exposre. Handle sed reagents as chemical waste and discard in accordance with applicable regional, national, and local laws and reglations. For additional environmental, health, and safety information, see the SDS at spport.illmina.com/sds.html. WARNING This set of reagents contains ß-mercaptoethanol. Perform the following procedre in a hood or well-ventilated area. Abot Reagents When mixing, mix thoroghly. Mix only the amonts of LNA1 and LNB1 reqired for the crrent experiment. Use a P1000 pipette to transfer LNB1 to LNA1. Store remaining LNA1 and LNB1 separately at their respective temperatres. Vortex LNB1 thoroghly before se, and make sre that it is resspended. Homogeneos resspension is essential for consistent clster density on the flow cell. Preparation 1 Prepare the following consmables. Reagent Storage Instrctions LNA1-25 C to -15 C Thaw at room temperatre. Let stand for 30 mintes to bring to room temperatre. Vortex to mix. Inspect in front of a light. Make sre that all precipitate has dissolved. LNB1 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. Vortex for at least 1 minte. Invert intermittently to resspend. Make sre that the bottom of the tbe is free of pellets. LNW1 2 C to 8 C Thaw at room temperatre. Let stand for 30 mintes to bring to room temperatre. LNS2 15 C to 30 C Vortex to mix. 2 Prepare fresh 0.1 N NaOH. 3 Label a new midi plate SGP. Procedre 1 Add 44 µl LNA1 per library to a new 15 ml conical tbe. 2 Use a P1000 pipette to resspend LNB1. 3 Transfer 8 µl LNB1 per library to the 15 ml conical tbe of LNA1. Invert to mix. 4 Add 45 µl LNA1/LNB1 to each well of the LNP plate. Each well contains 20 µl library. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 17

21 TrSeq Genotype Ne Reference Gide 5 Shake at 1800 rpm for 30 mintes. Shorter drations can affect library representation and clster density. 6 Place on a magnetic stand and wait ntil the liqid is clear (~2 mintes). 7 Remove and discard all spernatant. 8 Remove from the magnetic stand. 9 Wash two times as follows. a b c d Add 45 µl LNW1 to each library well. Shake at 1800 rpm for 5 mintes. Place on a magnetic stand and wait ntil the liqid is clear (~2 mintes). Remove and discard all spernatant. 10 Use a 20 µl pipette to remove residal LNW1 from each well. 11 Remove from the magnetic stand. 12 Add 30 µl fresh 0.1 N NaOH to each well. 13 Shake at 1800 rpm for 5 mintes. 14 Place on a magnetic stand and wait ntil the liqid is clear (~2 mintes). 15 Add 30 µl LNS2 to each well of the SGP plate. 16 Transfer 30 µl spernatant from each well of the LNP plate to the corresponding well of the SGP plate. 17 Centrifge the SGP plate at 1000 g for 1 minte. SAFE STOPPING POINT If yo are stopping, seal the plate and store at -25 C to -15 C for p to 30 days. Pool Libraries This step pools libraries by combining eqal volmes of normalized libraries in one tbe. Consmables PAL (Pooled Amplicon Library) barcode label Microcentrifge tbe RNase/DNase-free 8-tbe strips and caps Preparation 1 If the SGP plate was stored frozen, prepare as follows. a b c Thaw at room temperatre. Centrifge at 1000 g for 1 minte. Pipette to mix. 2 Label a new Eppendorf tbe PAL. Procedre 1 Centrifge the SGP plate at 1000 g for 1 minte. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 18

22 TrSeq Genotype Ne Reference Gide 2 Transfer 5 µl of each library to an 8-tbe strip, colmn by colmn. 3 Seal the plate and store at -25 C to -15 C. 4 Transfer the contents of the 8-tbe strip to the PAL tbe. Pipette to mix. 5 Denatre and dilte the library pool to the appropriate loading concentration for the seqencing rn. For instrctions, see the denatre and dilte libraries gide for yor instrment. SAFE STOPPING POINT If yo are stopping, cap the tbes and store at -25 C to -15 C for p to 7 days. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 19

23 Appendix A Spporting Information Spporting Information Introdction 20 Acronyms 20 Kit Contents 21 Consmables and Eqipment 23 Introdction The protocols described in this gide assme that yo have reviewed the contents of this appendix, confirmed kit contents, and obtained all reqired consmables and eqipment. Acronyms Acronym Definition ACP3 Amplicon Control Oligo Pool 3 CLP Cleanp Plate EDP Enhanced DNA Polymerase ELB Extension-Ligation Bffer ELE Extension-Ligation Enzyme EMM Enhanced Master Mix CAT TrSeq Genotype Ne Oligos HT1 Hybridization Bffer HYP Hybridization Plate LNA1 Library Normalization Additives 1 LNB1 Library Normalization Beads 1 LNP Library Normalization Plate LNS2 Library Normalization Storage Bffer 2 LNW1 Library Normalization Wash 1 OHS2 Oligo Hybridization for Seqencing Reagent 2 PAL Pooled Amplicon Library RS1 Resspension Soltion 1 RSB Resspension Bffer SGP Storage Plate SNP Single Ncleotide Polymorphism SPB Sample Prification Beads SS1 Sample Stabilization Soltion 1 SW1 Stringent Wash 1 Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 20

24 TrSeq Genotype Ne Reference Gide Kit Contents Make sre that yo have all the kit components identified in this section before starting the library prep procedres. The TrSeq Genotype Ne kit is provided in a cstom configration that incldes library prep reagents, index adapters, and seqencing reagents. Kit Name Catalog # TrSeq Genotype Ne (96 Samples) Library Prep Reagents Reagents for preparing TrSeq Genotype Ne libraries are provided in three DNA Amplicon Assay v2 boxes and one oligo box. Box 1 DNA Amplicon Assay v2 (16 samples), Store at -25 C to -15 C in the Pre-PCR Area Qantity Reagent Description 1 ACP3 Amplicon Control Oligo Pool 3 1 EDP Enhanced DNA Polymerase 1 ELB Extension-Ligation Bffer 1 ELE Extension-Ligation Enzyme 1 EMM Enhanced Master Mix Box 1 also contains the HYP barcode label. Box 2 DNA Amplicon Assay v2 (16 samples), Store in the Pre-PCR Area Qantity Reagent Description Storage Temperatre 1 LNB1 Library Normalization Beads 1 2 C to 8 C 1 OHS2 Oligo Hybridization for Seqencing Reagent 2 2 C to 8 C 1 RS1 Resspension Soltion 1 15 C to 30 C 1 SPB Sample Prification Beads 2 C to 8 C 1 SS1 Sample Stabilization Soltion 1 2 C to 8 C 1 SW1 Stringent Wash 1 2 C to 8 C Box 3 DNA Amplicon Assay v2, Store in the Post-PCR Area Qantity Reagent Description Storage Temperatre 1 HT1 Hybridization Bffer -25 C to -15 C 1 LNA1 Library Normalization Additives 1-25 C to -15 C 1 LNS2 Library Normalization Storage Bffer 2 15 C to 30 C 2 LNW1 Library Normalization Wash 1 2 C to 8 C 2 RSB Resspension Bffer 15 C to 30 C Box 3 also contains the CLP, LNP, SGP, PAL, and DAL barcode labels. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 21

25 TrSeq Genotype Ne Reference Gide TrSeq Genotype Ne Oligo Box, Store at -25 C to -15 C Qantity Reagent Description 1 CAT TrSeq Genotype Ne Oligos Index Adapters Index adapters for TrSeq Genotype Ne libraries are provided in the Nextera XT Index Kit v2 Sets A D. Combining all for sets achieves 384 index combinations. Nextera XT Index Kit v2 Set A (96 indexes, 192 samples), Store at -25 C to -15 C in the Pre-PCR Area Qantity Reagent Name 8 Index Adapters S502, S503, S505 S508, S510, and S Index Adapters N701 N707, N710 N712, N714, and N715 Nextera XT Index Kit v2 Set B (96 indexes, 192 samples), Store at -25 C to -15 C in the Pre-PCR Area Qantity Reagent Name 8 Index Adapters S502, S503, S505 S508, S510, and S Index Adapters N716, N718 N724, and N726 N729 Nextera XT Index Kit v2 Set C (96 indexes, 192 samples), Store at -25 C to -15 C in the Pre-PCR Area Qantity Reagent Name 8 Index Adapters S513, S515 S518, and S520 S Index Adapters N701 N707, N710 N712, N714, and N715 Nextera XT Index Kit v2 Set D (96 indexes, 192 samples), Store at -25 C to -15 C in the Pre-PCR Area Qantity Reagent Name 8 Index Adapters S513, S515 S518, and S520 S Index Adapters N716, N718 N724, and N726 N729 Index Adapter Replacement Caps, Store at 15 C to 30 C in the Pre-PCR Area Qantity Description 1 i7 Index Tbe Caps, Orange 1 i5 Index Tbe Caps, White Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 22

26 TrSeq Genotype Ne Reference Gide Seqencing Reagents A TrSeq Genotype Ne kit incldes the reagents and flow cell reqired for a seqencing rn on the MiniSeq, NextSeq, or MiSeq system. MiniSeq Reagents Qantity Description Storage Temperatre 1 MiniSeq Reagent Cartridge (150 cycles) -25 C to -15 C 1 MiniSeq Flow Cell 2 C to 8 C 1 Hybridization Bffer (HT1) -25 C to -15 C NextSeq Reagents Qantity Description Storage Temperatre 1 NextSeq 500 High Otpt Reagent Cartridge v2 (150 cycles) -25 C to -15 C 1 NextSeq 500 High Otpt Flow Cell v2 2 C to 8 C 1 NextSeq 500/550 Bffer Cartridge v2 15 C to 30 C 1 NextSeq Accessory Box v2-25 C to -15 C MiSeq Reagent Kit v3 Table 3 Box 1 of 2 Qantity Description Storage Temperatre 1 MiSeq v3 Reagent Tray (150 Cycles, PE) -25 C to -15 C 1 Hybridization Bffer (HT1) -25 C to -15 C Table 4 Box 2 of 2 Qantity Description Storage Temperatre 1 PE MiSeq Flow Cell 2 C to 8 C 1 Incorporation Bffer (PR2) 2 C to 8 C Consmables and Eqipment Make sre that yo have the reqired ser-spplied consmables and eqipment before starting the protocol. The protocol has been optimized and validated sing the items listed. Comparable performance is not garanteed when sing alternate consmables and eqipment. Use separate sets of consmables and eqipment for pre-pcr and post-pcr procedres. Different types of magnetic stands are needed for pre-pcr and post-pcr procedres. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 23

27 TrSeq Genotype Ne Reference Gide Consmables Consmable Spplier 10 N NaOH, moleclar biology grade¹ General lab spplier 20 µl barrier pipette tips General lab spplier 20 µl mltichannel pipettes General lab spplier 20 µl single channel pipettes General lab spplier 200 µl barrier pipette tips General lab spplier 200 µl mltichannel pipettes General lab spplier 200 µl single channel pipettes General lab spplier 1000 µl barrier pipette tips General lab spplier 1000 µl mltichannel pipettes General lab spplier 1000 µl single channel pipettes General lab spplier One of the following plate types: Hard-shell 96-well skirted PCR plates, low-profile, skirted Eppendorf 96-well twin.tec PCR plates, semiskirted 96-well storage plates, 0.8 ml (midi plate) Adhesive seal roller Conical tbes, 15 ml DNA moleclar weight markers Ethanol, 100% for moleclar biology Ice bcket Microcentrifge tbes Microseal 'B' adhesive seals PCR grade water RNase/DNase-free 8-tbe strips and caps One of the following library qality assessment methods: 4% Agarose gel Standard Sensitivity NGS Fragment Analysis Kit ( bp) DNA 1000 Kit [Optional] TrSeq Index Plate Fixtre Kit² One of the following sppliers, depending on plate type: Bio-Rad, catalog # HSP-9601 Fisher Scientific, catalog # E Fisher Scientific, catalog # AB-0859 or AB-0765 General lab spplier General lab spplier General lab spplier General lab spplier General lab spplier General lab spplier Bio-Rad, catalog # MSB-1001 General lab spplier General lab spplier One of the following sppliers, depending on method: General lab spplier Advanced Analytical Technologies, part # DNF-473 Agilent Technologies, catalog # Illmina, catalog # FC ¹ Prepare from tablets or se a standard soltion. ² A resable part for setting p index adapters. Eqipment Pre-PCR Eqipment Eqipment Iceless cooler for 96-well plates 96-well thermal cycler (with heated lid) See Thermal Cyclers. Spplier General lab spplier General lab spplier Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 24

28 TrSeq Genotype Ne Reference Gide Eqipment One of the following magnetic stands, depending on the type of PCR plate: DynaMag-96 Side Skirted Magnet (se with 96-well fllskirted PCR plates) DynaMag-96 Side Magnet (se with Eppendorf 96-well twin.tec PCR plates) Microplate centrifge Spplier Life Technologies: Catalog # Catalog # 12331D General lab spplier Post-PCR Eqipment Eqipment Magnetic stand-96 (se with midi 96-well storage plates) One of the following: BioShake iq high-speed thermal mixer BioShake XP high-speed lab shaker Microplate centrifge One of the following library qality assessment methods: Fragment Analyzer Atomated CE System 2100 Bioanalyzer Desktop System Gel electrophoresis spplies and apparats Agilent 2200 TapeStation Heat block for 1.5 ml centrifge tbes Spplier Life Technologies, catalog # AM10027 Q.Instrments: Order # Order # General lab spplier One of the following sppliers, depending on method: Advanced Analytical Technologies, part # FSv2-CE2 or FSv2-CE10 Agilent Technologies, catalog # G2940CA General lab spplier Agilent Technologies, catalog # G2964AA General lab spplier Thermal Cyclers Use the following recommended settings for selected thermal cycler models. Before performing library prep, validate any thermal cyclers not listed. NOTE The Bio-Rad thermal cyclers might provide sperior specificity. Thermal Cycler Block Type Ramp Rate Lid Temp Bio-Rad S1000 Standard 0.1 C Heated, Constant at 100 C Bio-Rad C1000 Standard 0.1 C Heated, Constant at 100 C Bio-Rad T100 Standard 0.1 C Heated, Constant at 100 C Applied Biosystems GeneAmp PCR System 9700 Applied Biosystems Veriti 96-Well Gold 1% Heated, Constant at 100 C Alloy 2% to 2.1% Heated, Constant at 100 C Block Rate Vessel Type -- Bio-Rad Hard-Shell 96-Well Skirted PCR Plates, low-profile, skirted -- Bio-Rad Hard-Shell 96-Well Skirted PCR Plates, low-profile, skirted -- Eppendorf twin.tec PCR Plate 96, semiskirted 9600 for HYB and EXT_LIG programs or MAX for PCR program Eppendorf twin.tec PCR Plate 96, semiskirted -- Eppendorf twin.tec PCR Plate 96, semiskirted Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 25

29 Technical Assistance For technical assistance, contact Illmina Technical Spport. Website: Illmina Cstomer Spport Telephone Nmbers Region Toll Free Regional North America Astralia Astria Belgim China Denmark Finland France Germany Hong Kong Ireland Italy Japan Netherlands New Zealand Norway Singapore Spain Sweden Switzerland Taiwan United Kingdom Other contries Safety data sheets (SDSs) Available on the Illmina website at spport.illmina.com/sds.html. Prodct docmentation Available for download in PDF from the Illmina website. Go to spport.illmina.com, select a prodct, then select Docmentation & Literatre. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 26

30 Docment # v00 Illmina 5200 Illmina Way San Diego, California U.S.A ILMN (4566) (otside North America) techspport@illmina.com For Research Use Only. Not for se in diagnostic procedres Illmina, Inc. All rights reserved.