Twist Human Core Exome EF Multiplex Complete Kit, 16 Samples PN

Transcription

1 Twist Human Core Exome Enrichment Kit Twist Human Core Exome EF Multiplex Complete Kit, 16 Samples PN Reagents for preparing 16 exome-enriched libraries ready for sequencing from human genomic DNA The Twist Human Core Exome EF Multiplex Complete Kit is used to generate exome libraries from human genomic DNA for sequencing on Illumina next generation sequencing (NGS) systems. The kit provides reagents needed to construct 16 libraries and perform two capture reactions, each with eight indexed libraries. The following kits are included: Twist Library Preparation EF Kit 1, 16 Samples and Twist Library Preparation Kit 2, 16 Samples PN , PN Contains reagents for enzymatic DNA fragmentation and adapter ligation. Twist Human Core Exome Multiplex Hybridization Kit, 16 Samples PN Contains biotinylated probes, hybridization buffer and PCR amplification primers. Twist Enrichment Reagents, 2 Reactions PN Contains reagents for enrichment and purification of targeted regions. Twist CD Index Adapter Set 1 16, 16 Samples PN Contains 16 indexed adapters for DNA library ligation. Twist Adapter-Specific Blockers 1 16, 2 Reactions PN Contains blockers to prevent nonspecific capture. This protocol also provides guidance on preparing exome libraries using the Twist Human Core Exome Multiplex Enrichment Kit 1 PN , Twist Human Core Exome Multiplex Enrichment Kit 2 PN , and the Twist Human Core Exome Multiplex Hybridization Kit 1 PN This product is for research use only. 1

2 Legal & Intended Use LEGAL This document may contain references to other third-party resources such as sources of information, hardware or software, products, or services and/or web sites owned or licensed by third parties. Twist Bioscience does not control or take responsibility for any third-party resources, including, without limitation, the accuracy, reliability, copyright compliance, compatibility, performance, legality, or any other aspect of third-party resources. The inclusion of such resources in this document does not imply endorsement by Twist Bioscience of any third-party resources. Certain processes described in this document may be subject to patent protection or licenses in local jurisdictions, including those owned or licensed by parties other than Twist Bioscience. Purchase of this product does not include a license to perform any such processes. Users of this product may therefore be required to obtain a patent license depending upon the particular application and country in which the product is used before performing such processes. INTENDED USE This product is for research use only. This product is not intended for the diagnosis, prevention, or treatment of a disease or condition. Twist Bioscience assumes no liability regarding use of the product for applications in which it is not intended. 2

3 Table of Contents TABLE OF CONTENTS Twist Human Core Exome EF Multiplex Complete Kit 1 Material Included in this Kit 4 Material to be Supplied by User 6 Equipment to be Supplied by User 7 General Notes and Precautions 8 Important Notes 8 Guidelines for gdna Samples 9 Twist Human Exome Enrichment Process Workflow 10 Fragment DNA, End Repair, da-tailing 11 Ligate Indexed Adapters and Purify 13 Pre-Capture PCR Amplify, Purify and QC 15 Pool Amplified Indexed Libraries in Set of 8 18 Hybridize Capture Probes with Pools 19 Bind Hybridized Targets to Streptavidin Beads 21 Post-Capture PCR Amplify, Purify and QC 23 Sequencing on the Illumina Platform 26 appendix a1 List of CD Index Sequences 27 appendix a2 Pooling Guidelines 28 appendix b Overview of Modular Kits 29 appendix C Guidance for Use of Modular Kits 30 3

4 Material Included in this Kit MATERIAL INCLUDED IN THIS KIT Please read product packaging and storage recommendations carefully and store components as recommended immediately upon arrival. COMPONENT QUANTITY SHIPPING & STORAGE 5x Fragmentation Enzyme PN Twist Library Preparation EF Kit 1, 16 Samples PN x Fragmentation Buffer PN DNA Ligation Mix PN DNA Ligation Buffer PN samples 20 C Amplification Primers, ILMN PN Twist Library Preparation Kit 2, 16 Samples PN DNA Purification Beads PN samples 2 8 C (Shipped at room temperature) Twist Human Core Exome Multiplex Hybridization Kit, 16 Samples PN Twist Adapter-Specific Blockers 1 16, 2 Reactions PN Exome Probes PN Amplification Primers, ILMN PN Hybridization Mix PN Blocker Solution PN Blocker Mix 1 16 PN Binding Buffer PN samples 20 C 2 reactions 20 C Twist Enrichment Reagents, 2 Reactions PN Wash Buffer 1 PN Wash Buffer 2 PN DNA Purification Beads PN reactions 2 8 C (Shipped at room temperature) Streptavidin Binding Beads PN

5 Material Included in this Kit, Continued MATERIAL INCLUDED IN THIS KIT, continued COMPONENT QUANTITY SHIPPING & STORAGE Indexed Adapters 01 PN Indexed Adapters 02 PN Indexed Adapters 03 PN Indexed Adapters 04 PN Indexed Adapters 05 PN Indexed Adapters 06 PN Indexed Adapters 07 PN Twist CD Index Adapter Set 1 16, 16 Samples PN Indexed Adapters 08 PN Indexed Adapters 09 PN Indexed Adapters 10 PN samples 20 C Indexed Adapters 11 PN Indexed Adapters 12 PN Indexed Adapters 13 PN Indexed Adapters 14 PN Indexed Adapters 15 PN Indexed Adapters 16 PN

6 Material to be Supplied by User MATERIAL TO BE SUPPLIED BY USER The following materials or their equivalent are required to generate exome libraries using the Twist Human Core Exome EF Multiplex Complete Kit. PRODUCT SUPPLIER/CATALOG NUMBER Ethanol Sigma-Aldrich, #E7023 Molecular biology grade water Fisher Scientific, #SH KAPA HiFi HotStart ReadyMix Kapa Biosystems, #KK mm Tris-HCl ph8 Fisher Scientific, #NC Buffer EB Qiagen, # mL microcentrifuge tubes VWR, # Thin-walled PCR 0.2-mL strip-tubes Eppendorf, # well thermal cycling plates VWR, # mL compatible magnetic stand Beckman Coulter, # well compatible magnetic plate Alpaqua, #A Qubit dsdna Broad Range Quantitation Assay Thermo Fisher Scientific, #Q32853 Qubit dsdna High Sensitivity Quantitation Assay Thermo Fisher Scientific, #Q32851 BioAnalyzer DNA 7500 Kit Agilent, # BioAnalyzer High Sensitivity DNA Kit Agilent, #

7 Equipment to be Supplied by User EQUIPMENT TO BE SUPPLIED BY USER The following equipment or their equivalent are required to generate exome libraries using the Twist Human Core Exome EF Multiplex Complete Kit. PRODUCT SUPPLIER/CATALOG NUMBER Pipettes and tips Rainin pipette and tips Vortex mixer Scientific Industries, #(G560)SI-0236 Benchtop mini-centrifuge for 0.2-mL tube Spectrafuge Mini-Centrifuge, #C1301 Thermomixer for 1.5-mL tube Eppendorf, # well thermal cycler with heated lid Thermo Fisher Scientific, # Lab shaker, rocker, rotator Southwest Science, #STR200-V Qubit 3.0 Fluorometer Thermo Fisher Scientific, #Q BioAnalyzer Agilent, #G2939BA DNA vacuum concentrator Thermo Fisher Scientific, #DNA

8 General Notes and Precautions & Important Notes WHAT HAS CHANGED The protocol version supports the updated kit and component names for the Twist Human Core Exome EF Multiplex Complete Kit PN GENERAL NOTES AND PRECAUTIONS Wear the appropriate protective equipment (lab coat, gloves, and protective glasses or goggles) at all times while performing this protocol and while handling the materials included in this kit. To achieve optimal results, read this document before performing the protocol, and follow the provided instructions. Twist cannot guarantee the performance of the Twist Human Core Exome EF Multiplex Complete Kit if modifications are made to the protocol. For guidance on preparing exome libraries using the Twist Human Core Exome Multiplex Enrichment Kit 1 PN , Twist Human Core Exome Multiplex Enrichment Kit 2 PN , and the Twist Human Core Exome Multiplex Hybridization Kit 1 PN , please refer to Appendix B on page 29. For technical support contact us at NGSsupport@twistbioscience.com IMPORTANT NOTES The library preparation step may yield more material than needed for the next step. Excess product may be stored at 20 C for later use. Test the compatibility of your thermal cycler and PCR tubes by incubating at 95 C for up to 5 minutes to ensure the PCR tubes do not crack under heat and pressure. Adjust the tightness of the thermal cycler lid and/or use a spacer specific to the thermal cycler model. Evaporation control is very important during the hybridization process. Test and validate the thermal cycler using water to determine evaporation retention of volumes under the workflow operating conditions required in the protocol. If evaporation loss is >10% during testing of hybridization conditions, make sure to limit evaporation by sealing tubes tightly, avoid bubbles while adding probe, and ensure firm downward pressure from thermal cycler lid. If thermal cycler lid is not adjustable, or otherwise cannot exert sufficient pressure on the tube lids, pressure can be applied using a spacer insert or compression mat. 96-well plates are not recommended for hybridization due to evaporation concerns. 8

9 Guidelines for gdna Samples GUIDELINES FOR gdna SAMPLES Correct input quantity is critical for achieving optimal yield and library fragment length. The recommended DNA input is 50 ng of purified gdna. Use the Thermo Fisher Scientific Qubit dsdna Broad Range Quantitation Assay to accurately quantify input purified gdna. Measuring DNA concentration by absorbance at 260 nm is not recommended. It is important to remove all cations and chelators from the starting gdna sample. The presence of cations and chelators may affect the initial fragmentation reaction. Input DNA should be suspended in Molecular Biology Grade Water, 10 mm Tris-HCl ph 8.0, or Buffer EB. 9

10 Twist Human Exome Enrichment Process Workflow TWIST HUMAN EXOME ENRICHMENT PROCESS WORKFLOW Perform Steps 1 5 on Day 1. Note that Step 5 requires an incubation time of 16 hours, so plan the experiment such that Steps 6 7 can be performed on Day 2. Genomic DNA samples (50ng DNA per sample, 8 samples per hyb) 1 Fragment DNA, repair ends, da-tailing PAGE 11 da-tailed DNA fragments 1 hour 2 Ligate indexed adaptors and purify PAGE 13 Indexed gdna libraries 1 hour DAY 1 3 Pre-capture PCR amplify, purify and QC PAGE 15 Amplified indexed libraries 1 hour STOPPING POINT 4 Pool 8 amplified indexed libraries PAGE 18 Indexed library pool 0.5 hours STOPPING POINT 5 Hybridize capture probes with the pool PAGE 19 Hybridized targets in solution 16.5 hours 6 Bind hybridized targets on streptavidin binding beads PAGE 21 Captured targets on beads 1.5 hours DAY 2 7 Post-capture PCR amplify, purify and QC PAGE 23 Exome-enriched libraries 1 hour STOPPING POINT 8 Ready for Sequencing PAGE 26 Libraries ready for sequencing on Illumina platform 10

11 Step FRAGMENT DNA, REPAIR ENDS, AND da-tailing Protocol utilizing the Twist Library Preparation EF Kit 1 PN Prepare 8 gdna samples in parallel for a single 8-plex capture reaction, or 16 gdna samples in parallel for two 8-plex capture reactions. Each sample should contain 50 ng of purified gdna. Before You Begin: Thaw 5x Fragmentation Enzyme on ice, and mix by flicking the tube with a finger. Thaw 10x Fragmentation Buffer and gdna samples on ice, then mix by pulse vortexing for 2 seconds. Prepare DNA and Thermal Cycler 1.1 Program a thermal cycler with the following conditions. Set the temperature of the heated lid to 70 C. Start the program to pre-chill the thermal cycler. TEMPERATURE TIME 1 Pre-chill 4 C HOLD 2 Fragmentation 32 C 22 minutes 3 Inactivation 65 C 30 minutes 4 Hold 4 C HOLD Use the Qubit dsdna Broad Range Quantitation Assay to determine the concentration of your gdna samples. Dilute gdna samples to 5 ng/µl with water. Add 10 µl of each diluted gdna sample into a clean thin-walled PCR 0.2-mL strip-tube or well of a 96-well thermal cycling plate, and place on ice. Mix the diluted gdna sample by flicking with a finger, and pulse-spin to ensure all solution is at the bottom of the tube. 11

12 Step Fragment DNA 1.5 Prepare the Enzymatic Fragmentation Mix in a 1.5-mL microcentrifuge tube on ice as indicated in the table below. Mix well by gentle pipetting. REAGENT VOLUME FOR 1 REACTION VOLUME FOR 8 REACTIONS (INCLUDING EXCESS) VOLUME FOR 16 REACTIONS (INCLUDING EXCESS) Water 25 µl µl 425 µl 10x Fragmentation Buffer 5x Fragmentation Enzyme 5 µl 42.5 µl 85 µl 10 µl 85 µl 170 µl TOTAL 40 µl 340 µl 680 µl 1.6 Add 40 µl of Enzymatic Fragmentation Mix (from Step 1.5), to each 10-µL gdna sample well or tube, and mix well by gentle pipetting. Keep the reaction on ice. 1.7 Pulse-spin the sample plate or tubes and immediately transfer to the pre-chilled thermal cycler. Proceed to the next cycling step at 32 C (refer to Step 1.1). NOTE: Prepare reagents for Step 2 while the thermal cycler program is in progress (see Step 2, Before You Begin). 1.8 When the thermal cycler program is complete and the sample block has returned to 4 C, remove the samples from the block and place on ice. Immediately proceed to Step 2. 12

13 Step LIGATE INDEXED ADAPTERS AND PURIFY Protocol utilizing the Twist Library Preparation EF Kit 1 PN and Twist CD Index Adapter Set 1 16 PN Before You Begin: Thaw Indexed Adapters, DNA Ligation Mix and DNA Ligation Buffer on ice in preparation for Step 2 while Step 1 is incubating. Prepare 80% ethanol (1 ml for each sample) for use in Step 2 and Step 3. Ligate Adapters 2.1 Equilibrate DNA Purification Beads at room temperature for at least 30 minutes prior to use in Step 2 and Step 3. Add 5.5 µl of Indexed Adapter into each sample well or tube (containing the da-tailed DNA fragments). Mix gently by pipetting, and keep on ice. NOTE: For each group of 8 samples, use either Indexed Adapters set or set Do not mix Indexed Adapters from different sets. 2.2 Prepare the Ligation Mix in a 1.5-mL microcentrifuge tube on ice as indicated in the table below. Mix well by gentle pipetting. REAGENT VOLUME FOR 1 REACTION VOLUME FOR 8 REACTIONS (INCLUDING EXCESS) VOLUME FOR 16 REACTIONS (INCLUDING EXCESS) Water 14.5 µl µl µl DNA Ligation Buffer 20 µl 170 µl 340 µl DNA Ligation Mix 10 µl 85 µl 170 µl TOTAL 44.5 µl µl µl 2.3 Add 44.5 µl of the Ligation Mix to the sample from step 2.1 and mix well by gentle pipetting. Pulse-spin to ensure all solution is at the bottom of the tube. 13

14 Ligate Adapters, continued 2.4 Incubate the ligation reaction at 20 C for 15 minutes in a thermal cycler. IMPORTANT: Turn off the heated lid or set to minimum temperature. Purify NOTE: Prepare reagents for Step 3 while the thermal cycler program is in progress (see Step 3, Before You Begin). 2.5 Vortex the pre-equilibrated DNA Purification Beads until homogenized. 2.6 Add 80 µl (0.8x) of homogenized DNA Purification Beads to each ligation sample from step 2.4, then mix well by vortexing. 2.7 Incubate the samples for 5 minutes at room temperature. 2.8 Place the samples on a magnetic plate for 1 minute. The DNA Purification Beads will form a pellet, leaving a clear supernatant. 2.9 Remove and discard the clear supernatant. Do not remove the plate or tubes from the magnetic plate Wash the bead pellet with 200 µl of freshly prepared 80% ethanol, incubate for 1 minute, then remove and discard the ethanol. Repeat this wash once, for a total of two washes, while keeping the samples on the magnetic plate Carefully remove all remaining ethanol with a 10-µL pipette, making sure not to disturb the bead pellet Air-dry the bead pellet on the magnetic plate for 5 10 minutes or until the bead pellet is dry. Do not overdry the bead pellet Remove the plate or tubes from the magnetic plate and add 17 µl of water to each sample. Mix by pipetting until homogenized and incubate at room temperature for 2 minutes Place the plate or tubes on a magnetic plate and let stand for 3 minutes or until the beads fully pellet Transfer 15 µl of clear supernatant containing the Ligated Indexed Libraries to a clean thin-walled PCR 0.2-mL strip-tube or well of a 96-well thermal cycling plate, making sure not to disturb the bead pellet. 14

15 Step PRE-CAPTURE PCR AMPLIFY, PURIFY AND QC Protocol utilizing the Twist Library Preparation Kit 2 PN Prepare Thermal Cycler and PCR Mix 3.1 Before You Begin: Thaw KAPA HiFi HotStart ReadyMix and Amplification Primers, ILMN on ice in preparation for Step 3 while Step 2 is incubating. Program a thermal cycler with the following conditions. Set the temperature of the heated lid to 105 C. TEMPERATURE TIME CYCLE NUMBER 1 Initialization 98 C 45 seconds 1 2 Denaturation 98 C 15 seconds Annealing 60 C 30 seconds Extension 72 C 30 seconds Final Extension 72 C 1 minute 1 Final Hold 4 C HOLD 3.2 Prepare the PCR Mix in a 1.5-mL microcentrifuge tube on ice as indicated in the table below. Mix well by gentle pipetting. REAGENT VOLUME FOR 1 REACTION VOLUME FOR 8 REACTIONS (INCLUDING EXCESS) VOLUME FOR 16 REACTIONS (INCLUDING EXCESS) Amplification Primers, ILMN KAPA HiFi HotStart ReadyMix 10 µl 85 µl 170 µl 25 µl µl 425 µl TOTAL 35 µl µl 595 µl PCR Amplify Add 35 µl of PCR Mix to the Ligated Indexed Libraries from Step 2.15 and mix well by gentle pipetting. Pulse-spin sample plate or tube and immediately transfer to the thermal cycler and start the cycling program. Remove sample from block when thermal cycler program is complete. 15

16 Step Purify Vortex pre-equilibrated DNA Purification Beads until homogenized. Add 50 µl (1x) of homogenized DNA Purification Beads to the PCR sample from Step 3.5 and mix well by vortexing. 3.8 Incubate the samples for 5 minutes at room temperature Place the samples on a magnetic plate for 1 minute. The DNA Purification Beads will form a pellet, leaving a clear supernatant. Remove and discard the clear supernatant. Do not remove the plate or tubes from the magnetic plate. Wash the bead pellet with 200 µl of freshly prepared 80% ethanol, incubate for 1 minute, then remove and discard the ethanol. Repeat this wash once, for a total of two washes, while keeping the sample on the magnetic plate. Carefully remove and discard all remaining ethanol with a 10 µl pipette, making sure not to disturb the bead pellet. Air-dry the bead pellet on the magnetic plate for 5 10 minutes or until the bead pellet is dry. Do not overdry the bead pellet. Remove the plate or tubes from the magnetic plate and add 22 µl of water to each sample. Mix by pipetting until homogenized and incubate at room temperature for 2 minutes. Place the plate or tubes on a magnetic plate and let stand for 3 minutes or until the beads fully pellet. Transfer 20 µl of clear supernatant containing the Amplified Indexed Libraries to a clean thinwalled PCR 0.2-mL strip-tube or well of a 96-well thermal cycling plate, making sure not to disturb the bead pellet. 16

17 Step 3.17 QC 3.17 Validate and quantify each library using the Agilent DNA 7500 Assay and a Thermo Fisher Scientific Qubit dsdna Broad Range Quantitation Assay. An ideal BioAnalyzer trace is shown below. Average fragment length should be between 375 bp and 425 bp using a range setting of 150 bp to 1000 bp. Ideally, final concentration values should be 80 ng/µl. NOTE: If average fragment length is not in the range of 375 bp and 425 bp, input DNA concentration may be inaccurate. Typically, excess DNA leads to shorter fragments and insufficient input DNA leads to longer fragments. The presence of cations and chelators may also affect the average fragment length. If neither of the above factors apply, optimize the 32 C fragmentation in Step 1.1. Time may be adjusted in 3 minute increments increase time to produce shorter fragments, and decrease time to produce longer fragments. NOTE: You can proceed with concentrations lower than 80 ng/µl, but low concentrations may reflect low efficiency in sample preparation and can result in low library diversity after capture. STOPPING POINT: If not proceeding immediately, the Amplified Indexed Libraries can be stored at 20 C. 17

18 Step POOL AMPLIFIED INDEXED LIBRARIES IN SETS OF 8 Pool Libraries 4.1 Each hybridization reaction requires a total of 1500 ng of indexed libraries, made by pooling equal amounts from 8 individual libraries. Ensure each Indexed Library Pool contains either Indexed Adapter set or set Using Indexed Adapters from two different sets in the same capture is not recommended. Calculate volume in µl needed for ng of each Amplified Indexed Library by dividing by the concentration values measured in ng/µl in Step NUMBER OF INDEXED LIBRARIES PER POOL AMOUNT OF EACH INDEXED LIBRARY PER POOL TOTAL MASS PER POOL ng 1500 ng 4.2 Transfer the calculated volumes from each Amplified Indexed Library to a clean thin-walled PCR 0.2-mL strip-tube. IMPORTANT: Do not use PCR plates for the hybridization preparation step, as excessive evaporation may occur during overnight incubation. 4.3 Pulse-spin the tube and dry the Indexed Library Pool using a SpeedVac system (or a similar evaporator device) using low or no heat. STOPPING POINT: If not proceeding immediately, the dried Indexed Library Pool sample can be stored at 20 C for up to 24 hours. 18

19 Step HYBRIDIZE CAPTURE PROBES WITH POOLS Protocol utilizing the Twist Human Core Exome Multiplex Hybridization Kit PN and Twist Adapter-Specific Blockers 1 16 PN IMPORTANT: Ensure the compatibility of your thermal cycler and PCR tubes with 95 C incubation before proceeding. See Important Notes, page 8 for details. Before You Begin: Thaw Exome Probes, Hybridization Mix, Blocker Solution and Blocker Mix 1 16 on ice. Once reagents are thawed, pulse-vortex for 2 seconds to mix components. Prepare Probes 5.1 Heat Hybridization Mix at 65 C for 10 minutes, or until all precipitate is dissolved, then cool to room temperature on the benchtop for 5 minutes. 5.2 Prepare Exome Probe solution in a thin-walled PCR 0.2-mL strip-tube as indicated in the table below. Mix by gentle pipetting. REAGENT VOLUME Hybridization Mix 20 µl Exome Probes 4 µl Water 3 µl TOTAL 27 µl NOTE: For users adding spike-in content, add 3 µl of spike-in probes in place of water. NOTE: Hybridization Mix is very viscous. Pipette slowly to ensure accurate pipetting. NOTE: Small white particles may be present in the Exome Probes. This will have no effect on the final capture product. 5.3 Heat Exome Probe solution to 95 C for 2 minutes in a thermal cycler with the lid at 105 C, then immediately cool on ice for 5 minutes. Equilibrate to room temperature on the benchtop for 5 minutes. 19

20 Step Prepare Probes, continued 5.4 Resuspend the dried Indexed Library Pool (from Step 4.3) while the Exome Probe solution is cooling by adding the reagents described below. Mix by pipetting. REAGENT VOLUME Dried Indexed Library Pool Blocker Solution 5 µl Blocker Mix µl TOTAL 13 µl Hybridize probes Heat the tube containing the resuspended Indexed Library Pool at 95 C for 5 minutes in a thermal cycler with the lid at 105 C, then allow it to cool to room temperature on the benchtop for no longer than 5 minutes. Carefully mix the Exome Probe solution by pipetting, then add all 27 µl to the tube containing the resuspended Indexed Library Pool. Mix the entire Capture Reaction thoroughly by gentle pipetting, making sure to not generate bubbles. Pulse-spin to ensure all solution is at the bottom of the tube(s). IMPORTANT: Make sure the tube is sealed tightly to prevent excess evaporation over the 16-hour incubation. See Important Notes on page 8 for more details. 5.7 Incubate the Hybridization Reaction at 70 C for 16 hours in a thermal cycler with the lid at 85 C. 20

21 Step BIND HYBRIDIZED TARGETS TO STREPTAVIDIN BEADS Protocol utilizing the Twist Enrichment Reagents PN Optimal performance is achieved when there is minimal evaporation during the 16-hour incubation. The performance of the kit cannot be guaranteed if the volume of the Hybridization Reaction is < 36 µl after incubation. See Important Notes on page 8 for more details. Before You Begin: Inspect Binding Buffer, Wash Buffer 1 and Wash Buffer 2 for precipitate. If precipitate is observed, heat buffer at 48 C until all precipitate is dissolved into solution. Prepare 700 µl of Wash Buffer 2 for each Hybridization Reaction, and preheat to 48 C. Equilibrate Streptavidin Binding Beads and DNA Purification Beads to room temperature for at least 30 minutes prior to use in Step 6 and Step 7. Prepare Beads Thaw KAPA HiFi HotStart ReadyMix and Amplification Primers on ice. Once reagents are thawed, mix by pulse vortexing for 2 seconds. Vortex pre-equilibrated Streptavidin Binding Beads until homogenized. Add 100 µl of Streptavidin Binding Beads to a clean 1.5-mL microcentrifuge tube. Prepare one tube for each Capture Reaction to be performed Add 200 µl of Binding Buffer to the tube(s) and mix by pipetting until homogenized. Place the tube(s) on a magnetic stand for 1 minute. Remove and discard the clear supernatant. Make sure to not disturb the bead pellet. Remove the tube from the magnetic stand. Repeat the wash (Steps 6.3 through 6.6) two additional times. After removing the clear supernatant from the third wash, add a final 200 µl of Binding Buffer and resuspend the beads by vortexing until homogenized. 21

22 Prepare Beads, continued 6.9 Bind Targets 6.10 After the 16-hour hybridization is complete, open thermal cycler lid and quickly transfer the full volume of each Hybridization Reaction (36-40 µl) from Step 5.7 into a corresponding tube of washed Streptavidin Binding Beads from Step 6.8. NOTE: Do not remove the tube(s) of Hybridization Reaction from the thermal cycler, or otherwise allow it to cool to less than 70 C before transferring the solution to the washed Streptavidin Binding Beads. Rapid transfer directly from the thermal cycler at 70 C is a critical step to achieve a low off-target rate. Mix the tube(s) of Hybridization Reaction with Streptavidin Binding Beads for 30 minutes at room temperature on a shaker, rocker, or rotator at a speed sufficient to keep the solution homogenized. NOTE: Aggressive mixing is not required. Beads need only be kept homogenized Remove the tube(s) containing the Hybridization Reaction with Streptavidin Binding Beads from the mixer and pulse-spin to ensure all solution is at the bottom of the tube(s). Place the tube(s) on a magnetic stand for 1 minute Remove and discard clear supernatant. Make sure to not disturb the bead pellet. Remove the tube(s) from the magnetic stand and add 200 µl of Wash Buffer 1. Mix by pipetting until homogenized. Pulse-spin to ensure all solution is at the bottom of the tube(s). Place the tube(s) on a magnetic stand for 1 minute. Remove and discard the clear supernatant. Make sure to not disturb bead pellet Remove the tube(s) from the magnetic stand and add 200 µl of 48 C pre-warmed Wash Buffer 2. Mix by pipetting until homogenized. Pulse-spin to ensure all solution is at the bottom of the tube(s) Incubate tube(s) from for 5 minutes at 48 C Place the tube(s) on a magnetic stand for 1 minute Remove and discard the clear supernatant. Make sure to not disturb bead pellet Repeat the wash (Steps ) two additional times, for a total of three washes After the final wash, remove all traces of supernatant, using a 10 µl pipette. Proceed immediately to the next step. Do not allow the beads to dry Remove the tube(s) from the magnetic stand and add 45 µl of water. Mix by pipetting until homogenized, then incubate the Streptavidin Binding Bead slurry on ice. 22

23 Step POST-CAPTURE PCR AMPLIFY, PURIFY AND QC Protocol utilizing the Twist Enrichment Reagents PN and the Twist Human Core Exome Multiplex Hybridization Kit PN Before You Begin: Prepare 500 µl of 80% ethanol for each Streptavidin Binding Bead slurry to be processed. Prepare Beads, Thermal Cycler, and PCR Mix 7.1 Program a thermal cycler with the following conditions. Set the temperature of the heated lid to 105 C. TEMPERATURE TIME CYCLE NUMBER 1 Initialization 98 C 45 seconds 1 2 Denaturation 98 C 15 seconds Annealing 60 C 30 seconds Extension 72 C 30 seconds Final Extension 72 C 1 minute 1 Final Hold 4 C HOLD If the Streptavidin Binding Bead slurry from Step 6.23 has visibly settled, mix by pipetting until homogenized. Transfer 22.5 µl of the Streptavidin Binding Bead slurry to a clean thin-walled PCR 0.2-mL striptube(s). Keep on ice until ready to use in next step. NOTE: The remaining 22.5 µl of water/streptavidin Binding Bead slurry can be stored at 20 C for future use. 23

24 Step Prepare Beads, Thermal Cycler, and PCR Mix, continued 7.4 Prepare a PCR mixture by adding the following reagents to the tube(s) containing the Streptavidin Binding Bead slurry, then mix by pipetting. REAGENT VOLUME Streptavidin Binding Bead Slurry 22.5 µl Amplification Primers, ILMN 2.5 µl KAPA HiFi HotStart ReadyMix 25 µl TOTAL 50 µl PCR Amplify Purify 7.7 Pulse-spin and transfer the tube(s) to the thermal cycler and start the cycling program. When the thermal cycler program is complete, remove the tube(s) from the block and immediately proceed to purification. Vortex pre-equilibrated DNA Purification Beads until homogenized. 7.8 Add 90 µl (1.8x) homogenized DNA Purification Beads to the tube(s) from step 7.6 and mix well by vortexing. NOTE: It is not necessary to recover supernatant or remove Streptavidin Binding Beads from the amplified PCR product. 7.9 Incubate for 5 minutes at room temperature Place the tube(s) on a magnetic plate for 1 minute Remove and discard the clear supernatant. Do not remove the tube(s) from the magnetic plate. 24

25 Step Purify, continued 7.12 Wash the DNA Purification Bead pellet with 200 µl of freshly prepared 80% ethanol for 1 minute, then remove and discard ethanol. Repeat this wash once, for a total of two washes, while keeping the tube on the magnetic plate Thoroughly remove all remaining ethanol with a 10 µl pipette, making sure to not disturb the bead pellet Air-dry the bead pellet on a magnetic plate for 5 10 minutes or until the bead pellet is dry. Do not overdry the bead pellet Remove the tube(s) from the magnetic plate and add 32 µl of water. Mix by pipetting until homogenized and incubate at room temperature for 2 minutes Place the tube(s) on a magnetic plate and let stand for 3 minutes or until the beads fully pellet QC Transfer 30 µl of clear supernatant containing the Whole Exome Capture Library to a clean thinwalled PCR 0.2-mL strip-tube, making sure not to disturb the bead pellet Validate and quantify each Whole Exome Capture Library using an Agilent BioAnalyzer High Sensitivity DNA Kit and a Thermo Fisher Scientific Qubit dsdna High Sensitivity Quantitation Assay. NOTE: If using the Agilent BioAnalyzer High Sensitivity DNA Kit, load 0.5 µl of the final sample. An ideal BioAnalyzer trace is shown below. Average fragment length should be between 375 bp and 425 bp using a range setting of 150 bp to 1000 bp. Ideally, final concentration values should be 15 ng/µl. STOPPING POINT: If not proceeding immediately to sequencing, the Whole Exome Capture Libraries can be stored at 20 C. 25

26 Step 8 8 SEQUENCING ON THE ILLUMINA PLATFORM Sequence the Whole Exome Capture Libraries on the Illumina HiSeq, NextSeq, or NovaSeq platforms using the cycles settings shown in the table below. RUN SEGMENT CYCLE NUMBER Read 1 76 or 101 Index 1 (i7) 8 Index 2 (i5) 8 Read 2 76 or 101 End of Workflow 26

27 Appendix A1 APPENDIX A1 LIST OF CD INDEX SEQUENCES Table A.1. Sequence of Compatible Barcodes PART NAME TWIST PART NUMBER INDEX 1 ADAPTER INDEX 2 ADAPTER NOVASEQ, MISEQ, HISEQ 2000/2500 INDEX 2 ADAPTER MINISEQ, NEXTSEQ, HISEQ 3000/4000 Indexed Adapters Indexed Adapters Indexed Adapters Indexed Adapters Indexed Adapters Indexed Adapters Indexed Adapters Indexed Adapters Indexed Adapters Indexed Adapters Indexed Adapters Indexed Adapters Indexed Adapters Indexed Adapters Indexed Adapters Indexed Adapters D701 D501 D501 ATTACTCG TATAGCCT AGGCTATA D702 D501 D501 TCCGGAGA TATAGCCT AGGCTATA D701 D502 D502 ATTACTCG ATAGAGGC GCCTCTAT D702 D502 D502 TCCGGAGA ATAGAGGC GCCTCTAT D703 D503 D503 CGCTCATT CCTATCCT AGGATAGG D704 D503 D503 GAGATTCC CCTATCCT AGGATAGG D703 D504 D504 CGCTCATT GGCTCTGA TCAGAGCC D704 D504 D504 GAGATTCC GGCTCTGA TCAGAGCC D703 D501 D501 CGCTCATT TATAGCCT AGGCTATA D704 D501 D501 GAGATTCC TATAGCCT AGGCTATA D703 D502 D502 CGCTCATT ATAGAGGC GCCTCTAT D704 D502 D502 GAGATTCC ATAGAGGC GCCTCTAT D701 D503 D503 ATTACTCG CCTATCCT AGGATAGG D702 D503 D503 TCCGGAGA CCTATCCT AGGATAGG D701 D504 D504 ATTACTCG GGCTCTGA TCAGAGCC D702 D504 D504 TCCGGAGA GGCTCTGA TCAGAGCC 27

28 Appendix A2 APPENDIX A2 POOLING GUIDELINES When pooling dual-indexed Whole Exome Capture Libraries, refer to the Illumina TruSeq pooling guidelines to avoid index read failure during sequencing. Sets of compatible adapter sets are shown below. For additional multiplexing options please refer to Illumina Index Adapters Pooling Guide at support.illumina.com. Table A.2. Compatible adapter sets D701 D702 D703 D704 D501 T1 T2 T9 T10 D502 T3 T4 T11 T12 D503 T13 T14 T5 T6 D504 T15 T16 T7 T8 End of Appendix A 28

29 Appendix B APPENDIX B OVERVIEW OF MODULAR KITS The Twist Human Core Exome EF Multiplex Complete Kit is offered in a modular format. These modules, sold separately, enable flexibility and seamless integration into your own custom workflow. Kit performance is validated with the complete kit, and partial adoption of the Twist validated workflow will require optimization. The following section provides recommendations for successfully performing target enrichment reactions using individual modules of the Twist kit in a partial workflow. Refer to Appendix C for guidance on preparing exome libraries using the modular kits. TWIST HUMAN CORE EXOME EF MULTIPLEX COMPLETE KIT, 16 SAMPLES PN Twist Library Preparation EF Kit 1, 16 Samples PN Twist Library Preparation Kit 2, 16 Samples PN TWIST HUMAN CORE EXOME MULTIPLEX ENRICHMENT KIT 1, 16 SAMPLES PN Twist CD Index Adapter Set 1 16, 16 Samples PN Twist Adapter-Specific Blockers 1 16, 2 Reactions PN TWIST HUMAN CORE EXOME MULTIPLEX ENRICHMENT KIT 2, 16 SAMPLES PN Twist Enrichment Reagents, 2 Reactions PN Twist Human Core Exome Multiplex Hybridization Kit, 16 Samples PN Figure B.1. Overview of the modular kits. End of Appendix B 29

30 Appendix C APPENDIX C GUIDANCE FOR USE OF MODULAR KITS TWIST HUMAN CORE EXOME EF MULTIPLEX COMPLETE KIT PN Included Components: Twist Library Preparation EF Kit 1 PN Twist Library Preparation Kit 2 PN Twist CD Index Adapter Set 1 16 PN Twist Adapter-Specific Blockers 1 16 PN Twist Human Core Exome Multiplex Hybridization Kit PN Twist Enrichment Reagents PN Protocol for using the complete kit as provided: Follow the full protocol provided in this manual. TWIST HUMAN CORE EXOME MULTIPLEX ENRICHMENT KIT 1 PN Included Components: Twist CD Index Adapter Set 1 16 PN Twist Adapter-Specific Blockers 1 16 PN Twist Human Core Exome Multiplex Hybridization Kit PN Twist Enrichment Reagents PN Protocol for using Twist reagents with in-house library preparation protocol: After adapter ligation, post-ligation amplification and purification, the amplified indexed libraries can be carried directly into Step 4: Pool Amplified Indexed Libraries. Note: Using Twist adapters with in-house protocol may require optimization. Follow the remaining steps in the protocol as stated. Technical Considerations: User chooses fragmentation method (sonication or enzyme-based), and supplies end-repair and da-tailing enzymes, and ligase. The recommended average target fragment size is 200 bp. Twist CD Index Adapter Set 1 16 PN are dual-indexed adapters compatible with TA-ligation, and supplied at a concentration of 10 µm. Adapters are not compatible with transposon-based fragmentation methods. Adapter concentration affects ligation efficiency and adapter-dimer formation and carry-over during the post-ligation purification. User is responsible for optimizing ligation protocol to achieve high ligation efficiency and few byproducts. Twist Adapter-Specific Blockers 1 16 PN are designed to block adapter sequences used in the Twist CD Index Adapter Set 1 16 module, and other factors that result in off-target capture. In this configuration, use of Twist adapters supplied in the kit during ligation is required to ensure blocker compatability. 30

31 Appendix C TWIST HUMAN CORE EXOME MULTIPLEX ENRICHMENT KIT 2 PN Included Components: Twist Human Core Exome Multiplex Hybridization Kit PN Twist Enrichment Reagents PN Protocol for performing ligation with custom adapters followed by hybridization and enrichment with Twist reagents: After adapter ligation, post-ligation amplification and purification, the amplified indexed libraries can be carried directly into Step 4: Pool Amplified Indexed Libraries. In Step 5: Hybridize Capture Probes with Pools, replace Blocker Solution with Cot-1 DNA, and Blocker Mix 1 16 with custom adapter blockers. If components total to < 40µL, replace any remaining volume with molecular grade water to a final volume of 40 µl. Follow the remaining steps in the protocol as stated. Technical Considerations: User chooses fragmentation method (sonication or enzyme-based), and supplies end-repair and da-tailing enzymes, and ligase. The recommended average target fragment size is 200 bp. User supplies blockers for use in hybridization and is responsible for optimizing the blocker formulation to ensure low off-target during capture. In a typical hybridization-based capture, two types of blockers are typically utilized: Human Cot-1 DNA: Utilized to reduce capture of repetitive sequences present in the human genome. A total loading of 5 µg of Human Cot-1 DNA is recommended for each hybridization reaction (Thermo Fisher Scientific, # or equivalent). Adapter Blockers: Utilized to prevent adapter cross-hybridization and improve on-target sequencing. Must be compatible with the user selected sequencing adapters. Twist Adapter-Specific Blockers 1 16 PN are designed to block adapter sequences used in the Twist CD Index Adapter Set 1 16 module. For other TruSeq -style adapters, Twist Universal Blockers PN are recommended. Twist Universal Blockers PN are compatible with TruSeq -style library kits from Illumina with single- and dualindexing schemes and various barcode/umi lengths. 31

32 Appendix C TWIST HUMAN CORE EXOME MULTIPLEX HYBRIDIZATION KIT PN Included Components: Exome Probes PN Hybridization Mix PN Amplification Primers, ILMN PN Protocol for using Twist probes with in-house library preparation and enrichment protocol: After adapter ligation, post-ligation amplification and purification, the amplified indexed libraries can be carried directly into Step 4: Pool Amplified Indexed Libraries. In Step 5: Hybridize Capture Probes with Pools, replace Blocker Solution with Cot-1 DNA, and Blocker Mix 1 16 with custom adapter blockers. If components total to < 40µL, replace any remaining volume with molecular grade water to a final volume of 40 µl. In Step 6: Bind Hybridized Targets on Streptavidin Binding Beads is replaced by user workflow and reagents. In Step 7: Post-capture PCR Amplify, Purify and QC is executed with the included Amplification Primers, ILMN PN and supplemented by user workflow and reagents. Technical Considerations: User chooses fragmentation method (sonication or enzyme-based), and supplies end-repair and da-tailing enzymes, and ligase. The recommended average target fragment size is 200 bp. Exome Probes PN have been optimized and validated for use in Hybridization Mix PN with the conditions specified in Step 5: Hybridize Capture Probes with Pools. The use of both supplied reagents is recommended. Probe performance may differ if modifications to the buffer or conditions are made and should be optimized accordingly. User supplies blockers in hybridization and is responsible for optimizing the blocker formulation to ensure low off-target during capture. In a typical hybridization-based capture, two types of blockers are typically utilized: Human Cot-1 DNA: Utilized to reduce capture of repetitive sequences present in the human genome. A total loading of 5 µg of Human Cot-1 DNA is recommended for each hybridization reaction (Thermo Fisher Scientific, # or equivalent). Adapter Blockers: Utilized to prevent adapter cross-hybridization and improve on-target sequencing. Must be compatible with the user selected sequencing adapters. Twist Adapter-Specific Blockers 1 16 PN are designed to block adapter sequences used in the Twist CD Index Adapter Set 1 16 module. For other TruSeq -style adapters, Twist Universal Blockers PN are recommended. Twist Universal Blockers PN are compatible with TruSeq -style library kits from Illumina with single- and dualindexing schemes and various barcode/umi lengths. Continued next page... 32

33 Appendix C User supplies streptavidin beads to bind hybridized targets and is responsible for ensuring appropriate amount of streptavidin beads are used to pull down all probes. 100 µl of Dynabeads MyOne Streptavidin T1 (Thermo Fisher Scientific #65601) or Dynabeads MyOne Streptavidin C1 (Thermo Fisher Scientific #65002) are recommended. Twist probes are DNA-based. Performing stringent wash at high temperature may result in significant loss of product, especially for AT-rich sequences. A temperature of 48 C or lower is recommended for any stringent wash set. End of Appendix C 33