NEBNext Direct Cancer HotSpot Panel

LIBRARY PREPARATION NEBNext Direct Cancer HotSpot Panel Instruction Manual NEB #E7000S/L/X 8/24/96 reactions Version 2.0 3/17 be INSPIRED drive DISCOVERY stay GENUINE i

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals. ISO 9001 Registered Quality Management ISO 14001 Registered Environmental Management ISO 13485 Registered Medical Devices This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc. For more information, please contact NEB s Global Business Development team at gbd@neb.com. This product is covered by one or more patents. This product is licensed from Bio-Rad Laboratories, Inc. under U.S. Pat. Nos. 6,627,424, 7,541,170, 7,670,808, 7,666,645 and corresponding patents in other countries for use only in; (a) standard (non-real time) PCR in the research field only, but not real-time PCR or digital PCR; (b) any in vitro diagnostics application, except for applications using real-time or digital PCR; and (c) any non-pcr applications in DNA sequencing, isothermal amplification and the production of synthetic DNA. AGILENT and BIOANALYZER are registered trademarks of Agilent Technologies, Inc. EPPENDORF and LOBIND are registered trademarks of Eppendorf AG. ILLUMINA, HISEQ and MISEQ are registered trademarks of Illumina, Inc. COVARIS is a registered trademark of Covaris, Inc.

NEBNext Direct Cancer HotSpot Panel Table of Contents: Kit Includes...2-7 Required Equipment/Materials Not Included...7-8 Guidelines....8 Applications....9 Introduction....10 Chapter 1 Protocol for NEBNext Direct Cancer HotSpot Panel...12 Chapter 2 Guidelines for Setting Up PCR Reactions...28 Chapter 3 Index Pooling Guidelines...31 Chapter 4 Guidelines for Running Samples on the Illumina MiSeq...40 Kit Components...43 Revision History....46 1

Kit Includes: Each kit contains sufficient reagents for preparation of up to 8 reactions (NEB #E7000S), 24 reactions (NEB #E7000L) and 96 reactions (NEB #E7000X). NEB #E7000S Box 1 of 2: Store at 20 C. 1. NEBNext Direct Cancer HotSpot Baits 2. NEBNext Direct Hybridization Additive 3. NEBNext Direct 3 Blunting Enzyme Mix 4. NEBNext Direct da-tailing Enzyme 5. NEBNext Direct 3 Adaptor 6. NEBNext Direct Ligase 7. NEBNext Direct 5 Blunting Enzyme Mix 8. NEBNext Direct 5 UMI Adaptor 9. NEBNext Direct Cleaving Enzyme Mix 10. NEBNext Direct Q5 Master Mix 11. NEBNext Direct FFPE Phosphorylation Enzyme 12. NEBNext Direct FFPE Phosphorylation Buffer D01 D08 NEBNext Direct Index Primer Mix D01-D08 1 2 3 4 5 11 6 7 8 9 10 12 D01 D02 D03 D04 D05 D06 D07 D08 2

Box 2 of 2: Store at 4 C. Do not freeze. 1. NEBNext Direct Bead Wash 1 (BW1)* 2. NEBNext Sample Purification Beads** 3. NEBNext Direct Streptavidin Beads 4. NEBNext Direct Hybridization Wash (HW) 5. NEBNext Direct Bead Prep Buffer 6. NEBNext Direct Hybridization Buffer 7. NEBNext Direct 3 Blunting Buffer 8. NEBNext Direct da-tailing Buffer 9. NEBNext Direct Adaptor Ligation Buffer 10. NEBNext Direct 5 Blunting Buffer 11. NEBNext Direct Cleaving Buffer 12. NEBNext Direct Bead Wash 2 (BW2) * This component should be stored at room temperature. Upon arrival, you may notice a precipitate, which is normal. Once BW1 reaches room temperature, gently invert the bottle ~10 times to dissolve the precipitate. Do not use (BW1) until all of the precipitate has dissolved. ** This component should be stored at room temperature. 1 3 4 5 6 7 8 9 2 Room temp. removable insert 10 11 12 3

NEB #E7000L Box 1 of 2: Store at 20 C. 1. NEBNext Direct Cancer HotSpot Baits 2. NEBNext Direct Hybridization Additive 3. NEBNext Direct 3 Blunting Enzyme Mix 4. NEBNext Direct da-tailing Enzyme 5. NEBNext Direct 3 Adaptor 6. NEBNext Direct Ligase 7. NEBNext Direct 5 Blunting Enzyme Mix 8. NEBNext Direct 5 UMI Adaptor 9. NEBNext Direct Cleaving Enzyme Mix 10. NEBNext Direct Q5 Master Mix 11. NEBNext Direct Q5 Master Mix 12. NEBNext Direct FFPE Phosphorylation Enzyme 13. NEBNext Direct FFPE Phosphorylation Buffer D01 D24 NEBNext Direct Index Primer Mix D01-D24 1 7 2 3 4 5 8 9 10 11 6 12 13 D01 D02 D03 D04 D05 D06 D07 D08 D09 D10 D11 D12 D13 D14 D15 D16 D17 D18 D19 D20 D21 D22 D23 D24 4

Box 2 of 2: Store at 4 C. Do not freeze. 1. NEBNext Direct Bead Wash 1 (BW1)* 2. NEBNext Sample Purification Beads** 3. NEBNext Direct Streptavidin Beads 4. NEBNext Direct Hybridization Wash (HW) 5. NEBNext Direct Bead Prep Buffer 6. NEBNext Direct Hybridization Buffer 7. NEBNext Direct 3 Blunting Buffer 8. NEBNext Direct da-tailing Buffer 9. NEBNext Direct Adaptor Ligation Buffer 10. NEBNext Direct 5 Blunting Buffer 11. NEBNext Direct Cleaving Buffer 12. NEBNext Direct Bead Wash 2 (BW2) * This component should be stored at room temperature. Upon arrival, you may notice a precipitate, which is normal. Once BW1 reaches room temperature, gently invert the bottle ~10 times to dissolve the precipitate. Do not use (BW1) until all of the precipitate has dissolved. ** This component should be stored at room temperature. 1 2 Room temp. removable insert 3 4 6 7 8 10 11 12 5 9 5

NEB #E7000X Box 1 of 2: Store at 20 C. 1. NEBNext Direct Cancer HotSpot Baits 2. NEBNext Direct Hybridization Additive 3. NEBNext Direct 3 Blunting Enzyme Mix 4. NEBNext Direct da-tailing Enzyme 5. NEBNext Direct 3 Adaptor 6. NEBNext Direct Ligase 7. NEBNext Direct Ligase 8. NEBNext Direct 5 Blunting Enzyme Mix 9. NEBNext Direct 5 UMI Adaptor 10. NEBNext Direct Cleaving Enzyme Mix 11. NEBNext Direct Q5 Master Mix 12. NEBNext Direct FFPE Phosphorylation Enzyme 13. NEBNext Direct FFPE Phosphorylation Buffer DHT1 DHT96 NEBNext Direct Index Primer Mix Plate 1 2 3 4 5 6 7 8 9 10 11 12 13 DHT1 DHT96 6

Box 2 of 2: Store at 4 C. Do not freeze. 1. NEBNext Direct Bead Wash 1 (BW1)* 2. NEBNext Sample Purification Beads** 3. NEBNext Sample Purification Beads** 4. NEBNext Direct Streptavidin Beads 5. NEBNext Direct Hybridization Wash (HW) 6. NEBNext Direct Bead Prep Buffer 7. NEBNext Direct Hybridization Buffer 8. NEBNext Direct 3 Blunting Buffer 9. NEBNext Direct da-tailing Buffer 10. NEBNext Direct Adaptor Ligation Buffer 11. NEBNext Direct 5 Blunting Buffer 12. NEBNext Direct Cleaving Buffer 13. NEBNext Direct Bead Wash 2 (BW2) * This component should be stored at room temperature. Upon arrival, you may notice a precipitate, which is normal. Once BW1 reaches room temperature, gently invert the bottle ~10 times to dissolve the precipitate. Do not use (BW1) until all of the precipitate has dissolved. ** This component should be stored at room temperature. 1 4 5 6 2 7 8 9 10 3 Room temp. removable insert 11 12 13 Required Equipment: Covaris Focused-ultrasonicator Thermocycler programmable to 100 μl Agilent Bioanalyzer or similar instrument Required Materials Not Included: Covaris microtubes or plate 1X TE buffer (10 mm Tris-HCl, ph 8.0, 1 mm EDTA) Molecular grade ethanol Molecular grade water 96-well PCR plates (or PCR strip tubes) 7

Required Materials Not Included (Cont.) Eppendorf DNA LoBind 2 ml tubes (VWR, cat#: 80077-234) Additional microcentrifuge or conical tubes to prepare master mixes 96-well plate magnet or PCR tube magnet Microcentrifuge tube magnet Agilent High Sensitivity DNA Kit (Agilent, cat#: 5067-4626) Guidelines: All incubations are performed in a thermocycler programmable to 100 μl. Note: Thermocyclers programmable to only 50 µl can give very different results. Perform all washes by pipetting up and down at least 10 times. Less pipetting can result in carryover of previous reagents. Pipette carefully to minimize bubble formation. Some foaming of the wash solutions is to be expected. All incubations can be performed uncapped except during FFPE phosphorylation (Step 2.1.4), hybridization (Step 2.2.5) and library amplification (Step 11.5). All incubations are performed with the thermocycler lid unheated and open except during FFPE phosphorylation (Step 2.1.4), hybridization (Step 2.2.5) and library amplification (Step 11.5). Take care to minimize bead loss during washes (excessive bead loss will result in lower yields). Warm all wash solutions and beads (Box 2) to room temperature before use. Bead Wash 1 (BW1) may contain a precipitate due to shipping at 4 C. This is normal and should go back into solution once the buffer reaches room temperature (at least 4 hours followed by gently inverting the bottle ~10 times). Do not use until all precipitate has dissolved. If necessary, incubate the bottle in a 25 C or 37 C incubator or water bath to dissolve the precipitate. If you are preparing more than one sample, we recommend making master mixes using ~10% more volume than required per reaction. A multichannel pipette (200 µl) may facilitate wash and mixing steps if preparing multiple samples. Note: Post-ligation Washes (Sections 7.2 and 9.2) contain different steps than the Post-reaction Washes. 8

Applications: The NEBNext Direct Cancer HotSpot Panel for Illumina is designed to enrich for DNA fragments from 190 common cancer targets from 50 genes for nextgeneration sequencing on the Illumina platform (Illumina, Inc). This kit contains the oligonucleotides, beads, enzymes and buffers required to convert the desired fragments into a sequence-ready library containing both sample indexes and unique molecular identifiers (UMI). The NEBNext Direct enrichment method can be performed within one day and is easily automated. Each kit component must pass rigorous quality control standards and is lot controlled both individually and as a set of reagents. Lot Control: The lots provided in the NEBNext Direct Cancer HotSpot Panel are managed separately and qualified by additional functional validation. Individual reagents undergo standard enzyme activity and quality control assays. Functionally Validated: Each set of reagents is functionally validated together through construction and sequencing of a target enriched DNA library on an Illumina sequencing platform. For 100 ng of NA19240 DNA input and an average coverage of 100X, 100% of the targets are covered. For larger volume requirements, customized and bulk packaging is available by purchasing through the OEM/Bulks department at NEB. Please contact OEM@ neb.com for further information. BED files: BED files for the NEBNext Direct Cancer HotSpot Panel are available for download at: www.neb.com/e7000, which can be found in the Other Tools & Resources tab. Available BED files include the baited and targeted regions, and one available in GRCh37/hg19 and GRCh38/hg38 compatible formats. 9

Introduction: Target enrichment, coupled with next generation sequencing (NGS), enables high throughput, deep sequencing of genomic regions of interest. NEBNext Direct is a novel, hybridization-based capture method offering significant advantages over traditional in-solution hybridization and multiplex PCR protocols. In the NEBNext Direct target enrichment approach (Figure 1), fragmented DNA is rapidly hybridized to biotinylated oligonucleotide baits that define the 3 end of each target of interest. The bait-target hybrids are bound to streptavidin beads and any 3 off target sequence is removed enzymatically. This combination of a short hybridization time with the enzymatic removal of 3 off target sequence enables greater sequencing efficiency relative to conventional hybridization-based enrichment methods. The trimmed targets are then converted into Illumina-compatible libraries that include unique molecular identifiers (UMI) and a sample barcode. Sequence-ready libraries are generated within one day. The procedure is compatible with most automated liquid handling instruments. The NEBNext Direct HotSpot Cancer Panel contains baits that capture both strands of DNA across 190 common cancer targets from 50 genes, encompassing approximately 40 kb of sequence and including over 18,000 COSMIC features. The panel is designed to generate targets of roughly 150 bp, compatible with PE75 Illumina sequencing. Targets include regions from the following cancer-related genes: ABL1 EGFR GNAQ KRAS PTPN11 AKT1 ERBB2 GNAS MET RB1 ALK ERBB4 HNF1A MLH1 RET APC EZH2 HRAS MPL SMAD4 ATM FBXW7 IDH1 NOTCH1 SMARCB1 BRAF FGFR1 IDH2 NPM1 SMO CDH1 FGFR2 JAK2 NRAS SRC CDKN2A FGFR3 JAK3 PDGFRA STK11 CSF1R FLT3 KDR PIK3CA TP53 CTNNB1 GNA11 KIT PTEN VHL For research use only, not intended for diagnostic use. 10

Figure 1. NEBNext Direct Workflow. 5 3 Target region 3 Genomic 5 DNA Fragmentation 5 3 3 5 Denaturation & probe hybridization 5 3 3 5 Biotin probe Biotin probe targets both strands (shown for 1 strand) 3 blunting of DNA 5 3 3 5 Enzymatic removal of off-target sequence Streptavidin bead da-tailing 5 A 3 3 5 Ligation of 3 adaptor 5 A 3 T 3 adaptor 5 blunting of DNA 5 3 A T Probes are extended to the 5 end of the randomly sheared fragment, creating a variable 5 end of the target read Ligation of 5 UMI adaptor 5 3 5 UMI adaptor A T 5 Unique Molecular Identifier (UMI) addition improves sensitivity Adaptor cleaving 5 3 A T 3 5 PCR amplification 5 3 A T Sequencing-ready fragment 3 5 Incorporated sample index 11

1 Protocol for NEBNext Direct Cancer HotSpot Panel NEBNext Direct Cancer HotSpot Panel Instruction Manual 12 12

CHAPTER 1 Symbols! This caution sign signifies a step in the protocol that has multiple paths leading to the same end point but is dependent on a user variable, like the amount of input DNA. SAFE STOP Stopping points in the protocol. Starting Material: 10 ng 1 µg DNA* *For detection of somatic variants we strongly recommend starting with a minimum of 100 ng DNA input.! If you are starting with cell free DNA, skip DNA Fragmentation and go directly to Hybridization in Section 2.2. 1. DNA Fragmentation with Covaris Shear input DNA in 50 μl (total volume) of 1X TE. Follow the Covaris recommendations for instrument and microtube setup using the 200 bp target size protocol for shearing in 50 μl. 2. Probe Hybridization! Follow Section 2.1 if using FFPE DNA. If you are not using FFPE DNA, skip to Section 2.2 (Hybridization). 2.1. FFPE Phosphorylation 2.1.1. Make a FFPE Phosphorylation master mix by mixing the following components for the appropriate number of reactions: REAGENT PER REACTION WITH 10% OVERAGE FFPE Phosphorylation Buffer 5 µl 5.5 µl FFPE Phosphorylation Enzyme 1 µl 1.1 µl Total 6 µl 6.6 µl 2.1.2. Gently mix the master mix by pipetting up and down 10 times. 2.1.3. Add 42 µl of fragmented FFPE DNA from Section 1 to a PCR tube/well. 2.1.4. Add 6 µl of FFPE Phosphorylation master mix. Gently mix by pipetting up and down 5 times. 2.1.5. Incubate at 37 C for 15 minutes with the heated lid set to 45 C. 13

14 CHAPTER 1 2.1.6. Briefly spin down the reaction to collect the sample at the bottom of the tube/well. Place the tubes on ice. 2.1.7. Proceed directly to Hybridization in Section 2.2. 2.2. Hybridization 2.2.1. Make a Hybridization master mix by adding the following components for the appropriate number of reactions: REAGENT PER REACTION WITH 10% OVERAGE Hybridization Buffer 47 µl 51.7 µl Hybridization Additive 20 µl 22 µl Cancer HotSpot Baits 5 µl 5.5 µl Total 72 µl 79.2 µl 2.2.2. Gently mix the master mix by pipetting up and down 10 times. 2.2.3. Add 48 μl of fragmented DNA from Section 1 or 48 µl phosphorylated FFPE DNA from Section 2.1 to a PCR tube/well. 2.2.4. Add 72 μl of Hybridization master mix to each sample for a final volume of 120 μl. Mix by pipetting up and down 10 times. Seal the PCR plate or cap tubes securely to avoid evaporation. 2.2.5. Run the following program with the heated lid set to 105 C and place the samples in the thermocycler when the block temperature reaches 95 C: 10 min @ 95 C 90 min @ 60 C Hold @ 60 C 2.2.6. While the samples are incubating, prepare Streptavidin beads (see Streptavidin Bead Preparation in Section 3). 2.2.7. After the incubation at 60 C and when Section 3 (Streptavidin Bead Preparation) is complete, unseal the tubes/wells, leave the samples on the thermocycler at 60 C with the lid open and proceed to Bead Binding in Section 4. 3. Streptavidin Bead Preparation 3.1. Warm Streptavidin beads to room temperature (~15 minutes). 3.2. Vortex the Streptavidin beads to resuspend. 3.3. For each reaction, 75 μl of beads are required (82.5 µl with 10% overage). In a 2 ml Eppendorf tube, add the appropriate volume of beads for the number of reactions performed.

E7000 NEBNEXT DIRECT CANCER HOTSPOT PANEL Note: Use multiple 2 ml tubes if performing more than 12 reactions. It is not recommended to exceed 1 ml of beads per 2 ml Eppendorf tube. 3.4. Place the tube(s) on a magnet and wait for the solution to clear (~1 minute). Remove the supernatant, and then remove the tube(s) from the magnet. 3.5. Add 150 μl of Hybridization Wash (HW) per reaction (165 µl with 10% overage) to the beads and resuspend by vortexing or pipetting. 3.6. Place the tube(s) on a magnet and wait for the solution to clear (~1 minute). Remove the supernatant, and then remove the tube(s) from the magnet. 3.7. Repeat steps 3.5 3.6 twice for a total of 3 washes. 3.8. Resuspend the beads in 30 μl of Bead Prep Buffer per reaction (33 µl with 10% overage). 3.9. Keep the beads at room temperature until probe hybridization (Section 2.2) is completed. Note: For Sections 4 10, the thermocycler lid should be open and unheated. The PCR tubes can remain uncapped for ease of mixing. However, if it is preferred, tubes can be capped, and then decapped prior to mixing. 4. Bead Binding 4.1. Bead Binding 4.1.1. Vortex the washed Streptavidin Beads (from Step 3.9) in Bead Prep Buffer to resuspend. 4.1.2. Add 30 μl of resuspended beads to each reaction (from Step 2.2.7) while the samples are on the thermocycler at 60 C, and then mix gently by pipetting up and down 10 times. 4.1.3. Change the thermocycler temperature to 48 C and incubate the reactions for 10 minutes. 4.1.4. Remove the samples from the thermocycler and place on a magnet. Wait for the solution to clear (~ 15 seconds), remove the supernatant, and then remove the samples from the magnet. 4.1.5. Add 150 μl of HW to each sample. Gently mix by pipetting up and down 10 times. Place the samples on a thermocycler set at 62 C (lid open) and incubate for 5 minutes. 4.1.6. Remove the samples from the thermocycler and place on a magnet. Wait for the solution to clear (~ 15 seconds), remove the supernatant and then remove the samples from the magnet. 15

CHAPTER 1 4.1.7. Repeat Steps 4.1.5 4.1.6 for a total of 2 washes at 62 C. 4.1.8. Add 150 μl of Bead Wash Buffer 2 (BW2) to each sample. Gently mix by pipetting up and down 10 times. Keep the samples at room temperature for up to 30 minutes and proceed directly to 3 Blunting of DNA in Section 5. SAFE STOP If you choose to stop the protocol at this point, place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. Resuspend the beads in 100 µl of 1X TE. DNA bound beads can be stored in 1X TE for up to 16 hours at 4 C. When ready to proceed with the protocol, place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant and then remove the samples from the magnet. Add 150 μl of BW2 to each reaction, mix gently by pipetting up and down 10 times, and then proceed directly to 3 Blunting of DNA in Section 5. 5. 3 Blunting of DNA 5.1. 3 Blunting 5.1.1. While the beads are suspended in BW2 buffer, make a 3 Blunting master mix by adding the following components in a sterile nuclease-free tube for the appropriate number of reactions: REAGENT PER REACTION WITH 10% OVERAGE 3 Blunting Buffer 97 µl 106.7 µl 3 Blunting Enzyme Mix 3 µl 3.3 µl Total 100 µl 110 µl 5.1.2. Gently mix the master mix by pipetting up and down 10 times and set aside. 5.1.3. Place the DNA bound beads on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. 5.1.4. Add 100 μl of 3 Blunting master mix (from Step 5.1.1.) to each sample. Gently mix by pipetting up and down 10 times. Incubate the samples at 37 C for 10 minutes on a thermocycler with the thermocycler lid open. 5.1.5. Proceed immediately with the Post-reaction Wash (Section 5.2). 16

5.2. Post-reaction Wash E7000 NEBNEXT DIRECT CANCER HOTSPOT PANEL 5.2.1. Place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. 5.2.2. Add 150 μl of Bead Wash Buffer 1 (BW1) to each sample. Gently mix by pipetting up and down 10 times. Place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant and then remove the samples from the magnet. 5.2.3. Add 150 μl of BW2 to each sample. Gently mix by pipetting up and down 10 times. Keep the samples at room temperature for up to 30 minutes and proceed directly to da-tailing in Section 6. SAFE STOP If you choose to stop the protocol at this point, place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. Resuspend the beads in 100 µl of 1X TE. DNA bound beads can be stored in 1X TE for up to 16 hours at 4 C. When ready to proceed with the protocol, place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant and then remove the samples from the magnet. Add 150 μl of BW2 to each reaction, mix gently by pipetting up and down 10 times, and then proceed directly to da-tailing in Section 6. 6. da-tailing 6.1 da-tailing 6.1.1. While the beads are suspended in BW2 buffer, make a da- Tailing master mix by adding the following components in a sterile nuclease-free tube for the appropriate number of reactions: REAGENT PER REACTION WITH 10% OVERAGE da-tailing Buffer 97 µl 106.7 µl da-tailing Enzyme 3 µl 3.3 µl Total 100 µl 110 µl 6.1.2. Gently mix the master mix by pipetting up and down 10 times and set aside. 17

CHAPTER 1 6.1.3. Place the DNA bound beads on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. 6.1.4. Add 100 μl of da-tailing master mix (from Step 6.1.1) to each sample. Gently mix by pipetting up and down 10 times. Incubate the reactions at 37 C for 10 minutes on a thermocycler with the thermocycler lid open. 6.1.5. Proceed immediately with the Post-reaction Wash (Section 6.2). 6.2 Post-reaction Wash 6.2.1. Place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. 6.2.2. Add 150 μl of BW1 to each reaction and then mix gently by pipetting up and down 10 times. Place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the reactions from the magnet. 6.2.3. Add 150 μl of BW2 to each sample. Gently mix by pipetting up and down 10 times. Keep the samples at room temperature for up to 30 minutes and proceed directly to 3 Adaptor Ligation in Section 7. SAFE STOP If you choose to stop the protocol at this point, place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. Resuspend the beads in 100 µl of 1X TE. DNA bound beads can be stored in 1X TE for up to 16 hours at 4 C. When ready to proceed with the protocol, place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant and then remove the samples from the magnet. Add 150 μl of BW2 to each reaction, mix gently by pipetting up and down 10 times, and then proceed directly to 3 Adaptor Ligation in Section 7. 18

7. 3 Adaptor Ligation 7.1. 3 Adaptor Ligation E7000 NEBNEXT DIRECT CANCER HOTSPOT PANEL 7.1.1. While the beads are suspended in BW2 buffer, make a 3 Adaptor Ligation master mix by adding the following components in a sterile nuclease-free tube for the appropriate number of reactions: REAGENT PER REACTION WITH 10% OVERAGE Adaptor Ligation Buffer 80 µl 88 µl 3 Adaptor 10 µl 11 µl Ligase 10 µl 11 µl Total 100 µl 110 µl 7.1.2. Gently mix the master mix by pipetting up and down 10 times and set aside. 7.1.3. Place the DNA bound beads on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. 7.1.4. Add 100 μl of 3 Adaptor Ligation master mix (from Step 7.1.1) to each sample. Gently mix by pipetting up and down 10 times. 7.1.5. Incubate the samples at 20 C for 15 minutes on a thermocycler with the thermocycler lid open. 7.1.6. Proceed immediately with the Post-ligation Wash (Section 7.2). 7.2. Post-ligation Wash Note: The following wash steps are different than the Post-reaction Washes. 7.2.1. Place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. 7.2.2. Add 150 μl of BW1 to each sample. Gently mix by pipetting up and down 10 times. Place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. 7.2.3. Repeat Step 7.2.2 for a total of 2 washes in BW1. 19

CHAPTER 1 7.2.4. Add 150 μl of BW2 to each sample. Gently mix by pipetting up and down 10 times. Keep the samples at room temperature for up to 30 minutes and proceed directly to 5 Blunting of DNA in Section 8. SAFE STOP If you choose to stop the protocol at this point, place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. Resuspend the beads in 100 µl of 1X TE. DNA bound beads can be stored in 1X TE for up to 16 hours at 4 C. When ready to proceed with the protocol, place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant and then remove the samples from the magnet. Add 150 μl of BW2 to each reaction, mix gently by pipetting up and down 10 times, and then proceed directly to 5 Blunting of DNA in Section 8. 8. 5 Blunting of DNA 8.1. 5 Blunting 8.1.1. While the beads are suspended in BW2 buffer, make a 5 Blunting master mix by adding the following components in a sterile nuclease-free tube for the appropriate number of reactions: REAGENT PER REACTION WITH 10% OVERAGE 5 Blunting Buffer 97 µl 106.7 µl 5 Blunting Enzyme Mix 3 µl 3.3 µl Total 100 µl 110 µl 8.1.2. Gently mix the master mix by pipetting up and down 10 times and set aside. 8.1.3. Place the DNA bound beads on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. 8.1.4. Add 100 μl of 5 Blunting master mix (from Step 8.1.1) to each sample. Gently mix by pipetting up and down 10 times. 8.1.5. Incubate the samples at 20 C for 10 minutes on a thermocycler with the thermocycler lid open. 8.1.6. Proceed immediately with the Post-reaction Wash (Section 8.2). 20

E7000 NEBNEXT DIRECT CANCER HOTSPOT PANEL 8.2. Post-reaction Wash 8.2.1. Place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. 8.2.2. Add 150 μl of BW1 to each sample. Gently mix by pipetting up and down 10 times. Place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. 8.2.3. Add 150 μl of BW2 to each sample. Gently mix by pipetting up and down 10 times. Keep the samples at room temperature for up to 30 minutes and proceed directly to 5 Adaptor Ligation in Section 9. SAFE STOP If you choose to stop the protocol at this point, place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. Resuspend the beads in 100 µl of 1X TE. DNA bound beads can be stored in 1X TE for up to 16 hours at 4 C. When ready to proceed with the protocol, place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. Add 150 μl of BW2 to each reaction, mix gently by pipetting up and down 10 times, and then proceed directly to 5 Adaptor Ligation in Section 9. 9. 5 Adaptor Ligation 9.1. 5 Adaptor Ligation 9.1.1. While the beads are suspended in BW2 buffer, make a 5 Adaptor Ligation master mix by adding the following components in a sterile nuclease-free tube for the appropriate number of reactions: REAGENT PER REACTION WITH 10% OVERAGE Adaptor Ligation Buffer 80 µl 88 µl 5 UMI Adaptor 10 µl 11 µl Ligase 10 µl 11 µl Total 100 µl 110 µl 9.1.2. Gently mix the master mix by pipetting up and down 10 times and set aside. 21

CHAPTER 1 9.1.3. Place the DNA bound beads on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. 9.1.4. Add 100 μl of 5 Adaptor Ligation master mix (from Step 9.1.1) to each sample. Gently mix by pipetting up and down 10 times. 9.1.5. Incubate the samples at 20 C for 20 minutes on a thermocycler with the thermocycler lid open. 9.1.6. Proceed immediately with the Post-ligation Wash (Section 9.2). 9.2. Post-ligation Wash Note: The following wash steps are different than the Post-reaction washes. 9.2.1. Place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. 9.2.2. Add 150 μl of BW1 to each sample. Gently mix by pipetting up and down 10 times. Place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. 9.2.3. Repeat Step 9.2.2 for a total of 2 washes in BW1. 9.2.4. Add 150 μl of BW2 to each sample. Gently mix by pipetting up and down 10 times. Keep the samples at room temperature for up to 30 minutes and proceed directly to Adaptor Cleaving in Section 10. SAFE STOP If you choose to stop the protocol at this point, place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. Resuspend the beads in 100 µl of 1X TE. DNA bound beads can be stored in 1X TE for up to 16 hours at 4 C. When ready to proceed with the protocol, place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant and then remove the samples from the magnet. Add 150 μl of BW2 to each reaction, mix gently by pipetting up and down 10 times, and then proceed directly to Adaptor Cleaving in Section 10. 22

10. Adaptor Cleaving 10.1. Cleaving E7000 NEBNEXT DIRECT CANCER HOTSPOT PANEL 10.1.1. While the beads are suspended in BW2 buffer, make a Cleaving master mix by adding the following components in a sterile nuclease-free tube for the appropriate number of reactions: REAGENT PER REACTION WITH 10% OVERAGE Cleaving Buffer 95 µl 104.5 µl Cleaving Enzyme Mix 5 µl 5.5 µl Total 100 µl 110 µl 10.1.2. Gently mix the master mix by pipetting up and down 10 times and set aside. 10.1.3. Place the DNA bound beads on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. 10.1.4. Add 100 μl of Cleaving master mix (from Step 10.1.1.) to each sample. Gently mix by pipetting up and down 10 times. 10.1.5. Incubate the samples at 37 C for 15 minutes on a thermocycler with the thermocycler lid open. 10.1.6. Proceed immediately with the Post-reaction Wash (Section 10.2). 10.2. Post-reaction Wash 10.2.1. Place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. 10.2.2. Add 150 μl of BW1 to each sample. Gently mix by pipetting up and down 10 times. Place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. 10.2.3. Add 150 μl of BW2 to each sample. Gently mix by pipetting up and down 10 times. Keep the samples at room temperature for up to 30 minutes and proceed directly to Library Amplification in Section 11. 23

CHAPTER 1 SAFE STOP If you choose to stop the protocol at this point, place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. Resuspend the beads in 100 µl of 1X TE. DNA bound beads can be stored in 1X TE for up to 16 hours at 4 C. When ready to proceed with the protocol, place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant and then remove the samples from the magnet. Add 150 μl of BW2 to each reaction, mix gently by pipetting up and down 10 times, and then proceed directly to Library Amplification in Section 11. 11. Library Amplification Note: Refer to Chapter 2 if using the NEBNext Direct Index Primer Mix Plate provided in NEB #E7000X. Refer to Chapter 3 for guidelines on index pooling. 11.1. Place the reactions on a magnet, wait for the solution to clear (~ 15 seconds), remove the supernatant then remove the reactions from the magnet. 11.2. Add 45 μl of molecular grade water to each reaction. Mix gently by pipetting up and down 10 times to completely resuspend the beads. 11.3. Add the following components to a sterile strip tube/well in a PCR plate: REAGENT PER REACTION Q5 Master Mix 50 µl Index Primer Mix 5 µl Resuspended beads (from Step 11.2) 45 µl Total 100 µl 11.4. Gently mix by pipetting up and down 10 times. Seal the PCR plate or cap tubes. 24

E7000 NEBNEXT DIRECT CANCER HOTSPOT PANEL 11.5. Run the following program with the heated lid set 105 C and place the samples in the thermocycler when the block temperature reaches 98 C (It is critical to ensure that the block temperature has reached 98 C before placing samples in the thermocycler): CYCLE STEP TEMP TIME CYCLES Initial Denaturation 98 C 30 seconds 1 Denaturation Annealing Extension 98 C 62 C 72 C 10 seconds 15 seconds 20 seconds 20 25* Final Extension 72 C 5 minutes 1 Hold 4 C *Follow the PCR cycle number recommendations listed in Table 11.1. Table 11.1: PCR Cycle Number Recommendations. INPUT DNA RECOMMENDED NUMBER OF PCR CYCLES 1,000 ng 20 500 ng 21 100 ng 23 10 ng 25 11.6. Proceed to Purify Amplified Fragments in Section 12. SAFE STOP PCR reactions with beads can be stored for up to 16 hours at 4 C. 12. Purify and Size Select Amplified Fragments 12.1. If you detect significant evaporation from the PCR reaction, bring the volume up to 100 µl with molecular grade water. 12.2. Vortex the Sample Purification Beads to resuspend. 12.3. Add 85 μl of Sample Purification Beads to the PCR reactions. Mix well by pipetting up and down at least 10 times. 12.4. Incubate for 10 minutes uncapped at room temperature. 12.5. Place the tubes/pcr plate on a magnet. After the solution is clear (about 2 minutes), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain the DNA targets (Caution: do not discard beads). 25

CHAPTER 1 12.6. Add 200 μl of freshly prepared (same day) 80% EtOH while the tubes/ plate are on the magnet. Incubate at room temperature for 30 seconds and then carefully remove and discard the supernatant. 12.7. Repeat Step 12.6 once for a total of 2 washes in 80% EtOH, ensuring that all of the supernatant is removed from each reaction. 12.8. Incubate the samples, uncapped (or unsealed), at 37 C for 5 minutes on a thermocycler with the thermocycler lid open to dry the beads. 12.9. Remove the tubes/plate from the thermocycler and resuspend the dry beads in 102 μl of water. Incubate for 2 minutes at room temperature. 12.10. Place the tubes/plate on a magnet and allow the solution to clear (about 2 minutes). 12.11. Transfer 100 μl of the eluted library to fresh tubes/plate and add 85 μl of Sample Purification Beads. Mix well by pipetting up and down at least 10 times. 12.12. Incubate for 10 minutes at room temperature. 12.13. Place the tubes/plate on a magnet. After the solution is clear (about 2 minutes), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain the DNA targets (Caution: do not discard beads). 12.14. Add 200 μl of freshly prepared (same day) 80% EtOH while the tubes/ plate are on the magnet. Incubate at room temperature for 30 seconds and then carefully remove and discard the supernatant. 12.15. Repeat Step 12.14 once for a total of 2 washes in 80% EtOH, ensuring that all of the supernatant is removed from each well. 12.16. Incubate the samples, uncapped (or unsealed), at 37 C for 2 minutes on a thermocycler with the thermocycler lid open to dry the beads. 12.17. Remove the tubes/plate from the thermocycler and resuspend the dry beads in 30 μl of 1X TE by gently pipetting (or gently vortex capped tubes/sealed plate, followed by a quick spin). Incubate for 2 minutes at room temperature. 12.18. Place the tubes on a magnet and allow the solution to clear (about 2 minutes). 12.19. Transfer 28 μl of the eluted library to a fresh tube. 12.20. Evaluate 1 μl of the eluted library on High Sensitivity Bioanalyzer Chip. 26

E7000 NEBNEXT DIRECT CANCER HOTSPOT PANEL Figure 1.1: Examples of libraries prepared with human DNA (NA19240). 10,380 7,000 3,000 2,000 1,000 700 600 500 400 300 200 150 100 35 1 2 Lane 1: Lane 2: DNA Ladder Library made with 100 ng human DNA (NA19240) 27

2 Guidelines for Setting Up PCR Reactions (NEB #E7000X only) NEBNext Direct Cancer HotSpot Panel Instruction Manual 28 28

CHAPTER 2 Setting up the PCR Reaction: Symbols! This caution sign signifies a step in the protocol that has multiple paths leading to the same end point but is dependent on a user variable, like the amount of input DNA. 1. PCR Amplification! For < 96 samples, follow the protocol in Section 1.1. For 96 samples, follow the protocol in Section 1.2. 1.1. Setting up the PCR reactions (< 96 samples) 1.1.1. Determine the number of libraries that will be amplified and pooled for subsequent sequencing. 1.1.2. Ensure that you choose a valid combination of barcode primers based on color balance guidelines (see Chapter 3). 1.1.3. Thaw the NEBNext Direct Index Primer Mix Plate for 10-15 minutes at room temperature. 1.1.4. Remove the hard plastic plate cover. Briefly centrifuge the plate (280 g for ~1 min) to collect all of the primer at the bottom of each well. 1.1.5. Orient the NEBNext Direct Index Primer Mix Plate as indicated in Figure 2.1 (red stripe towards the user). With a pipette tip, pierce the desired well(s) (Figure 2.1A) and transfer 5 µl of the primer mix required for the PCR reaction to the PCR plate/ tubes. It is important to change pipette tips before piercing a new well to avoid cross contamination of indexed primers. Alternatively, the wells can be pierced using the bottom of clean PCR strip tubes (see Figure 2.1B) prior to pipetting the primer mix. Use a new, clean strip tube for each new well to be pierced. Note: Each well contains the Universal Primer and the Index Primer. There is enough primer in each well for one PCR reaction. Do not reuse primer if the seal has been previously pierced, to avoid contamination with other indexed primers. 1.1.6. Proceed with the PCR reaction according to Chapter 1, Section 11. 29

CHAPTER 2 Figure 2.1: NEBNext Direct Index Primer Mix Plate. A B NEBNext Direct Index Primer Mix 1.2. Setting up the PCR reactions (96 samples) 1.2.1. Thaw the NEBNext Direct Index Primer Mix Plate for 10-15 minutes at room temperature. 1.2.2. Remove the hard plastic plate cover. Briefly centrifuge the plate (280 g for ~1 min) to collect all of the primer at the bottom of each well. 1.2.3. Orient the NEBNext Direct Index Primer Plate as indicated in Figure 2.1 (red stripe towards the user). With a pipette tip, pierce the wells (Figure 2.1A) and transfer 5 µl of the primer mix required for the PCR reaction to the PCR plate. It is important to change pipette tips before piercing a new well to avoid cross contamination of indexed primers. Alternatively, the wells can be pierced using the bottom of clean PCR strip tubes (see Figure 2.1B) prior to pipetting the primer mix. Use a new, clean strip tube for each new well to be pierced. Note: Each well contains the Universal Primer and the Index Primer. There is enough primer in each well for one PCR reaction. Do not reuse primer if the seal has been previously pierced to avoid contamination with other indexed primers. 1.2.4. Proceed with the PCR reaction according to Chapter 1, Section 11. 30

3 Index Pooling Guidelines NEBNext Direct Cancer HotSpot Panel Instruction Manual 31 31

CHAPTER 3 To download a sample sheet with the index sequences for use with the Illumina Experiment Manager (IEM), please go to our FAQs tab on www.neb. com/e7000 NEBNext Direct Cancer HotSpot Panel (NEB #E7000). For the HiSeq /MiSeq, Illumina uses a red laser/led to sequence bases A and C and a green laser/led to sequence bases G and T. For each cycle, both the red and the green channel need to be read to ensure proper image registration (i.e. A or C must be in each cycle, and G or T must be in each cycle). If this color balance is not maintained, sequencing the index read could fail. Tables 3.1, 3.2 and 3.3 list some valid combinations (up to 4-plex) that can be sequenced together. For combinations > 4 choose any 4-plex combination and add any other index as needed. Table 3.1 Valid index primer combinations for NEB #E7000S. PLEX INDEX PRIMER 2 D01 and D02 D03 and D04 D05 and D06 D07 and D08 3 D01, D02 and D03 D02, D03 and D04 D03, D04 and D05 D04, D05 and D06 D05, D06 and D07 D06, D07 and D08 4 D01, D02, D03 and D04 D01, D04, D05 and D08 D02, D03, D04 and D05 D02, D04, D06 and D07 D03, D04, D05 and D06 D03, D05, D07 and D08 D04, D05, D06 and D07 D04, D05, D07 and D08 D05, D06, D07 and D08 32

E7000 DIRECT CANCER HOTSPOT PANEL Table 3.2 Valid index primer combinations for NEB #E7000L. PLEX INDEX PRIMER 2 D01 and D02 D03 and D04 D05 and D06 D07 and D08 D09 and D10 D11 and D12 D17 and D23 3 D01, D02 and D03 D02, D05 and D16 D04, D05 and D06 D07, D08 and D09 D10, D11 and D12 D11, D12 and D14 D13, D17 and D22 D17, D23 and D24 4 D01, D02, D03 and D04 D02, D04, D06 and D07 D03, D04, D14 and D17 D05, D06, D07 and D08 D07, D08, D13 and D22 D08, D14, D21 and D22 D09, D10, D11 and D12 D13, D14, D16 and D17 D15, D17, D22 and D23 D16, D17, D20 and D24 33

CHAPTER 3 Table 3.3 Valid index primer combinations for NEB #E7000X. PLEX INDEX PRIMER (WELL POSITION) 2 DHT18 (B6) and DHT22 (B10) DHT37 (D1) and DHT42 (D6) DHT52 (E4) and DHT76 (G4) DHT68 (F8) and DHT95 (H11) 3 DHT4 (A4), DHT11 (A11) and DHT12 (A12) DHT15 (B3), DHT22 (B10) and DHT24 (B12) DHT25 (C1), DHT31 (C7) and DHT33 (C9) DHT37 (D1), DHT42 (D6) and DHT48 (D12) DHT49 (E1), DHT54 (E6) and DHT55 (E7) DHT64 (F4), DHT69 (F9) and DHT71 (F11) DHT76 (G4), DHT77 (G5) and DHT83 (G11) DHT87 (H3), DHT93 (H9) and DHT94 (H10) 4 DHT1 (A1), DHT2 (A2), DHT3 (A3) and DHT4 (A4) DHT5 (A5), DHT6 (A6), DHT8 (A8) and DHT10 (A10) DHT13 (B1), DHT14 (B2), DHT15 (B3) and DHT16 (B4) DHT17 (B5), DHT18 (B6), DHT19 (B7) and DHT20 (B8) DHT25 (C1), DHT26 (C2), DHT27 (C3) and DHT30 (C6) DHT28 (C4), DHT29 (C5), DHT32 (C8) and DHT35 (C11) DHT37 (D1), DHT38 (D2), DHT39 (D3) and DHT40 (D4) DHT45 (D9), DHT46 (D10), DHT47 (D11) and DHT48 (D12) DHT49 (E1), DHT50 (E2), DHT51 (E3) and DHT52 (E4) DHT56 (E8), DHT58 (E10), DHT59 (E11) and DHT60 (E12) DHT61 (F1), DHT62 (F2), DHT63 (F3) and DHT69 (F9) DHT64 (F4), DHT65 (F5), DHT66 (F6) and DHT67 (F7) DHT73 (G1), DHT74 (G2), DHT75 (G3) and DHT76 (G4) DHT80 (G8), DHT82 (G10), DHT83 (G11) and DHT84 (G12) DHT85 (H1), DHT86 (H2), DHT87 (H3) and DHT89 (H5) DHT91 (H7), DHT94 (H10), DHT95 (H11) and DHT96 (H12) 34

E7000 DIRECT CANCER HOTSPOT PANEL For combinations of valid indexes, ensure that you will have signal in both the red and green channels in each cycle. See examples below: GOOD PRIMER INDEX SEQUENCE PRIMER INDEX SEQUENCE DHT1 T T A C C G A C DHT41 G A C G T C A T DHT2 A G T G A C C T DHT42 C T T A C A G C DHT3 T C G G A T T C DHT43 T C C A T T G C DHT4 C A A G G T A C DHT44 A G C G A G A T BAD PRIMER INDEX SEQUENCE PRIMER INDEX SEQUENCE DHT9 C G C A A C T A DHT56 T A T G G C A C DHT10 C G T A T C T C DHT57 C T C G A A C A DHT11 G T A C A C C T DHT58 C A A C T C C A DHT12 C G G C A T T A DHT59 G T C A T C G T Tables 3.4, 3.5 and 3.6 lists each index sequence color coded to correspond to the red/green channel. Table 3.4 Index sequences for NEB #E7000S. INDEX PRIMER INDEX SEQUENCE READ D01 A T T A C T C G D02 T C C G G A G A D03 C G C T C A T T D04 G A G A T T C C D05 A T T C A G A A D06 G A A T T C G T D07 C T G A A G C T D08 T A A T G C G C 35

CHAPTER 3 Table 3.5 Index sequences for NEB #E7000L. INDEX PRIMER INDEX SEQUENCE READ D01 A T T A C T C G D02 T C C G G A G A D03 C G C T C A T T D04 G A G A T T C C D05 A T T C A G A A D06 G A A T T C G T D07 C T G A A G C T D08 T A A T G C G C D09 C G G C T A T G D10 T C C G C G A A D11 T C T C G C G C D12 A G C G A T A G D13 A G T C A A C A D14 A G T T C C G T D15 A T G T C A G A D16 C C G T C C A T D17 G T C C G C A C D18 G T G A A A C G D19 G T G G C C T T D20 G T T T C G G A D21 C G T A C G T A D22 G A G T G G A T D23 A C T G A T A T D24 A T T C C T T T 36

Table 3.6 Index sequences for NEB #E7000X E7000 DIRECT CANCER HOTSPOT PANEL INDEX PRIMER WELL POSITION INDEX SEQUENCE READ DHT1 A1 T T A C C G A C DHT2 A2 A G T G A C C T DHT3 A3 T C G G A T T C DHT4 A4 C A A G G T A C DHT5 A5 T C C T C A T G DHT6 A6 G T C A G T C A DHT7 A7 C G A A T A C G DHT8 A8 T C T A G G A G DHT9 A9 C G C A A C T A DHT10 A10 C G T A T C T C DHT11 A11 G T A C A C C T DHT12 A12 C G G C A T T A DHT13 B1 T C G T C T G A DHT14 B2 A G C C T A T C DHT15 B3 C T G T A C C A DHT16 B4 A G A C C T T G DHT17 B5 A G G A T A G C DHT18 B6 C C T T C C A T DHT19 B7 G T C C T T G A DHT20 B8 T G C G T A A C DHT21 B9 C A C A G A C T DHT22 B10 T T A C G T G C DHT23 B11 C C A A G G T T DHT24 B12 C A C G C A A T DHT25 C1 T T C C A G G T DHT26 C2 T C A T C T C C DHT27 C3 G A G A G T A C DHT28 C4 G T C G T T A C DHT29 C5 G G A G G A A T DHT30 C6 A G G A A C A C DHT31 C7 C A G T G C T T DHT32 C8 C T T G C T A G 37

CHAPTER 3 INDEX PRIMER WELL POSITION INDEX SEQUENCE READ DHT33 C9 T G G A A G C A DHT34 C10 A G C T A A G C DHT35 C11 G A A C G G T T DHT36 C12 G G A A T G T C DHT37 D1 T A C G G T C T DHT38 D2 C C A G T A T C DHT39 D3 T C T A C G C A DHT40 D4 G T A A C C G A DHT41 D5 G A C G T C A T DHT42 D6 C T T A C A G C DHT43 D7 T C C A T T G C DHT44 D8 A G C G A G A T DHT45 D9 C A A T A G C C DHT46 D10 A A G A C A C C DHT47 D11 C C A G T T G A DHT48 D12 T G G T G A A G DHT49 E1 A A G A C C G T DHT50 E2 T T G C G A G A DHT51 E3 G C A A T T C C DHT52 E4 G A A T C C G T DHT53 E5 C C G C T T A A DHT54 E6 T A C C T G C A DHT55 E7 G T C G A T T G DHT56 E8 T A T G G C A C DHT57 E9 C T C G A A C A DHT58 E10 C A A C T C C A DHT59 E11 G T C A T C G T DHT60 E12 G G A C A T C A DHT61 F1 C A G G T T C A DHT62 F2 G A A C G A A G DHT63 F3 C T C A G A A G DHT64 F4 C A T G A G C A 38

E7000 DIRECT CANCER HOTSPOT PANEL INDEX PRIMER WELL POSITION INDEX SEQUENCE READ DHT65 F5 G A C G A A C T DHT66 F6 A G A C G C T A DHT67 F7 A T A A C G C C DHT68 F8 G A A T C A C C DHT69 F9 G G C A A G T T DHT70 F10 G A T C T T G C DHT71 F11 C A A T G C G A DHT72 F12 G G T G T A C A DHT73 G1 T A G G A G C T DHT74 G2 C G A A T T G C DHT75 G3 G T C C T A A G DHT76 G4 C T T A G G A C DHT77 G5 T C C A C G T T DHT78 G6 C A A C A C A G DHT79 G7 G C C T T A A C DHT80 G8 G T A A G G T G DHT81 G9 A G C T A C C A DHT82 G10 C T T C A C T G DHT83 G11 G G T T G A A C DHT84 G12 G A T A G C C A DHT85 H1 T A C T C C A G DHT86 H2 G G A A G A G A DHT87 H3 G C G T T A G A DHT88 H4 A T C T G A C C DHT89 H5 A A C C A G A G DHT90 H6 G T A C C A C A DHT91 H7 G G T A T A G G DHT92 H8 C G A G A G A A DHT93 H9 C A G C A T A C DHT94 H10 C T C G A C T T DHT95 H11 C T T C G G T T DHT96 H12 C C A C A A C A 39

CHAPTER 4 4 Guidelines for Running Samples on the Illumina MiSeq NEBNext Direct Cancer HotSpot Panel Instruction Manual 40 40

E7000 DIRECT CANCER HOTSPOT PANEL 1. Guidelines for Running samples on the Illumina MiSeq The NEBNext Direct protocol incorporates Illumina adapter sequences; therefore, the libraries generated from this protocol may be sequenced on Illumina platforms including the MiSeq, NextSeq, and HiSeq Platforms. Here we describe the steps necessary to sequence NEBNext direct libraries on the Illumina MiSeq. If samples are run on the Illumina NextSeq, please note that while the i5 index is generated in the reverse complement orientation, no adjustments need to be made because the i5 UMI is a random sequence. 4.1. Follow the Illumina Miseq Reporter Software Guide to reconfigure the MiSeq reporter parameter CreateFastqForIndexReads to write both index reads to FASTQs. The sample barcode is in the i7 position and is sequenced as the index read 1. The UMI is in the i5 position and is sequenced as the index read 2. By default, the Illumina MiSeq is set to not generate FASTQs for index reads; therefore, it is necessary to override this setting in order to make use of these features. 4.2. Generate a MiSeq sample sheet with the following parameters: Workflow: Generate FASTQ Application: FASTQ only Assay: TruSeq HT Chemistry: Amplicon The remaining fields will require information specific to your experiment. The choice for read lengths will depend on the particular bait design for your experiment, but in general NEBNext Direct baits are designed for PE75 reads. PE150 reads can also be used to increase depth of coverage. For a sample sheet template, visit the NEBNext Direct website at: https://www.neb.com/e7000 - NEBNext Direct Cancer HotSpot Panel (NEB #E7000) 4.3. Pool, dilute and denature samples for an 8 pm final concentration following the MiSeq Denature and Dilute Libraries Guide and the NEB- Next Direct guidelines for pooling together multiple barcoded samples (https://www.neb.com/nebnext-direct/nebnext-direct-for-target-enrichment). The number of samples that can be pooled together will depend on the input amount, panel size, and number of reads required from each sample for the particular analysis being performed. 41