TruSeq Custom Amplicon Low Input Kit

Transcription

1 TrSeq Cstom Amplicon Low Inpt Kit Reference Gide Docment # v04 Jne 2017 For Research Use Only. Not for se in diagnostic procedres. ILLUMINA PROPRIETARY

2 TrSeq Cstom Amplicon Low Inpt Kit 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, DesignStdio, 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. For Research Use Only not for any clinical or therapetic se in hmans or animals. This prodct incldes 2800M Control DNA manfactred by Promega Corporation for distribtion by Illmina, Inc. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. ii

3 TrSeq Cstom Amplicon Low Inpt Kit Reference Gide Revision History Docment Date Description of Change Docment # v04 Docment # v03 Docment # v02 Docment # v01 Docment # v00 Jne 2017 March 2016 Febrary 2016 Janary 2016 October 2015 Added thermal cycler programs for the hybridize oligo pool step. Added reqirement for niqe index combinations in each pool of a dal-pool protocol. Clarified the procedre for qantifying and dilting DNA. Updated the step for removing nbond oligos: In the introdction, corrected the nmber of SW1 wash steps to three. In the procedre, specified the HYB plate and keeping the plate on the magnetic stand when washing beads. Added alternate dal-strand protocol. Updated PCR cycle nmber gidelines in Amplify Libraries. Modified the thermal cycler program in Hybridize Oligo Pool. Clarified ELE/ELB mixtre instrctions in Hybridize Oligo Pool. Corrected storage temperatre for LNW1. Corrected the volme of LNA1 to 44 µl in Normalize Libraries. Added temperatres to the thermal cycler program in Hybridize Oligo Pool. Modified the thermal cycler program in Hybridize Oligo Pool. Removed reference to obsolete Experienced User Cards and added reference to the Cstom Protocol Selector. Renamed and combined some procedres as needed to improve continity. Initial release Docment # v04 For Research Use Only. Not for se in diagnostic procedres. iii

4 Table of Contents Chapter 1 Overview 1 Introdction 1 DNA Inpt Recommendations 2 Additional Resorces 2 Chapter 2 Protocol 4 Introdction 4 Tips and Techniqes 4 Library Prep Workflow 6 Qantify and Dilte DNA 7 Hybridize Oligo Pool 8 Remove Unbond Oligos 11 Extend and Ligate Bond Oligos 12 Amplify Libraries 14 Clean Up Libraries 16 Normalize Libraries 18 Pool Libraries 21 Appendix A Spporting Information 22 Introdction 22 Acronyms 22 Kit Contents 23 Consmables and Eqipment 24 Thermal Cycler HYB Programs 27 Technical Assistance 30 Docment # v04 For Research Use Only. Not for se in diagnostic procedres. iv

5 Chapter 1 Overview Introdction 1 DNA Inpt Recommendations 2 Additional Resorces 2 Introdction This protocol explains how to prepare p to 96 niqely indexed paired-end libraries of genomic DNA (gdna) sing the Illmina TrSeq Cstom Amplicon Low Inpt Library Prep Kit. The kit spports seqencing targeted regions of the genome spanning pwards of 600 kb with p to 1536 amplicons in a single mltiplex reaction. This highly targeted approach allows a wide range of applications for discovering, validating, and screening genetic variants rapidly and efficiently. The TrSeq Cstom Amplicon Low Inpt protocol offers: Streamlined 96-well based workflow amenable to atomation. Mltiplexing capability to amplify p to 1536 amplicons in one reaction and seqence p to 96 samples in one rn. Fast and simple workflow to generate p to 1536 amplicons across 96 samples in one plate with less than 3 hors of hands-on time. The TrSeq Cstom Amplicon Low Inpt library prep spports: Qalified FFPE samples. Inpt DNA amont as low as 10 ng, depending on DNA qality. Cstomized design to create and manage projects sing Illmina DesignStdio software online for a range of amplicon sizes and reference genomes. Atomated data analysis to perform variant calling and analysis across all samples sing simple oninstrment, atomated analysis software. The convenience of a flly integrated DNA-to- data soltion inclding online probe design and ordering, assay, seqencing, atomated data analysis, and offline software for reviewing reslts. Alternate Dal Strand Protocol This gide incldes an alternate dal strand protocol that adds a second, mirrored set of complementary amplicons to target both DNA strands at all loci. The alternate protocol allows preparation of p to 96 niqely indexed paired-end libraries (48 libraries each in Pool A and Pool B) of gdna. It is recommended for formalin-fixed, paraffin-embedded (FFPE) samples. Yo can configre the dal-strand protocol in DesignStdio dring design configration or after design completion. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 1

6 TrSeq Cstom Amplicon Low Inpt Kit Reference Gide DNA Inpt Recommendations Qantify the inpt DNA and assess the DNA qality before beginning library prep. Greater inpt increases library yield and improves seqencing metrics, sch as specificity (percent aligned reads). Type of DNA Spported Amplicon Size (bp) DNA Qality Cq Inpt (ng) * Genomic DNA 150, 175, 250 High FFPE genomic DNA 150, 175 High -1 to 1 10 Medim > Low * Inpt amont depends on Cq. For more information, see the TrSeq FFPE DNA Library Prep QC Reference Gide (docment # v00). 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) and specifically and accrately qantify dsdna, even when many common contaminants are present. In contrast, 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. Assessing DNA Qality Use the TrSeq FFPE DNA Library Prep QC Kit (Illmina catalog # FC ) to determine FFPE DNA qality and inpt amonts for the TrSeq Cstom Amplicon Low Inpt libraries. Inpt DNA Diltion Qantify and dilte DNA to the desired inpt amont based on the DNA type and qality. For more information, see DNA Inpt Recommendations on page 2. Yo can dilte and store more than the reqired amont of DNA for later se. Dilte DNA in RS1 and SS1 and store as described in Qantify and Dilte DNA on page 7. Additional Resorces Visit the TrSeq Cstom Amplicon Low Inpt Kit spport page on the Illmina website for docmentation, software downloads, training resorces, and information abot compatible Illmina prodcts. The following docmentation is available for download from the Illmina website. Resorce Cstom Protocol Selector TrSeq Cstom Amplicon Low Inpt Kit Checklist (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. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 2

7 TrSeq Cstom Amplicon Low Inpt Kit Reference Gide Resorce TrSeq Cstom Amplicon Low Inpt Kit Consmables & Eqipment List (docment # ) TrSeq FFPE DNA Library Prep QC Reference Gide (docment # ) Description Provides an interactive checklist of ser-provided consmables and eqipment. Provides instrctions on how to determine the fragmentation stats and the amplification potential of FFPE-extracted gdna samples sing the TrSeq FFPE DNA QC Kit. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 3

8 Chapter 2 Protocol Introdction 4 Tips and Techniqes 4 Library Prep Workflow 6 Qantify and Dilte DNA 7 Hybridize Oligo Pool 8 Remove Unbond Oligos 11 Extend and Ligate Bond Oligos 12 Amplify Libraries 14 Clean Up Libraries 16 Normalize Libraries 18 Pool Libraries 21 Introdction This section describes the TrSeq Cstom Amplicon Low Inpt Kit protocol. Prepare at least eight samples at a time for the 16-sample kit. For the 96- sample kit, prepare at least 16 samples at a time. Follow the protocol in the order described sing the specified parameters. Before proceeding, confirm yor 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. Prepare for Pooling If yo plan to pool libraries, record information abot yor samples before beginning library prep. For more information, see the TrSeq Cstom Amplicon Low Inpt Kit spport page. 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 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 effective for thermal cycling. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 4

9 TrSeq Cstom Amplicon Low Inpt Kit Reference Gide 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 slowly. When mixing, mix thoroghly. If beads are aspirated into the 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. Leave 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 # v04 For Research Use Only. Not for se in diagnostic procedres. 5

10 TrSeq Cstom Amplicon Low Inpt Kit Reference Gide Library Prep Workflow The following figre illstrates the workflow for the TrSeq Cstom Amplicon Low Inpt Kit. Figre 1 TrSeq Cstom Amplicon Low Inpt Kit Workflow Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 6

11 TrSeq Cstom Amplicon Low Inpt Kit Reference Gide Qantify and Dilte DNA This step qantifies and diltes inpt DNA to the appropriate concentration in the reqired dilent for sbseqent steps. For FFPE samples, prepare dplicate or triplicate DNA samples for increased sensitivity in variant calling. Consmables RS1 (Resspension Soltion 1) SS1 (Sample Stabilization Soltion 1) Genomic DNA LoBind microcentrifge tbes Abot Reagents For later se, yo can dilte and store more DNA than is reqired. Dilte DNA in RS1 and SS1 as described in the procedre. Prepare aliqots of dilted DNA in LoBind 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 ng/µl in RS1, depending on the initial DNA concentration and the desired inpt DNA amont. Dilting to ng/µl is recommended for high-qality DNA. For medim- or low-qality DNA recommendations, see DNA Inpt Recommendations on page 2. 3 Reqantify the dilted DNA sing the same florometric qantification method. 4 Frther dilte DNA in a LoBind tbe as follows. For the dal strand protocol, prepare ~10% extra DNA to accont for pipetting errors. a b Dilte the desired inpt DNA amont in RS1 to reslt in a final volme of 4 µl. Add 1 µl SS1 to the 4 µl dilted DNA. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 7

12 TrSeq Cstom Amplicon Low Inpt Kit Reference Gide Example: If yo dilted high-qality DNA to 10 ng/µl in step 2, dilte the 10 ng/µl to 2.5 ng/µl in RS1 to reslt in 4 µl dilted DNA. Hybridize Oligo Pool This process 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 CAT (Cstom Amplicon Oligo Tbe) [Dal strand protocol] CAT A (Cstom Amplicon Oligo Tbe A) [Dal strand protocol] CAT B (Cstom Amplicon Oligo Tbe B) OHS2 (Oligo Hybridization for Seqencing 2) 2800M (Control DNA 2800M) ACP3 (Control Oligo Pool) RS1 (Resspension Soltion 1) HYP (Hybridization Plate) barcode label Dilted DNA 96-well PCR plate Microseal 'A' adhesive film LoBind microcentrifge tbes RNase/DNase-free eight-tbe strips and caps Prepare for a later procedre: ELB (Extension-Ligation Bffer) ELE (Extension-Ligation Enzyme) Prepare for a later procedre: 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 (or CAT A and CAT B for dal strand) Yo can dilte and store more than the reqired amont of CAT (or CAT A and B) to se later. Dilte CAT (or CAT A and B) in RS1 as described in the following procedre or Procedre for 48 Samples (Dal Strand) on page 10. Use a mltichannel pipette to dispense dilted CAT from a PCR eight-tbe strip that contains 70 µl in each tbe. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 8

13 TrSeq Cstom Amplicon Low Inpt Kit Reference Gide 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. 2800M Inclde the 2800M control DNA in every batch of samples being prepared. Use of this control allows Illmina Technical Spport to trobleshoot if assistance is needed. If this control is exclded from yor assay, assistance cannot be provided. ACP3 is specifically for se with the 2800M control 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. CAT or CAT A and CAT B (for dal strand) -25 C to -15 C Thaw at room temperatre for 30 mintes. Vortex to mix, and then centrifge briefly. ACP3-25 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. 2800M 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. Flick 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 a later procedre. Do not exceed 25 C. SW1 2 C to 8 C Let stand to bring to room temperatre in preparation for a later procedre. 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. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 9

14 TrSeq Cstom Amplicon Low Inpt Kit Reference Gide This HYB program is a general program. For specific programming instrctions, see Thermal Cycler HYB Programs on page Label a new 96- well PCR plate HYP. Procedre for 96 Samples 1 In a LoBind microcentrifge tbe, dilte 2.5 µl CAT with 2.5 µl RS1 per sample well. Plse vortex to mix, and then centrifge briefly. 2 In a LoBind microcentrifge tbe, dilte 2.5 µl ACP3 with 2.5 µl RS1. Plse vortex to mix, and then centrifge briefly. 3 In a LoBind microcentrifge tbe, dilte 2 µl 2800M with 2 µl RS1 and 1 µl SS1. Plse vortex to mix, and then centrifge briefly. 4 Add 5 µl dilted 2800M to one well of the HYP plate. 5 Add 5 µl dilted ACP3 to the well that contains dilted 2800M as an assay control. 6 Add 5 µl RS1 to 1 well as a no template control. 7 Add 5 µl dilted DNA to the remaining wells. 8 Add 5 µl dilted CAT to all wells except the well containing 2800M. 9 Add 15 µl OHS2 to each well. Using a P20 pipette, pipette slowly to mix. 10 If bbbles form, centrifge the plate at 100 g for 20 seconds. 11 Place on the preprogrammed thermal cycler and rn the HYB program. 12 Combine ELE and ELB as follows. [16-sample kit] Transfer 18 µl ELE to the ELB tbe. Flick and invert to mix. Do not vortex. [96-sample kit] Transfer 137 µl ELE to the ELB tbe. Flick and invert to mix. Do not vortex. NOTE Prepare the fll amont of the ELE/ELB mixtre for yor kit, even if yo are processing fewer than 16 or 96 samples. If necessary, store aliqots at -25 C to -15 C for p to 3 months. Do not allow more than six freeze-thaw cycles. 13 Place the ELB/ELE mixtre on ice. The mixtre is sed dring the procedre for extending and ligating bond oligos. Procedre for 48 Samples (Dal Strand) 1 In a LoBind microcentrifge tbe, dilte 2.5 µl CAT A with 2.5 µl RS1 per sample well. Plse vortex to mix, and then centrifge briefly. 2 In a LoBind microcentrifge tbe, dilte 2.5 µl CAT B with 2.5 µl RS1 per sample well. Plse vortex to mix, and then centrifge briefly. 3 In a LoBind microcentrifge tbe, dilte 2.5 µl ACP3 with 2.5 µl RS1. Plse vortex to mix, and then centrifge briefly. 4 In a LoBind microcentrifge tbe, diltedilte 2 µl 2800M with 2 µl RS1 and 1 µl SS1. Plse vortex to mix, and then centrifge briefly. 5 Add 5 µl dilted 2800M to one well of the HYP plate. 6 Add 5 µl dilted ACP3 to the well that contains dilted 2800M as an assay control. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 10

15 TrSeq Cstom Amplicon Low Inpt Kit Reference Gide 7 Add 5 µl RS1 to one well as a no template control. 8 Add 5 µl dilted DNA to wells on the left half of the plate, starting with colmn 1. 9 Add 5 µl dilted DNA to wells on the right half of the plate, starting with colmn 7. Do not add DNA to the well containing 2800M. 10 Add 5 µl dilted CAT A to each well containing DNA intended for CAT A. 11 Add 5 µl dilted CAT B to each well containing DNA intended for CAT B. 12 Add 15 µl OHS2 to each well. Pipette slowly to mix sing a P20 pipette. 13 If bbbles form, centrifge the plate at 100 g for 20 seconds. 14 Place on the preprogrammed thermal cycler and rn the HYB program. 15 Combine ELE and ELB as follows. [16 samples] Transfer 18 µl ELE to the ELB tbe. Flick and invert to mix. Do not vortex. [96 samples] Transfer 137 µl ELE to the ELB tbe. Flick and invert to mix. Do not vortex. NOTE Prepare the fll amont of the ELE/ELB mixtre for yor kit, even if yo are processing fewer than 16 or 96 samples. If necessary, store aliqots at -25 C to -15 C for p to 3 months. Do not allow more than six freeze-thaw cycles. 16 Place the ELB/ELE mixtre on ice. The mixtre is sed dring the procedre for extending and ligating 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. 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. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 11

16 TrSeq Cstom Amplicon Low Inpt Kit Reference Gide 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. 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 seqences reqired for amplification. Consmables ELB/ELE mixtre Microseal 'A' adhesive seal LoBind microcentrifge tbe Prepare for a later procedre: EDP (Enhanced DNA Polymerase) EMM (Enhanced Master Mix) Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 12

17 TrSeq Cstom Amplicon Low Inpt Kit Reference Gide Prepare for a later procedre: Index i7 adapters (A7XX) Index i5 adapters (A5XX) 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 fll amont of the mixtre even if processing fewer than 16 or 96 samples. If needed, store aliqots at -25 C to -15 C for p to 3 months. Do not allow more than six freeze-thaw cycles. 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 Thaw at room temperatre for 20 mintes. Vortex each tbe to mix. Centrifge briefly sing a 1.7 ml Eppendorf tbe. Place on ice for a later procedre. 1 Remove 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. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 13

18 TrSeq Cstom Amplicon Low Inpt Kit Reference Gide 6 Combine EDP and EMM in a LoBind microcentrifge tbe as follows. Nmber of Samples EDP (µl) EMM (µl) Volmes inclde an additional 10%. 7 Pipette the EDP/EMM mixtre to mix, and then centrifge briefly. Place on ice for the next step. 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. Each sample mst have a niqe index combination, inclding samples in separate pools for a dal pool design. Make sre that none of the samples in the CAT A pool have the same index combination as the samples in the CAT B pool. Consmables EDP/EMM mixtre Index i7 adapters (A7XX) Index i5 adapters (A5XX) Microseal 'A' adhesive film Microseal 'B' adhesive seal NOTE Use Microseal 'A' when sealing the plate before placing on the thermal cycler. Use Microseal 'B' for shaking, centrifging, and long-term storage. Preparation 1 Save the following PCR program on a thermal cycler. Use the following table to determine the nmber of cycles. PCR cycles are based on 10 ng inpt DNA and the nmber of amplicons in the CAT. 95 C for 3 mintes X cycles of: 98 C for 20 seconds 67 C for 20 seconds 72 C for 40 seconds 72 C for 1 minte Hold at 10 C Plexity Nmber of PCR Cycles (X) ¹, ² < 96 amplicons amplicons amplicons 28 Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 14

19 TrSeq Cstom Amplicon Low Inpt Kit Reference Gide Plexity Nmber of PCR Cycles (X) ¹, ² amplicons amplicons³ 25 1 Add one cycle for FFPE samples and one cycle for < 10 ng DNA inpt. ² To achieve desired library yield and specificity, optimize the PCR cycle nmber for yor oligo pool. ³ Alignment specificity might be redced for high-plexity panels designed with larger amplicon sizes. NOTE Process the 2800M/ACP3 control sing the same conditions as 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. 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. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 15

20 TrSeq Cstom Amplicon Low Inpt Kit Reference Gide 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. 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 per well fresh 80% ethanol 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. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 16

21 TrSeq Cstom Amplicon Low Inpt Kit Reference Gide 2 Transfer 45 µl of the spernatant from each well of the HYP plate to the corresponding well of the CLP plate. Transfer as few beads as possible. 3 Add 36 µl SPB to each well of the CLP plate. 4 Shake the plate at 1800 rpm for 2 mintes. 5 Incbate at room temperatre for 5 mintes. 6 Centrifge at 280 g for 1 minte. 7 Place on a magnetic stand and wait ntil the liqid is clear (~2 mintes). 8 Remove and discard all spernatant from each well. 9 Wash two times as follows. a b c Add 200 µl freshly prepared 80% EtOH to each sample well. Incbate on the magnetic stand for 30 seconds. Remove and discard all spernatant from each well. 10 Using a 20 µl pipette, remove residal EtOH from each well. 11 Remove from the magnetic stand and air-dry for 5 mintes. 12 Add 25 µl RSB to each well. 13 Shake the plate at 1800 rpm for 2 mintes. 14 Make sre that all beads are resspended. If necessary, pipette to mix and repeat the shaking step. 15 Incbate at room temperatre for 2 mintes. 16 Centrifge at 280 g for 1 minte. 17 Place on a magnetic stand and wait ntil the liqid is clear (~2 mintes). 18 Transfer 20 µl prified library from each well of the CLP plate to the corresponding well of the LNP plate. 19 From the remaining liqid in the CLP plate, rn an aliqot of the samples and control on 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 more than samples, 2 µl on an Advanced Analytical Fragment Analyzer sing the Standard Sensitivity NGS Fragment Analysis Kit. For 2 96 samples, 1 µl on an Agilent 2200 TapeStation sing the D1000 ScreenTape assay. Table 1 Expected PCR Prodct Sizes Amplicon Size PCR Prodct Size 150 bp ~280 bp 175 bp ~310 bp 250 bp ~350 bp 2800M ~310 bp Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 17

22 TrSeq Cstom Amplicon Low Inpt Kit Reference Gide Figre 3 Bioanalyzer Trace Example A B C Marker Expected PCR Prodct for 200 bp amplicons(~350 bp) Marker Figre 4 Fragment Analyzer Example (Showing Expected 2800M/ACP3 PCR Prodct) A B C Marker Expected PCR Prodct for 200 bp amplicons(~350 bp) 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. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 18

23 TrSeq Cstom Amplicon Low Inpt Kit Reference Gide 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) 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 wellventilated 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. Make sre that LNB1 is resspended before se. 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. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 19

24 TrSeq Cstom Amplicon Low Inpt Kit Reference Gide Reagent Storage Instrctions 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 If frozen, thaw at room temperatre for 20 mintes. Vortex to mix. 2 Prepare fresh 0.1 N NaOH. 3 Label a new 96-well 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. 5 Shake at 1800 rpm for 30 mintes. Drations other than 30 mintes 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 the LNP plate 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 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. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 20

25 TrSeq Cstom Amplicon Low Inpt Kit Reference Gide Pool Libraries This step pools libraries by combining eqal volmes of normalized libraries in one tbe. NOTE With the dal strand protocol, two libraries represent each sample and are generated from CAT A and CAT B. To analyze the reslts of each sample, the CAT A and CAT B libraries for each sample mst be rn together on the same flow cell. Consmables PAL (Pooled Amplicon Library) barcode label Microcentrifge tbe RNase/DNase-free eight-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 at 1000 g for 1 minte. 2 Transfer 5 µl of each library to an eight-tbe strip, colmn by colmn. 3 Seal the plate and store at -25 C to -15 C. 4 Transfer the contents of the eight-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 # v04 For Research Use Only. Not for se in diagnostic procedres. 21

26 Appendix A Spporting Information Spporting Information Introdction 22 Acronyms 22 Kit Contents 23 Consmables and Eqipment 24 Thermal Cycler HYB Programs 27 Introdction The protocols described in this gide assme that yo have reviewed the contents of this appendix, confirmed yor kit contents, and obtained all the reqired consmables and eqipment. Acronyms Acronym 2800M Definition Control DNA 2800M ACP3 Amplicon Control Oligo Pool 3 CAT CAT A CAT B CLP EDP ELB ELE EMM HT1 HYP Cstom Amplicon Oligo Tbe Cstom Amplicon Oligo Tbe A Cstom Amplicon Oligo Tbe B Clean-p Plate Enhanced DNA Polymerase Extension- Ligation Bffer Extension- Ligation Enzyme Enhanced Master Mix Hybridization Bffer 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 SPB SGP Resspension Bffer Sample Prification Beads Storage Plate SS1 Sample Stabilization Soltion 1 SW1 Stringent Wash 1 Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 22

27 TrSeq Cstom Amplicon Low Inpt Kit Reference Gide Kit Contents Make sre that yo have all the reagents identified in this section before proceeding to the library preparation procedres. A TrSeq Cstom Amplicon Low Inpt Kit and a TrSeq Cstom Amplicon Index Kit are reqired. Kit Name Catalog # TrSeq Cstom Amplicon Low Inpt Kit (16 samples) TrSeq Cstom Amplicon Low Inpt Kit (96 samples) TrSeq Cstom Amplicon Index Kit (96 indexes, 384 samples) FC FC FC TrSeq Cstom Amplicon Low Inpt Kit Contents (16 samples, FC ) (96 samples, FC ) Box 1, Store in the Pre-PCR Area Qantity Reagent Description Storage Temperatre 1 ACP3 Amplicon Control Oligo Pool 3-25 C to -15 C 1 ELE Extension- Ligation Enzyme -25 C to -15 C 1 ELB Extension- Ligation Bffer -25 C to -15 C 1 EDP Enhanced DNA Polymerase -25 C to -15 C 1 EMM Enhanced Master Mix -25 C to -15 C M 2800M Control DNA 2 C to 8 C This box also contains the HYP barcode label. Box 2 Qantity Reagent Description Storage Area Storage Temperatre 1 SS1 Sample Stabilization Soltion 1 Pre-PCR 2 C to 8 C 1 SPB Sample Prification Beads Pre-PCR 2 C to 8 C 1 OHS2 Oligo Hybridization for Seqencing Reagent 2 Pre-PCR 2 C to 8 C 1 SW1 Stringent Wash 1 Pre-PCR 2 C to 8 C 1 RS1 Resspension Soltion 1 Pre-PCR 15 C to 30 C 1 LNB1 Library Normalization Beads 1 Post-PCR 2 C to 8 C Box 3, 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 LNW1 Library Normalization Wash 1 2 C to 8 C 1 LNS2 Library Normalization Storage Bffer 2 15 C to 30 C 1 RSB Resspension Bffer 15 C to 30 C Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 23

28 TrSeq Cstom Amplicon Low Inpt Kit Reference Gide This box also contains plate barcode labels. TrSeq Cstom Amplicon Index Kit (96 indexes, 384 samples) (FC ) Box 1, Store at -25 C to -15 C in the Pre-PCR Area Qantity Reagent Description 1 A501 i5 Index Adapter 1 A502 i5 Index Adapter 1 A503 i5 Index Adapter 1 A504 i5 Index Adapter 1 A505 i5 Index Adapter 1 A506 i5 Index Adapter 1 A507 i5 Index Adapter 1 A508 i5 Index Adapter 1 A701 i7 Index Adapter 1 A702 i7 Index Adapter 1 A703 i7 Index Adapter 1 A704 i7 Index Adapter 1 A705 i7 Index Adapter 1 A706 i7 Index Adapter 1 A707 i7 Index Adapter 1 A708 i7 Index Adapter 1 A709 i7 Index Adapter 1 A710 i7 Index Adapter 1 A711 i7 Index Adapter 1 A712 i7 Index Adapter Index Adapter Replacement Caps, Store at 15 C to 30 C in the Pre-PCR Area Qantity Description 1 i5 Index Tbe Caps, White 1 i7 Index Tbe Caps, Orange 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 a dedicated set of consmables and eqipment for pre-pcr and post-pcr procedres. Different magnetic stands are needed for pre-pcr and post-pcr procedres. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 24

29 TrSeq Cstom Amplicon Low Inpt Kit 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 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 LoBind microcentrifge tbes, 1.5 ml Eppendorf, part # Microseal 'A' film Microseal 'B' adhesive seals PCR grade water RNase/DNase-free eight-tbes strips and caps TrSeq FFPE DNA Library Prep QC Kit 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 Bio-Rad, catalog # MSA-5001 Bio-Rad, catalog # MSB-1001 General lab spplier General lab spplier Illmina catalog # FC 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. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 25

30 TrSeq Cstom Amplicon Low Inpt Kit Reference Gide Eqipment Pre-PCR Eqipment Eqipment Iceless cooler for 96-well plates 96-well thermal cycler (with heated lid) See Thermal Cyclers. One of the following magnets, 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 General lab spplier General lab 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 Bio-Rad DNA Engine Tetrad 2 Block Type Ramp Rate Lid Temp Standard 0.1 C Heated, Constant at 100 C Bio-Rad S1000 Standard 0.1 C 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 Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 26

31 TrSeq Cstom Amplicon Low Inpt Kit Reference Gide Thermal Cycler Block Type Ramp Rate Lid Temp 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 -- 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 Thermal Cycler HYB Programs The thermal cycler program for the hybridize oligo pool step varies by thermal cycler model. The following tables provide the programs for selected models. Use these tables as a reference when saving the HYB program. Bio-Rad DNA Engine Tetrad 2, S1000, C1000, and T100 Step Ramp Speed Increment ( C) Temperatre ( C) Hold mintes C/s seconds 3 Go to step 2 for 59x C/s minte 5 Go to step 4 for 19x C/s mintes 7 Go to step 6 for 9x C/s mintes Applied Biosystems GeneAmp PCR System 9700 Initial hold at 95 C for 3 mintes. Select 41- temperatre, 2-cycle protocol. Becase the minimm cycle nmber is two, make sre that yo inclde the pase step to prevent the thermal cycler from retrning to 95 C. Set the temperatre and time as listed in the following table. Modify the ramp rate to 1% per temperatre. Add a pase at the end of the cycle. Remove the HYB plate dring the pase step. When performing the protocol, set the time to 90 mintes so that the HYB plate does not remain at 40 C for more than 10 mintes. Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 27

32 TrSeq Cstom Amplicon Low Inpt Kit Reference Gide Step Ramp Speed (%) Temperatre ( C) Hold 1 None 95 3 mintes seconds seconds seconds seconds minte minte minte minte minte minte mintes mintes mintes mintes mintes mintes mintes mintes mintes mintes mintes mintes mintes mintes mintes mintes mintes mintes mintes mintes mintes mintes mintes mintes mintes mintes mintes mintes Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 28

33 TrSeq Cstom Amplicon Low Inpt Kit Reference Gide Step Ramp Speed (%) Temperatre ( C) Hold mintes mintes Pase 99:59 Applied Biosystems Veriti 96-Well Thermal Cycler Step Ramp Speed Increment ( C) Temperatre ( C) Hold mintes 2 2.1% seconds 3 Go to step 2 for 59x 4 2% minte 5 Go to step 4 for 19x 6 2.1% mintes 7 Go to step 6 for 9x 8 2% mintes Docment # v04 For Research Use Only. Not for se in diagnostic procedres. 29