Nextera XT DNA Library Prep Kit

Transcription

1 Nextera XT DNA Library Prep Kit Reference Gide Docment # v02 April 2017 For Research Use Only. Not for se in diagnostic procedres. ILLUMINA PROPRIETARY

2 Nextera XT DNA Library Prep 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, HiSeq, MiSeq, Nextera, TrSeq, and the streaming bases design are registered or pending trademarks of Illmina, Inc. and/or its affiliate(s) in the U.S. and/or other contries. All other names, logos, and other trademarks are the property of their respective owners. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. ii

3 Nextera XT DNA Library Prep Reference Gide Revision History Docment Date Description of Change Docment # v02 Docment # v01 April 2017 Janary 2016 Added the following information: Spported genome size of < 5 Mb. The ratio of absorbance that indicates contaminants. Recommendations for PCR amplicons. AMPre XP bead recommendations for rns cycles. Reagent and library volmes in the PCR plate after the tagmentation and amplification steps. Beckman Colter Genomics item # A63880 for Agencort AMPre XP, 5 ml. Illmina catalog # PE and # FC for the TrSeq Dal Index Seqencing Primer Box. Added the following technical notes to the list of additional resorces: Best Practices for Standard and Bead-Based Normalization in Nextera XT DNA Library Preparation Kits (Pb. No ) Nextera XT Library Prep: Tips and Trobleshooting (Pb. No ) Consolidated steps in the pool libraries procedre. Identified the NaOH consmable as moleclar biology grade. Specified the se of moleclar- grade water or 10 mm Tris-HCl, ph to dilte starting material for DNA qality assessment. Specified proceeding immediately when tagmentation is complete so that netralization occrs while the transposome is active. Specified a thaw time of 20 mintes for NPM (Nextera PCR Master Mix). Updated the normalize libraries procedre to apply to varios sample nmbers, not only 96. Updated TCY plate to Hard-Shell 96-well PCR plate, skirted. Updated magnetic stand spplier to Thermo Fisher Scientific, Corrected the catalog nmbers for Nextera kits provided in the introdction. Corrected the illstration showing how the Nextera assay works. Updated design of workflow diagram. Renamed and combined some procedres as needed to improve continity. Simplified consmables information at the beginning of each section. Revised step-by-step instrctions to be more sccinct. Removed reference to obsolete Experienced User Cards and added reference to the Cstom Protocol Selector. Clarified AMPre XP bead recommendation for nonamplicon applications. See Clean Up Libraries. Added information abot normalizing low yield libraries. See Normalize Libraries. Corrected index adapter labels on the assay diagram Rev. E Janary 2015 Corrected kit contents for Nextera XT DNA Library Preparation Index Kit v2 Set A (FC ) to inclde index N715. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. iii

4 Nextera XT DNA Library Prep Reference Gide Docment Date Description of Change Rev. D September Rev. C October 2012 Added info for new index kits that enable preparation of p to 384 indexed paired-end libraries. Updated DNA Inpt Recommendations for dilting starting material and the potential reslts of incomplete tagmentation. Added new Nextera XT Qality Metrics with new information on how to trobleshoot flctations in clster density. Removed Dal Indexing Principle and Low Plexity Pooling Gidelines sections. This information can be fond in the Nextera Low-Plex Pooling Gidelines Tech Note on the Nextera XT DNA Library Prep Kit spport page. References to read lengths on the MiSeq were pdated for v3 chemistry. Added instrctions for alternate tip if processing fewer than 24 samples while transferring LNB1 beads in Library Normalization. Added NaOH 1N ph > 12.5 to the Consmables and Eqipment list as a ser-spplied consmable. Removed Tween 20 from Consmables and Eqipment list. Consmable not sed in protocol. Modifications were added in PCR Clean-Up for 2x300 rns on the MiSeq. New section for clstering samples on the HiSeq, HiScanSQ, and GAIIx. See Clstering Samples for HiSeq, HiScanSQ, and GAIIx. The Dal Indexing Principle section listed incorrect catalog nmbers for the Nextera XT Index kits. The correct catalog nmbers are now listed. Added emphasis on making sre the NT (Netralize Tagment Bffer) and LNS1 (Library Normalization Storage Bffer 1) reagents are at room temperatre before se in the protocol. Removed reference to Tris-Cl 10 mm, ph8.5 with 0.1% Tween 20 from the User-Spplied Consmables table becase it is not sed in this library preparation Rev. B Jly 2012 Added emphasis on making sre the NT (Netralize Tagment Bffer) and LNS1 (Library Normalization Storage Bffer 1) reagents are at room temperatre before se in the protocol. Removed reference to Tris-Cl 10 mm, ph8.5 with 0.1% Tween 20 from the User-Spplied Consmables table becase it is not sed in this library preparation Rev. A May 2012 Initial release. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. iv

5 Table of Contents Chapter 1 Overview 1 Introdction 1 DNA Inpt Recommendations 1 Additional Resorces 2 Chapter 2 Protocol 3 Introdction 3 Tips and Techniqes 3 Library Prep Workflow 5 Tagment Genomic DNA 6 Amplify Libraries 7 Clean Up Libraries 9 Check Libraries 11 Normalize Libraries 11 Pool Libraries 13 Appendix A Spporting Information 15 Introdction 15 How the Nextera XT Assay Works 15 Nextera XT Qality Metrics 16 Acronyms 17 Nextera XT DNA Library Prep Kit 17 Consmables and Eqipment 20 Technical Assistance 22 Docment # v02 For Research Use Only. Not for se in diagnostic procedres. v

6 Chapter 1 Overview Introdction 1 DNA Inpt Recommendations 1 Additional Resorces 2 Introdction This protocol explains how to prepare p to 384 indexed paired-end libraries from DNA for sbseqent seqencing on Illmina seqencing systems. Reagents provided in the Nextera XT Library Prep Kit and Nextera XT Index Kit are sed to fragment DNA and add adapter seqences onto the DNA template. The kit has the following featres: Uses tagmentation, an enzymatic reaction, to fragment DNA and add partial adapter seqences in only 5 mintes. Master mixed reagents redce reagent containers, pipetting, and hands-on time. Only 1 ng inpt DNA is needed. Nextera XT typically spports genomes that are < 5 Mb while Nextera DNA typically spports genomes > 5 Mb. Table 1 Example Applications for Nextera Kits Nextera XT (FC , FC ) Small genomes, amplicons, plasmids PCR amplicons (> 300 bp)* Plasmids Microbial genomes (eg, Prokaryotes, archaea) Concatenated amplicons Doble-stranded cdna Single-cell RNA-Seq Nextera DNA (FC , FC ) Large or complex genomes Hman genomes Nonhman mammalian genomes (eg, mose, rat, bovine) Plant genomes (eg, Arabidopsis, maize, rice) Invertebrate genomes (eg, Drosophila) * Using a > 300 bp amplicon size ensres even coverage across the length of the DNA fragment. For more information, see PCR Amplicons on page 2. DNA Inpt Recommendations The Nextera XT protocol is optimized for 1 ng of inpt DNA.Qantify the starting material before preparing libraries. Dilte starting material in moleclar-grade water or 10 mm Tris HCl, ph Inpt DNA Qantification The enzymatic DNA fragmentation sed for this protocol is more sensitive to DNA inpt compared to mechanical fragmentation. Sccess depends on accrate qantification of inpt DNA. Use a florometric-based method to qantify inpt DNA. For example, if yo se the Qbit dsdna BR Assay system, se 2 µl of each DNA sample with 198 µl of the Qbit working soltion. Avoid methods that measre total ncleic acid, sch as NanoDrop or other UV absorbance methods. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 1

7 Nextera XT DNA Library Prep Reference Gide Assess DNA Qality UV absorbance is a common method for assessing the qality of a DNA sample. The ratio of absorbance at 260 nm to absorbance at 280 nm is sed as an indication of sample prity. This protocol is optimized for DNA with absorbance ratio vales of , which indicates a pre DNA sample. Target a 260/230 ratio of Vales otside this range indicate the presence of contaminants. For a complete list of contaminants, inclding sorces, avoidance, and effects on the library, see Nextera XT Library Prep: Tips and Trobleshooting (Pb. No ). Dilte the starting material in moleclar-grade water or 10 mm Tris-HCl, ph Incomplete tagmentation can case library preparation failre, poor clstering, or an nexpectedly high scaffold nmber. PCR Amplicons The PCR amplicon mst be > 300 bp. Shorter amplicons can be lost dring the library cleanp step. Tagmentation cannot add an adapter directly to the distal end of a fragment, so a drop in seqencing coverage of ~50 bp from each distal end is expected. To ensre sfficient coverage of the amplicon target region, design primers to extend beyond the target region by 50 bp per end. Additional Resorces Visit the Nextera XT DNA Library Prep 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 Nextera XT DNA Library Prep Kit Checklist (docment # ) Nextera Low-Plex Pooling Gidelines (Pb. No ) Best Practices for Standard and Bead-Based Normalization in Nextera XT DNA Library Preparation Kits (Pb. No ) Nextera XT Library Prep: Tips and Trobleshooting (Pb. No ) 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 the protocol steps. The checklist is intended for experienced sers. Provides pooling gidelines and dal indexing strategies for Nextera XT library prep. Provides best practices for bead-based normalization of Nextera XT libraries. Provides best practices for addressing ndertagmentation, sample contaminants, and other problems that can occr when preparing Nextera XT libraries. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 2

8 Chapter 2 Protocol Introdction 3 Tips and Techniqes 3 Library Prep Workflow 5 Tagment Genomic DNA 6 Amplify Libraries 7 Clean Up Libraries 9 Check Libraries 11 Normalize Libraries 11 Pool Libraries 13 Introdction This chapter describes the Nextera XT DNA Library Prep Kit protocol. Review Best Practices before proceeding. See Additional Resorces on page 2 for information on accessing Best Practices on the Illmina website. Before proceeding, confirm kit contents and make sre that yo have the reqired eqipment and consmables. See Spporting Information on page 15. Follow the protocols in the order shown, sing the specified volmes and incbation parameters. Prepare for Pooling If yo plan to pool libraries, record information abot yor samples before beginning library prep. For more information, see the Nextera XT DNA Library Prep Kit spport page. Review Nextera Low-Plex Pooling Gidelines (Pb. No ) when preparing libraries for Illmina seqencing systems that reqire balanced index combinations. Tips and Techniqes Unless a safe stopping point is specified in the protocol, proceed immediately to the next step. Avoiding Cross-Contamination When adding or transferring samples, change tips between each sample. When adding adapters or primers, change tips between each row and each colmn. Remove nsed index adapter tbes from the working area. Sealing the Plate Always seal the 96-well plate before the following steps in the protocol: Shaking steps Vortexing steps Centrifge steps Thermal cycling steps 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 and easy to ct when sing fewer than 96 wells. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 3

9 Nextera XT DNA Library Prep 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. Handling Beads Pipette bead sspension 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 magnet for the plate. Dispense liqid so that beads on the side of the wells are wetted. Keep the plate on the magnet ntil the instrctions specify to remove it. Do not agitate the plate while on the magnetic stand. Do not distrb the bead pellet. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 4

10 Nextera XT DNA Library Prep Reference Gide Library Prep Workflow The following diagram illstrates the workflow sing a Nextera XT DNA Library Prep Kit for eight samples. Safe stopping points are marked between steps. Figre 1 Nextera XT Workflow Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 5

11 Nextera XT DNA Library Prep Reference Gide Tagment Genomic DNA This step ses the Nextera transposome to tagment gdna, which is a process that fragments DNA and then tags the DNA with adapter seqences in a single step. Consmables ATM (Amplicon Tagment Mix) TD (Tagment DNA Bffer) NT (Netralize Tagment Bffer) gdna (0.2 ng/µl per sample) Hard-Shell 96-well PCR plate, skirted Microseal 'B' adhesive seals Preparation 1 Prepare the following consmables: Item Storage Instrctions gdna -25 C to -15 C Thaw on ice. Invert the thawed tbes 3 5 times, and then centrifge briefly. ATM -25 C to -15 C Thaw on ice. Invert the thawed tbes 3 5 times, and then centrifge briefly. TD -25 C to -15 C Thaw on ice. Invert the thawed tbes 3 5 times, and then centrifge briefly. NT 15 C to 30 C Check for precipitates. If present, vortex ntil all particlates are resspended. 2 Save the following tagmentation program on the thermal cycler: Choose the preheat lid option 55 C for 5 mintes Hold at 10 C Procedre 1 Add the following volmes in the order listed to each well of a new Hard-Shell skirted PCR plate. Pipette to mix. TD (10 µl) Normalized gdna (5 µl) 2 Add 5 µl ATM to each well. Pipette to mix. 3 Centrifge at 280 g at 20 C for 1 minte. 4 Place on the preprogrammed thermal cycler and rn the tagmentation program. When the sample reaches 10 C, immediately proceed to step 5 becase the transposome is still active. 5 Add 5 µl NT to each well. Pipette to mix. 6 Centrifge at 280 g at 20 C for 1 minte. 7 Incbate at room temperatre for 5 mintes. The PCR plate contains 25 µl tagmented and netralized gdna, all of which is sed in the next step. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 6

12 Nextera XT DNA Library Prep Reference Gide Amplify Libraries This step amplifies the tagmented DNA sing a limited-cycle PCR program. PCR adds the Index 1 (i7), Index 2 (i5), and fll adapter seqences to the tagmented DNA from the previos step. The index primers and Nextera PCR Master Mix are added directly to the 25 μl of tagmented gdna from the previos step. The adapters and seqences are reqired for clster formation. Use the fll amont of recommended inpt DNA and the specified nmber of PCR cycles, which helps ensre high-qality seqencing reslts. When planning the index scheme for libraries, se the same index Index 1 (i7) index in each colmn of the PCR plate. This scheme allows se of a mltichannel pipette to transfer indexes from the tbes to the plate. See Additional Resorces on page 2 for information on accessing the tech note on low-plex pooling. Consmables NPM (Nextera PCR Master Mix) Index 1 primers (N7XX) Index 2 primers (S5XX) TrSeq Index Plate Fixtre Microseal 'A' film Preparation 1 Prepare the following consmables: Item Storage Instrctions Index adapters (i5 and i7) -25 C to -15 C Only prepare adapters being sed. Thaw at room temperatre for 20 mintes. Invert each tbe to mix. Centrifge briefly. NPM -25 C to -15 C Thaw on ice for 20 mintes. 2 Save the following program on the thermal cycler: Choose the preheat lid option. 72 C for 3 mintes 95 C for 30 seconds 12 cycles of: 95 C for 10 seconds 55 C for 30 seconds 72 C for 30 seconds 72 C for 5 mintes Hold at 10 C Procedre 1 [24 libraries] Arrange the index primers in the TrSeq Index Plate Fixtre as follows. Arrange Index 1 (i7) adapters in colmns 1 6 of the TrSeq Index Plate Fixtre. Arrange Index 2 (i5) adapter in rows A D of the TrSeq Index Plate Fixtre. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 7

13 Nextera XT DNA Library Prep Reference Gide Figre 2 Setp of TrSeq Index Plate Fixtre for 24 Libraries A B C D Rows A D: Index 2(i5) adapters(white caps) Colmns 1 6: Index 1(i7) adapters(orange caps) TrSeq Index Plate Fixtre Hard-Shell PCR plate 2 [96 libraries] Arrange the index primers in the TrSeq Index Plate Fixtre as follows. Arrange Index 1 (i7) adapters in colmns 1 12 of the TrSeq Index Plate Fixtre. Arrange Index 2 (i5) adapter in rows A H of the TrSeq Index Plate Fixtre. Figre 3 Setp of TrSeq Index Plate Fixtre for 96 Libraries A B Rows A H: Index 2(i5) adapters(white caps) Colmns 1 12: Index 1(i7) adapters(orange caps) Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 8

14 Nextera XT DNA Library Prep Reference Gide C D TrSeq Index Plate Fixtre Hard-Shell PCR plate 3 Using a mltichannel pipette, add 5 µl of each Index 1 (i7) adapter down each colmn. Replace the cap on each i7 adapter tbe with a new orange cap. 4 Using a mltichannel pipette, add 5 µl of each Index 2 (i5) adapter across each row. Replace the cap on each i5 adapter tbe with a new white cap. 5 Add 15 µl NPM to each well containing index adapters. Pipette to mix. 6 Centrifge at 280 g at 20 C for 1 minte. 7 Place on the preprogrammed thermal cycler and rn the PCR program. The volme is 50 µl. 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 AMPre XP beads to prify the library DNA and remove short library fragments. Consmables RSB (Resspension Bffer) AMPre XP beads Freshly prepared 80% ethanol (EtOH) 96-well midi plate Hard-Shell 96-well PCR plate, skirted Abot Reagents The AMPre XP beads are a ser-spplied consmable. Vortex AMPre XP beads before each se. Vortex AMPre XP beads freqently to make sre that beads are evenly distribted. Always prepare fresh 80% ethanol for wash steps. Ethanol can absorb water from the air, impacting yor reslts. Preparation 1 Prepare the following consmables: Item Storage Instrctions RSB -25 C to -15 C Thaw at room temperatre. RSB can be stored at 2 C to 8 C after the initial thaw. AMPre XP Beads 2 C to 8 C Let stand on the benchtop for 30 mintes to bring to room temperatre. 2 Prepare fresh 80% ethanol from absolte ethanol. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 9

15 Nextera XT DNA Library Prep Reference Gide Procedre 1 Centrifge at 280 g at 20 C for 1 minte. 2 Transfer 50 µl PCR prodct from each well of the PCR plate to corresponding wells of a new midi plate. NOTE The ratio of PCR prodct to volme of beads is 3:2. For example, 50 µl PCR prodct to 30 µl AMPre. If yo pll less than 50 µl of PCR prodct, adjst yor ratio of AMPre beads accordingly. 3 Add 30 µl AMPre XP beads to each well. Smaller amplicons in Nextera XT library preps typically yield smaller insert size ranges. To maximize recovery of smaller fragments from the bead cleanp step, se the following conditions. Inpt Size (bp) AMPre XP Recommendation AMPre XP Volme (µl) x AMPre XP 90 > x AMPre XP (0.5x AMPre XP for 2 x 250 cycles) * 30 (25 µl for 2 x 250 cycles)* gdna or other genomic inpt 0.6x AMPre XP 30 *Applicable only to the MiSeq or HiSeq 2500 sing HiSeq Rapid v2 reagents. 4 Shake at 1800 rpm for 2 mintes. 5 Incbate at room temperatre for 5 mintes. 6 Place on a magnetic stand and wait ntil the liqid is clear (~2 mintes). 7 Remove and discard all spernatant from each well. 8 Wash two times as follows. a b c Add 200µlfresh 80% EtOHtoeach well. Incbate on the magnetic stand for 30 seconds. Remove and discard all spernatant from each well. 9 Using a 20 µl pipette, remove residal 80% EtOH from each well. 10 Air-dry on the magnetic stand for 15 mintes. 11 Remove from the magnetic stand. 12 Add 52.5 µl RSB to each well. 13 Shake at 1800 rpm for 2 mintes. 14 Incbate at room temperatre for 2 mintes. 15 Place on a magnetic stand and wait ntil the liqid is clear (~2 mintes). 16 Transfer 50 µl spernatant from the midi plate to a new Hard-Shell PCR plate. SAFE STOPPING POINT If yo are stopping, seal the plate and store at -25 C to -15 C for p to 7 days. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 10

16 Nextera XT DNA Library Prep Reference Gide Check Libraries 1 Rn 1 µl of ndilted library on an Agilent Technology 2100 Bioanalyzer sing a High Sensitivity DNA chip. The following figre shows example traces of libraries sccessflly seqenced on a HiSeq 2500 system. Typical libraries show a broad size distribtion of ~ bp, as shown in the top panel. Varios libraries can be seqenced with average fragment sizes as small as 250 bp or as large as 1500 bp. Figre 4 Library Size Distribtions of Control gdna Normalize Libraries This process normalizes the qantity of each library to ensre more eqal library representation in the pooled library. NOTE Manally normalize libraries when the final library yield is less than nm. Bead-based normalization on low yield libraries can reslt in overly dilted samples and low seqencing yields. For more information, see Best Practices for Standard and Bead-Based Normalization in Nextera XT DNA Library Preparation Kits (Pb. No ). Consmables LNA1 (Library Normalization Additives 1) LNB1 (Library Normalization Beads 1) LNW1 (Library Normalization Wash 1) LNS1 (Library Normalization Storage Bffer 1) 0.1 N NaOH (fewer than 7 days old) (3 ml per 96 samples) 96-well midi plate Hard-Shell 96-well PCR plate, skirted 15 ml conical tbe Microseal 'B' adhesive seals Abot Reagents Vortex LNA1 vigorosly to make sre that all precipitates have dissolved. Inspect in front of a light. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 11

17 Nextera XT DNA Library Prep Reference Gide Vortex LNB1 vigorosly, with intermittent inversion (at least 1 minte). Repeat ntil all beads are resspended and no beads are present at the bottom of the tbe when it is inverted. Always se a wide-bore pipette tip for LNA1. Mix only the reqired amonts of LNA1 and LNB1 for the crrent experiment. Store the remaining LNA1 and LNB1 separately at the recommended temperatres. Aspirate and dispense beads slowly de to the viscosity of the soltion. 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. Preparation 1 Prepare the following consmables: Item Storage Instrctions LNA1-25 C to -15 C Prepare nder a fme hood. Bring to room temperatre. Use a 20 C to 25 C water bath as needed. LNB1 2 C to 8 C Bring to room temperatre. Use a 20 C to 25 C water bath as needed. LNW1 2 C to 8 C Bring to room temperatre. Use a 20 C to 25 C water bath as needed. LNS1 Room temperatre Bring to room temperatre. Procedre 1 Transfer 20 µl spernatant from the Hard-Shell PCR plate to a new midi plate. 2 Add 44 µl LNA1 per sample to a new 15 ml conical tbe. Calclate abot 5% extra sample to accont for sample loss de to pipetting. For example: for 96 samples, add 4.4 ml LNA1 to the tbe (100 samples 44 µl = 4.4 ml). 3 Thoroghly resspend LNB1. Pipette to mix. 4 Transfer 8 µl LNB1 per sample (inclding the 5% extra) to the 15 ml conical tbe containing LNA1. Invert to mix. For example: for 96 samples, transfer 800 µl LNB1 to the tbe of LNA1 (100 samples 8 µl = 800 µl). 5 Por the bead mixtre into a trogh. 6 Add 45 µl combined LNA1 and LNB1 to each well containing libraries. 7 Shake at 1800 rpm for 30 mintes. 8 Place on a magnetic stand and wait ntil the liqid is clear (~2 mintes). 9 Remove and discard all spernatant from each well. 10 Wash two times as follows. a b Add 45 µllnw1toeach well. Shake at 1800rpm for 5mintes. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 12

18 Nextera XT DNA Library Prep Reference Gide c d Place on a magnetic stand and wait ntilthe liqid isclear (~2mintes). Remove and discard all spernatant from each well. 11 Add 30 µl 0.1 N NaOH to each well. 12 Shake at 1800 rpm for 5 mintes. 13 Dring the 5 minte eltion, label a new 96-well PCR plate SGP for storage plate. 14 Add 30 µl LNS1 to each well of the SGP plate. Set aside. 15 After the 5 minte eltion, make sre that all samples in the midi plate are resspended. Pipette to mix or lightly tap the plate on the bench. 16 Shake at 1800 rpm for 5 mintes. 17 Place on a magnetic stand and wait ntil the liqid is clear (~2 mintes). 18 Transfer the spernatant from the midi plate to the SGP plate. 19 Centrifge at 1000 g for 1 minte. NOTE After denatration, the libraries are single-stranded DNA, which resolves poorly on an agarose gel or Bioanalyzer chip. For qality control, se the doble-stranded DNA saved from step 16 of the cleanp procedre. SAFE STOPPING POINT If yo are stopping, seal the plate and store at -25 C to -15 C for p to 7 days. Pool Libraries Pooling libraries combines eqal volmes of normalized libraries in a single tbe. After pooling, dilte and heat-denatre the library pool before loading libraries for the seqencing rn. Consmables Adhesive PCR foil seal Eppendorf LoBind microcentrifge tbes PCR eight-tbe strip Preparation 1 To prepare for the seqencing rn, begin thawing reagents according to the instrctions for yor instrment. 2 If the SGP plate was stored frozen at -25 C to -15 C, thaw at room temperatre. Pipette to mix. Procedre 1 Centrifge at 1000 g at 20 C for 1 minte. 2 Label a new Eppendorf tbe PAL. 3 Transfer 5 µl of each library from the SGP plate to the PAL tbe. Invert to mix. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 13

19 Nextera XT DNA Library Prep Reference Gide 4 Dilte pooled libraries to the loading concentration for yor seqencing system. For instrctions, see the denatre and dilte libraries gide for yor system. 5 Store nsed pooled libraries in the PAL tbe and SGP plate at -25 C to -15 C for p to 7 days. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 14

20 Appendix A Spporting Information Spporting Information Introdction 15 How the Nextera XT Assay Works 15 Nextera XT Qality Metrics 16 Acronyms 17 Nextera XT DNA Library Prep Kit 17 Consmables and Eqipment 20 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. How the Nextera XT Assay Works The Nextera XT DNA Library Prep Kit ses an engineered transposome to tagment genomic DNA, which is a process that fragments DNA and then tags the DNA with adapter seqences in one step. Limited-cycle PCR ses the adapters to amplify the insert DNA. The PCR step also adds index adapter seqences on both ends of the DNA, which enables dal-indexed seqencing of pooled libraries on Illmina seqencing platforms. A B C Nextera XT transposome with adapters combined with template DNA Tagmentation to fragment and add adapters Limited-cycle PCR to add index adapter seqences Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 15

21 Nextera XT DNA Library Prep Reference Gide Nextera XT Qality Metrics Two factors can case clster density flctations in libraries prepared with the Nextera XT DNA Library Prep Kit: An average sample size that is too large or too small after tagmentation. A final sample concentration that is too low de to a low yield when starting the bead-based normalization step. To trobleshoot flctations in clster density, consider checking library size and library concentration. For more information, see Nextera XT Library Prep: Tips and Trobleshooting (Pb. No ). Check Library Size Larger molecles clster less efficiently than smaller molecles. If the fragment size after tagmentation is larger than expected, low clster nmbers are possible. The inverse is also tre. The average expected library size after tagmentation is between 400 bp and 1.2 kb. Check the library size with a high sensitivity Bioanalyzer trace after the PCR cleanp step. Look for a long low platea. Alternatively, PCR-amplify the library with qpcr primers and rn the prodct on an agarose gel. The seqence for these primers is available in the Seqencing Library qpcr Qantification Gide (docment # ). Short libraries indicate too little inpt DNA Reqantify the inpt DNA with a florometric method. Start with 10% 25% more inpt DNA. If the library peak is below 400 bp and yo want to contine with this library, dilte the library frther. Long libraries indicate too mch inpt DNA or the presence of inhibitors Start with less inpt DNA, make sre that the inpt DNA is free from inhibitors, and repeat the qantification step. For more information on library diltion, see the denatre and dilte libraries gide for yor seqencing system. Check Library Concentration Bead-based normalization is most efficient when the library yield after amplification is nm, or higher. Measre library concentration sing high sensitivity dsdna Qbit after library cleanp, and measre library size with a Bioanalyzer to calclate molarity. If yo are starting with high-qality DNA and see low yield after library cleanp, there are possible isses with AMPre cleanp or the amplification step. If reslts show either condition, confirm proper storage of the PCR master mix at -25 C to -15 C in a no-frost freezer. Confirm minimal freeze-thaw cycles. The following resorces are available on the Illmina website: Best practices for bead handling From the Nextera XT DNA Library Prep Kit spport page, select the Best Practices tab and review Handling Magnetic Beads. Online training modle Review section 2.4 of the TrSeq: Sample Prification Bead Size Selection and Best Practices, which is a short training with gidance on bead handling. To access this training, select the Training tab on the Nextera XT DNA Library Prep Kit spport page. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 16

22 Nextera XT DNA Library Prep Reference Gide Acronyms Acronym Definition ATM Amplicon Tagment Mix CAA Clean Amplified Plate CAN Clean Amplified NTA Plate LNA1 Library Normalization Additives 1 LNB1 Library Normalization Beads 1 LNS1 Library Normalization Storage Bffer 1 LNW1 Library Normalization Wash 1 LNP Library Normalization Plate NT Netralize Tagment Bffer NPM Nextera PCR Master Mix NTA Nextera XT Tagment Amplicon Plate PAL Pooled Amplicon Library RSB Resspension Bffer SGP Storage Plate TD Tagment DNA Bffer Nextera XT DNA Library Prep Kit The Nextera XT DNA Library Prep Kit is packaged in either 96-index (384 samples) or 24-index (96 samples) boxes, and shipped on dry ice nless otherwise specified. Certain kit components are stored at a temperatre that differs from the shipping temperatre. Make sre that yo store kit components at the specified storage temperatres. Each kit has a corresponding index kit that contains 96 indexes or 24 indexes. Combining Nextera XT Index Kit v2 Sets A D achieves 384 index combinations. Seqencing primers provided in TrSeq v3 reagent kits are not compatible with Nextera XT libraries. Ths, seqencing Nextera XT libraries on a HiSeq 2500 sing TrSeq v3 reagents reqires the seqencing primers provided in the Illmina TrSeq Dal Index Seqencing Primer Box. This box is provided in a paired-end (PE ) and single-read (FC ) version. One box is needed for each rn. Nextera XT DNA Library Prep Kit - 96 Indexes Consmable Catalog # Nextera XT DNA Library Prep Kit Nextera XT Index Kit (96 Indexes, 384 Samples) Nextera XT Index Kit v2 Set A (96 Indexes, 384 Samples)* Nextera XT Index Kit v2 Set B (96 Indexes, 384 Samples)* Nextera XT Index Kit v2 Set C (96 Indexes, 384 Samples)* Nextera XT Index Kit v2 Set D (96 Indexes, 384 Samples)* TrSeq Index Plate Fixtre Kit FC FC FC FC FC FC FC * Combining Sets A D achieves 384 index combinations. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 17

23 Nextera XT DNA Library Prep Reference Gide Nextera XT DNA Library Prep Kit, 96 Indexes (FC ) 96-Index Kit Contents, Box 1 Qantity Acronym Reagent Name Storage Temperatre 1 ATM Amplicon Tagment Mix, 96 rxn -25 C to -15 C 2 TD Tagment DNA Bffer -25 C to -15 C 1 NPM Nextera PCR Master Mix -25 C to -15 C 4 RSB Resspension Bffer -25 C to -15 C 1 LNA1 Library Normalization Additives 1-25 C to -15 C 2 LNW1 Library Normalization Wash 1 2 C to 8 C 1 HT1 Hybridization Bffer -25 C to -15 C 96-Index Kit Contents, Box 2 Qantity Acronym Reagent Name Storage Temperatre 1 NT Netralize Tagment Bffer Room temperatre 1 LNB1 Library Normalization Beads 1 2 C to 8 C 1 LNS1 Library Normalization Storage Bffer 1 Room temperatre Index Kit (96 Indexes) (FC ) Qantity Reagent Name Storage Temperatre 8 tbes Index Primers, S502 to S508, and S C to -15 C 12 tbes Index Primers, N701 to N C to -15 C Index Kit v2 Set A (FC ) Qantity Reagent Name Storage Temperatre 8 tbes Index Primers, S502, S503, S505 to S508, S510, and S C to -15 C 12 tbes Index Primers, N701 to N707, N710 to N712, N714, and N C to -15 C Index Kit v2 Set B (FC ) Qantity Reagent Name Storage Temperatre 8 tbes Index Primers, S502, S503, S505 to S508, S510, and S C to -15 C 12 tbes Index Primers, N716, N718 to N724, and N726 to N C to -15 C Index Kit v2 Set C (FC ) Qantity Reagent Name Storage Temperatre 8 tbes Index Primers, S513, S515 to S518, and S520 to S C to -15 C 12 tbes Index Primers, N701 to N707, N710 to N712, N714, and N C to -15 C Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 18

24 Nextera XT DNA Library Prep Reference Gide Index Kit v2 Set D (FC ) Qantity Reagent Name Storage Temperatre 8 tbes Index Primers, S513, S515 to S518, and S520 to S C to -15 C 12 tbes Index Primers, N716, N718 to N724, and N726 to N C to -15 C Nextera XT DNA Library Prep Kit - 24 Indexes Consmable Catalog # Nextera XT DNA Library Prep Kit Nextera XT Index Kit (24 Indexes, 96 Samples) TrSeq Index Plate Fixtre Kit FC FC FC Nextera XT DNA Library Prep Kit, 24 Indexes (FC ) 24-Index Kit Contents, Box 1 Qantity Acronym Reagent Name Storage Temperatre 1 ATM Amplicon Tagment Mix, 24 rxn -25 C to -15 C 1 TD Tagment DNA Bffer -25 C to -15 C 1 NPM Nextera PCR Master Mix -25 C to -15 C 1 RSB Resspension 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 HT1 Hybridization Bffer -25 C to -15 C 24-Index Kit Contents, Box 2 Qantity Acronym Reagent Name Storage Temperatre 1 NT Netralize Tagment Bffer Room temperatre 1 LNB1 Library Normalization Beads 1 2 C to 8 C 1 LNS1 Library Normalization Storage Bffer 1 Room temperatre Index Kit (24 Indexes) (FC ) Qantity Reagent Name Storage Temperatre 4 tbes Index Primers, S502 to S504, and S C to -15 C 6 tbes Index Primers, N701 to N C to -15 C TrSeq Index Plate Fixtre Kit Each kit contains two index plate fixtres to help arrange index primers before dispensing to the 96-well plate dring library amplification. The Index Plate Fixtre can be sed with both the 24- sample kit and 96-sample kit. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 19

25 Nextera XT DNA Library Prep Reference Gide Consmable Catalog # TrSeq Index Plate Fixtre Kit FC 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. Consmables Consmable Spplier 10 µl pipette tips General lab spplier 10 µl mltichannel pipettes General lab spplier 10 µl single channel pipettes General lab spplier 1000 µl pipette tips General lab spplier 1000 µl mltichannel pipettes General lab spplier 1000 µl single channel pipettes General lab spplier 200 µl pipette tips General lab spplier 200 µl mltichannel pipettes General lab spplier 200 µl single channel pipettes General lab spplier 96-well storage plates, rond well, 0.8 ml (midi plate) Agencort AMPre XP, 60 ml kit or 5 ml kit Distilled water Ethanol 200 proof (absolte) for moleclar biology (500 ml) Microseal 'A' film Microseal 'B' adhesive seals NaOH 1 N, ph > 12.5, moleclar biology grade RNase/DNase-free mltichannel reagent reservoirs, disposable Ultrapre water Hard-Shell 96-well PCR plates Eqipment Fisher Scientific, catalog # AB-0859 Beckman Colter Genomics, item # A63881 (60 ml) Beckman Colter Genomics, item # A63880 (5 ml) General lab spplier Sigma-Aldrich, prodct # E7023 Bio-Rad, catalog # MSA-5001 Bio-Rad, catalog # MSB-1001 General lab spplier VWR, catalog # General lab spplier Bio-Rad, catalog # HSP-9601 Eqipment Spplier High-Speed microplate shaker VWR, catalog # (110 V/120 V) VWR, catalog # (230 V) Magnetic stand-96 Microplate centrifge Vortexer Thermo Fisher Scientific, catalog # AM10027 General lab spplier General lab spplier Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 20

26 Nextera XT DNA Library Prep Reference Gide Thermal Cyclers Use the following recommended settings for selected thermal cycler models. Before performing library prep, validate any thermal cyclers not listed. Thermal Cycler Temp Mode Lid Temp Vessel Type Bio-Rad DNA Engine Tetrad 2 Calclated Heated, Constant at 100 C MJ Research DNA Engine Tetrad Eppendorf Mastercycler Pro S Calclated Heated Plate Gradient S, Simlated Tbe Heated Polypropylene plates and tbes Plate Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 21

27 Technical Assistance For technical assistance, contact Illmina Technical Spport. Website: Illmina Cstomer Spport Telephone Nmbers Region Toll Free Regional North America Astralia Astria Belgim China Denmark Finland France Germany Hong Kong Ireland Italy Japan Netherlands New Zealand Norway Singapore Spain Sweden Switzerland Taiwan United Kingdom Other contries Safety data sheets (SDSs) Available on the Illmina website at spport.illmina.com/sds.html. Prodct docmentation Available for download in PDF from the Illmina website. Go to spport.illmina.com, select a prodct, then select Docmentation & Literatre. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 22

28 Docment # v02 Illmina 5200 Illmina Way San Diego, California U.S.A ILMN (4566) (otside North America) techspport@illmina.com For Research Use Only. Not for se in diagnostic procedres Illmina, Inc. All rights reserved.