Nextera DNA Flex Library Prep

Transcription

1 Nextera DNA Flex Library Prep Reference Gide Docment # v03 October 2018 For Research Use Only. Not for se in diagnostic procedres. ILLUMINA PROPRIETARY

2 Nextera DNA Flex 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, AND WILL VOID ANY WARRANTY APPLICABLE TO THE PRODUCT(S). 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. All trademarks are the property of Illmina, Inc. or their respective owners. For specific trademark information, see Docment # v03 For Research Use Only. Not for se in diagnostic procedres. ii

3 Nextera DNA Flex Library Prep Reference Gide Revision History Docment Date Description of Change Docment # v03 Docment # v02 Docment # v01 Docment # v00 October 2018 Jne 2018 April 2018 October 2017 Updated Index Adapter terminology. Updated to inclde IDT for Illmina -Nextera DNA UD Indexes Set A (96 Indexes, 96 Samples). Updated dilting to starting concentration information. Added clarification in regards to ordering index adapters. Added additional resorce information for Uniqe Dal Indexes. Added catalog nmber information for IDT for Illmina -Nextera DNA UD Indexes Set A (96 Indexes, 96 Samples) and Axygen 1.7 ml MaxyClear Snaplock Microcentrifge Tbes. Updated storage information for Lysis Reagent Kit. Clarified PCR Amplicons information. Clarified instrctions when safe stopping is an option. Moved recommended read lengths for each system to the spport site. Moved blood and lysis consmables to their own table. Revised step-by-step instrctions to be more sccinct. Reorganized the following content to improve continity: Rearranged DNA inpt recommendations. Moved information on blood and saliva lysis preparation and procedres. Added information abot PCR Amplicons. Replaced references to the Nextera DNA Flex Pooling Gide (docment # ) with the Index Adapters Pooling Gide (docment # ). Pooling information is consolidated into the Index Adapters Pooling Gide. Initial release. Docment # v03 For Research Use Only. Not for se in diagnostic procedres. iii

4 Table of Contents Chapter 1 Overview 1 Introdction 1 DNA Inpt Recommendations 1 Blood and Saliva Inpt Recommendations 2 Sample Inpt Recommendations 2 PCR Amplicons 3 Additional Resorces 3 Chapter 2 Protocol 4 Introdction 4 Nextera DNA Flex Library Prep Workflow 5 Tips and Techniqes 6 Tagment Genomic DNA 8 Post Tagmentation Cleanp 9 Amplify Tagmented DNA 10 Clean Up Libraries 12 Pool Libraries 14 Check Library Qality (Optional) 14 Dilte Libraries to the Starting Concentration 16 Chapter 3 Seqencing 18 Appendix 3 Spplemental Procedres 19 Introdction 19 Blood Lysis (Optional) 20 Saliva Lysis (Optional) 22 Appendix A Spporting Information 24 Introdction 24 How the Nextera DNA Flex Assay Works 25 Prodct Contents 26 Consmables and Eqipment 29 Acronyms 30 Technical Assistance 31 Docment # v03 For Research Use Only. Not for se in diagnostic procedres. iv

5 Chapter 1 Overview Introdction 1 DNA Inpt Recommendations 1 Blood and Saliva Inpt Recommendations 2 Sample Inpt Recommendations 2 PCR Amplicons 3 Additional Resorces 3 Introdction This protocol explains how to prepare p to 96 indexed paired-end libraries from genomic DNA for sbseqent seqencing on an Illmina seqencing system. The Nextera DNA Flex Library Prep workflow: Uses an enzymatic reaction, called tagmentation, to fragment DNA and add adapter seqences in only 15 mintes Innovates sample normalization at inpts > 100 ng Streamlines sample pooling and seqencing Redces excessive pipetting and overall hands-on time, while optimizing se of consmables by sing master mix reagents Generates libraries from as little as 1 ng inpt Prepares libraries directly from blood or saliva samples DNA Inpt Recommendations The Nextera DNA Flex Library Prep protocol is compatible with DNA inpts ranging from ng, or higher. For hman DNA samples and other large complex genomes, the recommended minimm DNA inpt is between ng. For small genomes, the DNA inpt amont can be redced to as low as 1 ng (modifying the PCR cycling conditions accordingly). Protocols specific to blood and saliva are inclded in the section of this gide. The blood protocol reqires the Flex Lysis Reagent Kit, which is not provided with the Nextera DNA Flex Library Prep Kit. This kit is sold separately refer to Illmina catalog # DNA Inpt < 100 ng If yo are sing less than 100 ng DNA inpt, the qantification of the initial DNA sample to determine the nmber of PCR cycles reqired is recommended. In this case, becase final libraries yields from low inpts are not normalized by this library prep protocol, qantification and normalization of libraries before seqencing is recommended. DNA Inpt ng For DNA inpts between ng, accrate qantification of the initial DNA sample is not reqired, and normalization of the final yield is expected. DNA Inpt Qantification When inpt is < 100 ng, se a florometric-based method to qantify DNA inpt. Avoid methods that measre total ncleic acid, sch as NanoDrop or other UV absorbance methods. Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 1

6 Nextera DNA Flex Library Prep Reference Gide If yo se the Qbit dsdna BR/HS Assay Kit, se 2 µl DNA sample with 198 µl Qbit Working Soltion. Assess DNA Qality UV absorbance is a common method sed for assessing the qality of a DNA sample. The ratio of absorbance at 260 nm to absorbance at 280 nm provides an indication of sample prity. This protocol is optimized for DNA with 260/280 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 that interfere with tagmentation and adversely impacts the final library yield. For a complete list of contaminants, inclding sorces, avoidance, and effects on the library preparation, see the Nextera XT Trobleshooting Technical Note. CAUTION Incomplete tagmentation cased by contaminants can reslt in library preparation failre, poor clstering, or low qality seqencing reslts. Blood and Saliva Inpt Recommendations The Nextera DNA Flex protocol is compatible with fresh whole blood (reqires the Flex Lysis Reagent Kit) and saliva sample inpts. For information abot protocols specific to blood and saliva, see Blood Lysis (Optional) on page 20 or Saliva Lysis (Optional) on page 22. When starting with 10 µl of liqid whole blood in EDTA tbes or 30 µl of saliva in Oragene tbes, expect normalization of libraries eqal to that observed when sing 100 ng gdna inpt. Blood and saliva are heterogeneos sample types, therefore the ability of Nextera DNA Flex to generate normalized libraries depends on the total amont of DNA obtained from the lysed sample. The following factors can adversely affect normalization of library independent of kit performance: Viscosity of the saliva samples Blood sample age Storage conditions Underlying medical conditions affecting white blood cell conts Sample Inpt Recommendations The Nextera DNA Flex workflow is compatible with blood and saliva samples when sing the following: Illmina Blood Lysis Protocol (blood) with Flex Lysis Reagent Kit Illmina Saliva Lysis Protocol (saliva) The recommended PCR cycles for the BLT PCR program are adjsted based on sample inpt concentration and qality. For more information, see Amplify Tagmented DNA on page 10. Table 1 DNA Inpt Recommendations Total DNA Inpt (ng) Qantification of Inpt DNA Recommended Normalized Library Yield Yes No Yes Blood/Saliva No Yes No Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 2

7 Nextera DNA Flex Library Prep Reference Gide PCR Amplicons The PCR amplicons mst be > 150 bp. Sample bead prification cleans p amplicons greater than 500 bp. Therefore, Illmina recommends that amplicons < 500 bp ndergo a 1.8 x sample prification bead volme ratio to spernatant dring Clean Up Libraries on page 12. 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 The Nextera DNA Flex Library Prep spport page on the Illmina website provide additional resorces. These resorces inclde software, training, compatible prodcts, best practices, and the following docmentation. Always check the spport pages for the latest versions. Resorce Cstom Protocol Selector Index Adapter Pooling Gide (docment # ) Nextera DNA Flex Library Prep Checklist (docment # ) Nextera DNA Flex Library Prep Consmables and Eqipment List (docment # ) Uniqe Dal Index Spport page Illmina Free Adapter Blocking Reagent (docment # ) Illmina Adapter Seqences (docment # ) Illmina Experiment Manager Gide (docment # ) Off-Instrment Analysis Gide Analysis Software Gide Description 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 pooling gidelines and dal indexing strategies for sing the Nextera DNA Flex Library Prep kit. Provides a checklist of the protocol steps. The checklist is intended for experienced sers. Provides an interactive checklist of ser-provided consmables and eqipment. Provides additional resorces and information abot Uniqe Dal Indexes and how to set p yor instrment for seqencing rns with regard to UDIs. Provides protocol to block excess free adapter, minimize potential index hopping levels, and enhance data qality. Provides the ncleotide seqences that comprise Illmina oligoncleotides sed in Illmina seqencing technologies. Provides information abot creating and editing appropriate sample sheets for Illmina seqencing systems and analysis software and record parameters for yor sample plate. Provides an overview of the Local Rn Manager software, instrctions for sing software featres, and instrctions for installing analysis modles on the instrment compter. Information abot the BaseSpace seqencing data analysis tool that also enables yo to organize samples, libraries, pools, and seqencing rns in a single environment. Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 3

8 Chapter 2 Protocol Introdction 4 Nextera DNA Flex Library Prep Workflow 5 Tips and Techniqes 6 Tagment Genomic DNA 8 Post Tagmentation Cleanp 9 Amplify Tagmented DNA 10 Clean Up Libraries 12 Pool Libraries 14 Check Library Qality (Optional) 14 Dilte Libraries to the Starting Concentration 16 Introdction This chapter describes the Nextera DNA Flex Library Prep protocol. Review Best Practices before proceeding. See Additional Resorces on page 3 for information on how to access Nextera DNA Flex Library Prep 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 24. 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 DNA Flex Library Prep spport page. Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 4

9 Nextera DNA Flex Library Prep Reference Gide Nextera DNA Flex Library Prep Workflow Figre 1 Nextera DNA Flex Library Prep Workflow Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 5

10 Nextera DNA Flex Library Prep Reference Gide 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 or reagent master mixes, change tips between each sample. When adding index adapters with a mltichannel pipette, change tips between each row or each colmn. If sing a single channel pipette, change tips between each sample. If sing index adapter tbes, only open one index adapter at a time to prevent misplacing caps. Remove nsed index adapter tbes from the working area. Preparing Nextera Uniqe Dal (UD) Indexes Plate Each index well is for single se only. Prepare Nextera UD Indexes plate as follows. Centrifge at 1000 x g for 1 minte to settle liqid away from the seal. If processing < 96 samples, pierce the foil seal on the index adapter plate with a new pipette tip for each well for only the nmber of samples being processed. If processing 96 samples, align a new Eppendorf 96-well PCR plate above the index adapter plate and press down to pnctre the foil seal on all 96 wells. Discard the empty Eppendorf plate sed to pnctre the foil seal. Sealing the Plate Always seal the 96-well plate with the adhesive seal sing a rbber roller to cover the plate before the following steps in the protocol: Shaking steps Thermal cycling steps Microseal 'B' adhesive seals are effective at -40 C to 110 C, and sitable for skirted or semiskirted PCR plates. Microseal 'B' seals can be sed for thermal cycling or short-term storage. Microseal F adhesive foils are effective at temperatres down to -70 C and are recommended for longterm storage of the 96-well plates containing the final libraries. Handling Bead-Linked Transposomes (BLT) Store the BLT stock tbe pright in the refrigerator so that the beads are always sbmerged in the bffer. Vortex the BLT stock tbe thoroghly ntil the beads are resspended. To avoid resettling the beads, centrifgation before pipetting is not recommended. If beads are adhered to the side or top of a 96-well plate, centrifge at 280 g for 3 seconds, and then pipette to resspend. When washing beads: Use the appropriate magnetic stand for the plate. Keep the plate on the magnetic stand ntil the instrctions specify to remove it. Do not agitate the plate while it is on the magnetic stand. Do not distrb the bead pellet. Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 6

11 Nextera DNA Flex Library Prep Reference Gide If beads are aspirated into pipette tips, dispense back into the plate on the magnetic stand and wait ntil the soltion is clear (~2 mintes). Dispense tagment wash bffer (TWB) directly onto the beads. If liqid becomes adhered to the side or top of the tbe or well, centrifge at 280 g for 3 seconds to pll volme into soltion. Handling Tagment Wash Bffer (TWB) Pipette slowly to minimize foaming. Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 7

12 Nextera DNA Flex Library Prep Reference Gide Tagment Genomic DNA This step ses the Bead-Linked Transposomes (BLT) to tagment DNA. This process fragments and tags the DNA with adapter seqences. Consmables BLT TB1 (Tagmentation Bffer 1) Nclease-free water 96-well PCR plate Microseal 'B' adhesive seal 1.7 ml microcentrifge tbes 8-tbe strip Pipette tips 20 µl mltichannel pipettes 200 µl mltichannel pipettes Abot Reagents BLT mst be stored at temperatres above 2 C. Do not se BLT that has been stored below 2 C. Preparation 1 Prepare the following consmables: Item Storage Instrctions BLT 2 C to 8 C Bring to room temperatre. Vortex to mix. Do not centrifge before pipetting. TB1-25 C to -15 C Bring to room temperatre. Vortex to mix. 2 Save the following TAG program on the thermal cycler: Choose the preheat lid option and set to 100 C Set the reaction volme to 50 µl 55 C for 15 mintes Hold at 10 C Procedre 1 Add 2 30 µl DNA to each well of a 96-well PCR plate so that the total inpt amont (ng) is within the desired range. See DNA Inpt Recommendations on page 1 for information on inpt and PCR range options. 2 If DNA volme < 30 µl, add nclease-free water to the DNA samples to bring the total volme to 30 µl. 3 Vortex BLT vigorosly for 10 seconds to resspend. Repeat as necessary. 4 Combine the following volmes to prepare tagmentation master mix. The volme reqired per reaction is: BLT (11 µl) TB1 (11 µl) Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 8

13 Nextera DNA Flex Library Prep Reference Gide 5 Vortex the tagmentation master mix thoroghly to resspend. 6 Divide the tagmentation master mix volme eqally into an 8-tbe strip. 7 Using fresh tips with a 200 µl mltichannel pipette, transfer 20 μl tagmentation master mix to each well of the plate containing a sample. Pipette to resspend. Discard tips after each dispense. Fresh tips are reqired to avoid contamination across wells. Discard the 8-tbe strip once the tagmentation master mix has been dispensed. 8 Seal the plate with Microseal 'B', place on the preprogrammed thermal cycler, and rn the TAG program. Post Tagmentation Cleanp This step washes the adapter-tagged DNA on the BLT before PCR amplification. Consmables TSB (Tagment Stop Bffer) TWB (Tagment Wash Bffer) 96-well plate magnet Microseal 'B' adhesive seal Pipette tips 20 µl mltichannel pipettes 200 µl mltichannel pipettes Preparation 1 Prepare the following consmables: Item Storage Instrctions TSB 15 C to 30 C If precipitates are observed, heat at 37 C for 10 mintes, and then vortex ntil precipitates are dissolved. Use at room temperatre. TWB 15 C to 30 C Use at room temperatre. Procedre 1 Add 10 µl TSB to the tagmentation reaction. 2 Slowly pipette the entire volme to resspend the beads. 3 Seal the plate and incbate at 37 C for 15 mintes on a thermal cycler with heated lid. Set the thermal cycler lid at 100 C Set the reaction volme to 60 µl Hold at 10 C 4 Place the plate on the magnetic stand and wait ntil soltion is clear (~3 mintes). 5 Using a mltichannel pipette, remove and discard spernatant. 6 Wash two times as follows: NOTE Minimize the potential of TWB foaming dring the tagmentation wash by sing a deliberately slow pipetting techniqe to avoid incorrect volme aspiration and incomplete mixing. Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 9

14 Nextera DNA Flex Library Prep Reference Gide a b c d Remove the sample plate from the magnetic stand and add 100 µl TWB directly onto the beads. Pipette slowly ntil beads are flly resspended. Place the plate on the magnetic stand and wait ntil the soltion is clear (~3 mintes). Using a mltichannel pipette, remove and discard spernatant. 7 Remove the plate from the magnetic stand and add 100 μl TWB. 8 Pipette each sample well slowly to resspend the beads. 9 Seal the plate and place on the magnetic stand ntil the soltion is clear (~3 mintes). Keep on the magnetic stand ntil step 3 in Amplify Tagmented DNA on page 10. The TWB remains in the wells to prevent overdrying of the beads. Amplify Tagmented DNA This step amplifies the tagmented DNA sing a limited-cycle PCR program. The PCR step adds Index 1 (i7) adapters, Index 2 (i5) adapters, and seqences reqired for seqencing clster generation. The index adapter plate is ordered separately from the library prep components. For a list of compatible index adapters for se with this protocol, see Index Kit Contents on page 27. Consmables EPM (Enhanced PCR Mix) Index adapters (tbes or plate) Nclease-free water Microseal 'B' adhesive seal 1.7 ml microcentrifge tbes Pipette tips 20 µl mltichannel pipettes 200 µl mltichannel pipettes Preparation 1 Prepare the following consmables: Item Storage Instrctions EPM -25 to-15 C Thaw on ice. Invert to mix, then briefly centrifge. Index Adapters (tbes or plate) -25 C to-15 C Thaw at room temperatre. For index tbes: Vortex to mix, then centrifge briefly. For index adapter plates: Spin briefly before se. Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 10

15 Nextera DNA Flex Library Prep Reference Gide 2 Seal the plate and incbate on a thermal cycler sing the following settings: Choose the preheat lid option and set to 100 C 68 C, 3 min 98 C, 3 min Repeat cycling conditions below for the total nmber of cycles listed in DNA Volme-Based Recommendations : 98 C, 45 sec 62 C, 30 sec 68 C, 2 min 68 C, 1 min 10 C hold Table 2 PCR Cycle Recommendations Total DNA Inpt (ng) Recommended # of PCR Cycles Blood/Saliva 5 Procedre 1 Prepare PCR master mix in a 1.7 ml microcentrifge tbe. For each reaction se: EPM (22 µl) Nclease-free water (22 µl) 2 Vortex and centrifge the PCR master mix at 280 g for 10 seconds. 3 With the plate on the magnetic stand, se a 20 µl mltichannel pipette to remove and discard spernatant. Foam that remains on the well walls does not adversely affect the library. 4 Remove the plate from the magnet. 5 Immediately add 40 µl of the PCR master mix to each sample well. 6 Immediately pipette to mix ntil the beads are thoroghly resspended. Alternatively, seal the plate and se a plate shaker at 1600 rpm for 1 minte. 7 Add the appropriate index adapters to each sample. For low-plexity conditions, refer to the Index Adapters Pooling Gide (docment # ). For tbes, open only one index adapter tbe at a time to prevent misplacing caps; alternatively, se fresh caps after opening each tbe. For plates, each well of the index plate is for a single se only. Index Kit Type Kit Configration Volme of Index Adapter per Sample 24 plex (dal index) Individal tbes 5 µl i5 adapter 5 µl i7 adapter 96 plex (dal index) 96-well plate 10 µl primer mix Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 11

16 Nextera DNA Flex Library Prep Reference Gide 8 Using a pipette set to 40 µl, pipette 10 times to mix. 9 Seal the plate with Microseal 'B', and centrifge at 280 g for 30 seconds to collect contents at the bottom of the wells. 10 Place on the thermal cycler and rn the BLT PCR program. 11 Remove the plate from the thermal cycler when the PCR program completes. 12 Centrifge at 280 g for 1 minte to collect contents at the bottom of the well. SAFE STOPPING POINT If yo are stopping, seal the plate with a Microseal 'B' adhesive seal, and store at 2 C to 8 C for p to 3 days. Clean Up Libraries This step prifies the amplified libraries throgh a doble-sided bead prification procedre. Consmables Sample Prification Beads (SPB) Freshly prepared 80% ethanol (EtOH) RSB (Resspension Bffer) 96-well 0.8 ml Polypropylene Deepwell Storage Plate (midi plate) (2) 96-well PCR plate Microseal 'B' adhesive seal Microseal 'F' foil seals 1.7 ml microcentrifge tbes Nclease-free water NOTE Use Microseal 'F' when sealing the plate for long-term storage. Use Microseal 'B' for other steps that reqire a sealed plate or long-term storage. Abot Reagents SPB Mst be at room temperatre before se Vortex before each se Vortex freqently to make sre that beads are evenly distribted Aspirate and dispense slowly de to the viscosity of the soltion Preparation 1 Prepare the following consmables: Item Storage Instrctions SPB 2 C to 8 C Let stand at room temperatre for 30 mintes. Vortex and invert to mix. RSB -25 C to -15 C Thaw and bring to room temperatre. Vortex to mix. Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 12

17 Nextera DNA Flex Library Prep Reference Gide Procedre 1 Place the plate on the magnetic stand and wait ntil the liqid is clear (~5 mintes). 2 Transfer 45 µl spernatant from each well to the corresponding well of a fresh midi plate. 3 Vortex and invert SPB mltiple times to resspend. 4 Combine the following volmes in a 1.7 ml microcentrifge tbe to create a master mix of dilted SPB. For smaller PCR amplicons refer to PCR Amplicons on page 3 for the SPB to spernatant ratio recommendation. SPB (45 µl) Nclease-free water (40 µl) 5 Thoroghly vortex the dilted SPB. 6 Pipette mix a minimm of 10 times or ntil thoroghly mixed. 7 Add 85 µl dilted SPB to each well containing spernatant. 8 Pipette 10 times to mix, or seal the plate and se a plate shaker at 1600 rpm for 1 minte. 9 Incbate the sealed plate at room temperatre for 5 mintes. 10 Place on the magnetic stand and wait ntil spernatant is clear (~5 mintes). 11 Dring incbation, thoroghly vortex the SPB (ndilted stock tbe), and then add 15 µl to each well of a new midi plate. 12 Transfer 125 µl spernatant from the first midi plate to the second midi plate (containing the 15 µl of SPB). 13 Pipette 10 times to mix. 14 Apply the seal and incbate at room temperatre for 5 mintes. 15 Place on a magnet and wait ntil clear (~5 mintes). 16 Withot distrbing the beads, remove and discard spernatant. 17 Wash two times as follows: a b c With the plate on the magnet, add 200 µl of fresh 80% ethanol withot mixing. Incbate for 30 seconds. Withot distrbing the beads, remove and discard spernatant. 18 Use a 20 µl pipette to remove and discard residal EtOH from the wells. 19 Air-dry on the magnetic stand (~5 mintes). 20 Remove from the magnetic stand and add 32 µl RSB to the beads. 21 Pipette to resspend. 22 Incbate at room temperatre for 2 mintes. 23 Place on the magnetic stand and wait ntil the liqid is clear (~ 2 mintes). 24 Transfer 30 µl spernatant to a new 96-well PCR plate. SAFE STOPPING POINT If yo are stopping, seal the plate with a Microseal 'F' foil seal and store at -25 C to -15 C for p to 30 days. Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 13

18 Nextera DNA Flex Library Prep Reference Gide Pool Libraries When the DNA inpt is ng, qantifying and normalizing individal libraries generated in the same experiment is not necessary. However, the final yield of libraries generated in separate experiments might vary slightly. To achieve optimal clster density, pool eqal library volmes and qantify the pool before seqencing. DNA Inpts of ng 1 Combine 5 l each library (p to 96 libraries) in a 1.7 ml microcentrifge tbe. 2 Vortex to mix, and then centrifge. 3 Qantify the library pool sing a dsdna florescent dye method, sch as Qbit or PicoGreen. For DNA Inpts of < 100 ng 1 Qantify each library individally sing Qbit or PicoGreen. Check Library Qality (Optional) 1 Rn 1 l library or pooled libraries on one of the following instrments: Fragment Analyzer with the HS-NGS High Sensitivity 474 kit. Agilent 2100 Bioanalyzer with a High Sensitivity DNA kit. The following figres show typical library size profiles with an average fragment size of 600 bp when analyzed with a size range of bp. Figre 2 Example Fragment Analyzer Trace Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 14

19 Nextera DNA Flex Library Prep Reference Gide Figre 3 Example Agilent 2100 Bioanalyzer Trace Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 15

20 Nextera DNA Flex Library Prep Reference Gide Dilte Libraries to the Starting Concentration This step diltes libraries to the starting concentration for yor seqencing system. After dilting to the starting concentration, libraries are ready to be denatred and dilted to the final loading concentration. For seqencing, Illmina recommends the following read lengths: Table 3 Recommended Read Length on Illmina Systems with Nextera CD Indexes (8 base pair index codes) Seqencing System NovaSeq 6000, HiSeq X, HiSeq 3000 and HiSeq 4000, NextSeq 500 and NextSeq 550, MiSeq, MiniSeq, iseq 100 Read Length 2 x 151 HiSeq 2000, HiSeq 2500 (high otpt) 2 x 126 HiSeq 2500 (rapid rn) 2 x 101 If yo are sing IDT for Illmina-Nextera DNA UD Indexes, it is important to know that they se 10 base pair index codes that differ from the Nextera DNA CD Indexes which se 8 base pair index codes. This change in base pair index codes can reqire adjstments to yor seqencing rn set p. For information on setp, see 1 Calclate the molarity vale of the library or pooled libraries sing the following formla. For libraries qalified on a Bioanalyzer, se the average size obtained for the library. For all other qalification methods, se 350 bp as the average library size. 2 Using the molarity vale, calclate the volmes of RSB and library needed to dilte libraries to the starting concentration for yor system. Seqencing System Starting Concentration (nm) Final Loading Concentration (pm) HiSeq 2500 and HiSeq 2000 (high otpt modes) HiSeq 2500 System (rapid rn mode) HiSeq 4000 and HiSeq iseq 100 System MiniSeq System MiSeq NextSeq 550 and NextSeq NovaSeq Dilte libraries sing RSB: Libraries qantified as a pool Dilte the pool to the starting concentration for yor system. Libraries qantified individally Dilte each library to the starting concentration for yor system. Add 10 µl each dilted library to a tbe to create a pool. 4 Follow the denatre and dilte instrctions for yor system, dilting to the final loading concentration listed in the preceding table. For the iseq 100 System, see the system gide for diltion instrctions (libraries are atomatically denatred). For the NovaSeq 6000, see the system gide for pool and denatre instrctions. Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 16

21 Nextera DNA Flex Library Prep Reference Gide For the HiSeq 4000 and HiSeq 3000, see the cbot 2 or cbot system gide for reagent preparation instrctions. For all other systems, see the denatre and dilte libraries gide. The final loading concentrations are a starting point and general gideline. Optimize concentrations for yor workflow and qantification method over sbseqent seqencing rns or by flow cell titration. Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 17

22 Chapter 3 Seqencing Nextera DNA Flex spports read lengths p to 2 x 151 cycles. However, if yo are sing IDT for Illmina- Nextera DNA UD Indexes, it is important to know that they se 10 base pair index codes that differ from the Nextera DNA CD Indexes which se 8 base pair index codes. This change in base pair index codes can reqire adjstments to yor seqencing rn set p. For information on setp, see For the recommended read length for each seqencer, see Prodct Compatibility on the Nextera DNA Flex Spport Page. Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 18

23 Nextera DNA Flex Library Prep Reference Gide Appendix 3 Spplemental Procedres Spplemental Procedres Introdction 19 Blood Lysis (Optional) 20 Saliva Lysis (Optional) 22 Introdction The contents of this section provide instrctions for optional procedres within the Nextera DNA Flex Library Prep workflow. Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 19

24 Blood Lysis (Optional) Use this protocol when performing the Nextera DNA Flex Library Prep workflow sing blood sample inpts with the Flex Lysis Reagent Kit. This protocol has been validated sing fresh whole blood collected in EDTA collection tbes. Store the blood at 4 C and process it within 3 days. NOTE The se of frozen blood has not been validated and therefore cannot be recommended. CAUTION Blood is a potential sorce of infectios diseases. Follow site-specific procedres to ensre the safe handling of blood samples. Dring the lysis protocol, make sre that the entire blood sample is flly lysed (brown in color following the heat incbation step) before proceeding to sbseqent steps. Consmables EDTA collection tbes (for blood sample collection) Sample Prification Beads BLB (Blood Lysis Bffer) PK1 (Proteinase K) Nclease-free water Freshly prepared 80% ethanol (EtOH) 96-well PCR plate Abot Reagents SPB Is inclded in the Nextera DNA Flex Library Prep Mst be at room temperatre before se Vortex before each se Vortex freqently to make sre that beads are evenly distribted Aspirate and dispense slowly de to the viscosity of the soltion Preparation 1 Prepare the following consmables. Item Storage Instrctions BLB 15 C to 30 C* If frozen, thaw at room temperatre. If precipitates are observed, heat at 37 C for 10 mintes and vortex ntil resspended. SPB 2 C to 8 C** Let stand for 30 mintes to bring to room temperatre. PK1-25 C to -15 C Place on ice ntil needed. *BLB is shipped -25 C to -15 C bt stored at 15 C to 30 C. **SPB is inclded in the Nextera DNA Flex Library Prep Kit. 2 Save the following BLP program on the thermal cycler: Choose the preheat lid option and set to 100 C 56 C for 10 mintes Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 20

25 Nextera DNA Flex Library Prep Reference Gide Procedre 1 Prepare a lysis master mix. For each reaction se: BLB (7 µl) PK1 (2 µl) Nclease-free water (31 µl) 2 Invert the EDTA tbe 10 times to mix. 3 Transfer 10 µl blood from the tbe to one well of a 96-well PCR plate. 4 Vortex and centrifge the lysis master mix. 5 Add 40 µl master mix to each sample. 6 Vortex and invert SPB mltiple times to resspend. 7 Add 20 µl SPB to the sample well. 8 Using a pipette set to 50 µl, slowly pipette 10 times to mix. 9 Seal the plate, place on the preprogrammed thermal cycler, and rn the BLP program. 10 Place on a magnetic stand and wait 5 mintes. The soltion will not become clear de to the dark brown color of the blood from the lysis reaction. The beads migrate after 5 mintes. 11 Withot distrbing the beads, remove and discard spernatant. 12 If beads are aspirated into pipette tips, dispense back to the plate on the magnetic stand, and wait ntil the soltion is clear (~2 mintes). 13 Add 150 µl of fresh 80% EtOH to the well. 14 Pipette to remove all EtOH. 15 Incbate on the magnetic stand for 30 seconds. 16 Use a 20 µl pipette to remove and discard all residal EtOH. 17 Remove the plate from the magnetic stand. 18 Add 30 µl nclease-free water and pipette to resspend. 19 Proceed immediately to step 3 of Tagment Genomic DNA Procedre on page 8 or stop and store the sample bead mixtre. SAFE STOPPING POINT If yo are stopping, seal the plate with a Microseal 'B' adhesive seal, and store at 2 C to 8 C for p to 3 days. Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 21

26 Saliva Lysis (Optional) Use this protocol when performing the Nextera DNA Flex Library Prep workflow sing saliva sample inpts. This protocol is validated for saliva collected only in Oragene DNA Saliva collection tbes. The saliva is mixed with the Oragene DX Soltion contained in the collection tbe, making it stable at room temperatre. This protocol is expected to generate > 100 ng of DNA otpt at the end of the saliva lysis step. WARNING Saliva is a potential sorce of infectios diseases. Follow site-specific procedres to ensre the safe handling of saliva samples. Consmables Oragene DNA collection tbes (for saliva sample collection) Sample Prification Beads Nclease-free water Freshly prepared 80% ethanol (EtOH) 96-well PCR plate Abot Reagents SPB Is inclded in the Nextera DNA Flex Library Prep Mst be at room temperatre before se Vortex before each se Vortex freqently to make sre that beads are evenly distribted Aspirate and dispense slowly de to the viscosity of the soltion Preparation 1 Prepare the following consmables. Item Storage Instrctions Saliva samples in Oragene DNA collection tbes Room temperatre Any time after sample collection, incbate for a minimm of 1 hor at 50 C in water or an air incbator (as recommended by DNA Genotek) to lyse the cells. Following heat treatment, store at room temperatre. For information on long-term storage of Oragene/saliva samples at room temperatre and garantees, see the DNA Genotek website. SPB 2 C to 8 C* Let stand for 30 mintes to bring to room temperatre. *SPB is inclded in the Nextera DNA Flex Library Prep Kit. Procedre 1 For each sample, add 20 µl nclease-free water to one well of a 96-well PCR plate. 2 Vortex the heat-treated Oragene DNA collection tbe. 3 Transfer 30 µl sample from the tbe to the well containing water. Slowly pipette to mix. For viscos samples, se a wide-bored pipette tip for more accrate pipetting. 4 Vortex and invert SPB mltiple times to resspend. Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 22

27 Nextera DNA Flex Library Prep Reference Gide 5 Add 20 µl SPB to the sample well. 6 Using a pipette set to 50 µl, slowly pipette 10 times to mix. 7 Incbate at room temperatre for 5 mintes. 8 Place on a magnetic stand and wait 5 mintes. 9 Withot distrbing the beads, remove and discard spernatant. 10 If beads are aspirated into pipette tips, dispense back to the plate on the magnetic stand, and wait ntil the liqid is clear (~2 mintes). 11 Dispense 150 µl fresh 80% EtOH onto the SPB pellet and incbate for 30 seconds. 12 Incbate on the magnetic stand for 30 seconds. 13 Pipette to remove and discard the EtOH. 14 Use a 20 µl pipette to remove and discard all residal EtOH. 15 Remove the plate from the magnetic stand. 16 Add 30 µl nclease-free water and pipette to resspend. 17 Proceed immediately to step 3 of Tagment Genomic DNA Procedre on page 8 or stop and store the sample bead mixtre. SAFE STOPPING POINT If yo are stopping, seal the plate with a Microseal 'B' adhesive seal, and store at 2 C to 8 C for p to 3 days. Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 23

28 Appendix A Spporting Information Spporting Information Introdction 24 How the Nextera DNA Flex Assay Works 25 Prodct Contents 26 Consmables and Eqipment 29 Acronyms 30 Introdction The protocol described in this gide assmes that yo have reviewed the contents of this section, confirmed workflow contents, and obtained all reqired consmables and eqipment. Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 24

29 Nextera DNA Flex Library Prep Reference Gide How the Nextera DNA Flex Assay Works The Nextera DNA Flex library prep kit ses an innovative, bead-based transposome complex to tagment genomic DNA. This tagmentation is done by fragmenting and adding adapter tag seqences in a single reaction step. After it is satrated with inpt DNA, the bead-based transposome complex fragments a set nmber of DNA molecles. This fragmentation provides flexibility to se a wide DNA inpt range to generate normalized libraries of consistent tight fragment size distribtion. Following tagmentation, a limited-cycle PCR step adds Nextera DNA Flex-specific index adapter seqences to the ends of a DNA fragment. This step enables capability across all Illmina seqencing platforms. A sbseqent Sample Prification Beads (SPB) cleanp step then prifies libraries for se on an Illmina seqencer. The doble-stranded DNA library is denatred before hybridization of the biotin probe oligoncleotide pool. Figre 4 Nextera DNA Flex Workflow Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 25

30 Nextera DNA Flex Library Prep Reference Gide Prodct Contents Some reagents are shipped at temperatres that differ from the storage temperatre. Always store reagents at the recommended storage temperatre. Nextera DNA Flex Library Prep Kit Configrations Make sre that yo have all the reagents identified in this section before proceeding to the library preparation procedres. Certain components of the kit are stored at a temperatre that differs from the shipping temperatre. Store kit components at the temperatre specified. Each Nextera DNA Flex Library Prep Kit reqires one of the following Nextera DNA Flex Index Kits to complete the protocol, regardless of the sample pooling level sed for seqencing. Nextera DNA Flex Library Prep - 24 Samples Box 1 of 3 Qantity Reagent Description Storage Temperatre 1 SPB Sample Prification Beads 2 C to 8 C 1 TSB Tagment Stop Bffer Room temperatre 1 TWB Tagment Wash Bffer Room temperatre Box 2 of 3 Qantity Reagent Description Storage Temperatre 1 RSB Resspension Bffer -25 C to -15 C 1 TB1 Tagmentation Bffer 1-25 C to -15 C 1 EPM Enhanced PCR Mix -25 C to -15 C Box 3 of 3 Qantity Reagent Description Storage Temperatre 1 BLT Bead-Linked Transposomes 2 C to 8 C Nextera DNA Flex Library Prep - 96 Samples Box 1 of 3 Qantity Reagent Description Storage Temperatre 1 SPB Sample Prification Beads 2 C to 8 C 4 TSB Tagment Stop Bffer Room temperatre 1 TWB Tagment Wash Bffer Room temperatre Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 26

31 Nextera DNA Flex Library Prep Reference Gide Box 2 of 3 Qantity Reagent Description Storage Temperatre 1 RSB Resspension Bffer -25 C to -15 C 4 TB1 Tagmentation Bffer 1-25 C to -15 C 4 EPM Enhanced PCR Mix -25 C to -15 C Box 3 of 3 Qantity Reagent Description Storage Temperatre 4 BLT Bead-Linked Transposomes 2 C to 8 C Index Kit Contents The following library prep and index adapter components are available to spport the Nextera DNA Flex Library Prep workflow. Depending on the nmber of samples for yor experiment, order one catalog nmber for the library prep component and one catalog nmber for the index adapter component. The IDT for Illmina-Nextera DNA UD Indexes se 10 base pair index codes that differ from the Nextera DNA CD Indexes which se 8 base pair index codes. This change in base pair index codes can reqire adjstments to yor seqencing rn set p. For information on setp, see Each Nextera DNA Flex Library Prep kit reqires one of the following Nextera DNA Flex Index Kits to complete the protocol, regardless of the sample pooling level sed for seqencing. These index kits are not interchangeable with the Nextera DNA or Nextera XT Index Kits. Component Catalog # Nextera DNA CD Indexes (24 Indexes, 24 Samples) Nextera DNA CD Indexes (96 Indexes, 96 Samples) IDT for Illmina -Nextera DNA UD Indexes Set A (96 Indexes, 96 Samples) Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 27

32 Nextera DNA Flex Library Prep Reference Gide 24 Dal Index (Tbe Format) - 24 Samples Qantity Index Name Description Storage Temperatre 1 H503 DNA Adapter -25 C to -15 C 1 H505 DNA Adapter -25 C to -15 C 1 H506 DNA Adapter -25 C to -15 C 1 H517 DNA Adapter -25 C to -15 C 1 H710 DNA Adapter -25 C to -15 C 1 H705 DNA Adapter -25 C to -15 C 1 H706 DNA Adapter -25 C to -15 C 1 H707 DNA Adapter -25 C to -15 C 1 H711 DNA Adapter -25 C to -15 C 1 H714 DNA Adapter -25 C to -15 C Qantity Consmable Catalog # 1 Bag of 48 Index Adapter replacement caps, orange Bag of 32 Index Adapter replacement caps, white Dal Index (Plate Format) - 96 Samples Qantity Description Storage Temperatre 1 96 Dal Adapter Index Plate -25 C to -15 C Blood Lysis Kit Contents Confirm that all reagents identified in this section are available before proceeding to the library preparation procedres. Certain components of the kit are stored at a temperatre that differs from the shipping temperatre. Store kit components at the temperatre specified. Consmable Catalog # Nextera DNA Flex Library Prep Flex Lysis Reagent Kit Flex Lysis Reagent Kit Qantity Acronym Description Storage Temperatre 4 BLB* Blood Lysis Bffer Room temperatre 4 PK1 Proteinase K -25 C to -15 C *These reagents are shipped at -25 C to -15 C. Promptly store reagents at the indicated tbe temperatre to ensre proper performance. NOTE Sample Prification Beads are not inclded in this kit, however sfficient SPB to rn the blood lysis workflow are inclded in the 24-plex and 96-plex library kits. Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 28

33 Nextera DNA Flex Library Prep Reference Gide Consmables and Eqipment Confirm that all reqired ser-spplied consmables and eqipment are present and available 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 20 µl pipette tips General lab spplier 20 µl mltichannel pipettes General lab spplier 20 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 1000 µl pipette tips General lab spplier 1000 µl single channel pipettes General lab spplier 96-well storage plates, rond well, 0.8 ml (midi plate) xs Fisher Scientific, catalog # AB-0859 Hard-Shell 96-well PCR plates Bio-Rad, catalog # HSP ml Microcentrifge Tbes General lab spplier Microseal 'B' adhesive seals Bio-Rad, catalog # MSB-1001 Microseal 'F' foil seals Bio-Rad, catalog # MSF-1001 RNase/DNase-free mltichannel reagent reservoirs, disposable VWR, catalog # Ethanol 200 proof (absolte) for moleclar biology (500 ml) Sigma-Aldrich, prodct # E7023 Nclease-free water General lab spplier Qbit dsdna HS Assay Kit ThermoFisher Scientific, catalog # Q32851 or Q32854 Qant-iT PicoGreen dsdna Assay Kit ThermoFisher Scientific, catalog # P11496 [Optional] Agilent High Sensitivity DNA Kit Agilent, catalog # [Optional] High Sensitivity NGS Fragment Analysis Kit Advanced Analytical, catalog # DNF-474 Consmables for Blood and Saliva Inpt Consmable Spplier [Blood] Flex Lysis Reagent Kit Illmina, catalog # [Blood] EDTA Blood Collection tbes Becton Dickinson [Saliva] Oragene DNA Collection Kit for Saliva DNA Genotek, catalog # OGR-500 or OGD-510 Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 29

34 Nextera DNA Flex Library Prep Reference Gide Eqipment Eqipment Magnetic Stand-96 Microplate centrifge Microcentrifge Vortexer Qbit Florometer 3.0 [Optional] 2100 Bioanalyzer System [Optional] Fragment Analyzer Analytical Spplier Thermo Fisher Scientific, catalog # AM10027 General lab spplier General lab spplier General lab spplier ThermoFisher Scientific, catalog # Q33216, Q33217 or Q33218 Agilent, catalog # G2940CA Advanced Analytical Thermal Cyclers Use the recommended settings for the selected thermal cycler models listed. Before performing library prep, validate any thermal cyclers not listed. Thermal Cycler Temp Mode Lid Temp Vessel Type Bio-Rad C-1000 Toch thermal cycler Calclated Heated Plate Bio-Rad DNA Engine Tetrad 2 Calclated Heated Polypropylene plates and tbes MJ Research DNA Engine Tetrad Calclated Heated Plate Eppendorf Mastercycler Pro S Gradient S, Simlated Tbe Heated Plate Acronyms Acronym Definition BLB Blood Lysis Bffer BLT Bead Linked Transposome EPM Enhanced PCR Mix EtOH Ethanol PK1 Proteinase K RSB Resspension Bffer SPB Sample Prification Beads TB1 Tagmentation Bffer 1 TSB Tagment Stop Bffer TWB Tagment Wash Bffer Docment # v03 For Research Use Only. Not for se in diagnostic procedres. 30

35 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 # v03 For Research Use Only. Not for se in diagnostic procedres. 31

36 Docment # v03 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.