TruSeq DNA PCR-Free. Reference Guide. For Research Use Only. Not for use in diagnostic procedures. Document # v00 October 2017

Transcription

1 TrSeq DNA PCR-Free Reference Gide Docment # v00 October 2017 For Research Use Only. Not for se in diagnostic procedres. ILLUMINA PROPRIETARY

2 TrSeq DNA PCR-Free 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. Illmina, BaseSpace, 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 # v00 For Research Use Only. Not for se in diagnostic procedres. ii

3 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 Prepare for Pooling 5 Fragment DNA 5 Repair Ends and Select Library Size 8 Adenylate 3 Ends 11 Ligate Adapters 12 Check Libraries 15 Normalize and Pool Libraries 18 Appendix A Spporting Information 21 Prodct Contents 21 Inline Control DNA [Optional] 22 Consmables and Eqipment 23 Index Adapter Seqences 25 Acronyms 26 Technical Assistance 27 Docment # v00 For Research Use Only. Not for se in diagnostic procedres. iii

4 Chapter 1 Overview Introdction 1 DNA Inpt Recommendations 1 Additional Resorces 2 Introdction This protocol explains how to prepare p to 96 libraries starting from genomic DNA (gdna) sing the Illmina TrSeq DNA PCR-Free library prep workflow. The goal is to add adapter seqences to DNA fragment ends to create indexed libraries for single-read or paired-end seqencing. The TrSeq DNA PCR-Free Library Prep workflow protocol incldes the following featres. Streamlined workflow: Size-selection beads and master-mixed reagents redce reagent containers and pipetting. Universal adapter to prepare DNA libraries for single-read, paired-end, and indexed seqencing. One workflow with options for processing low sample (LS) and high sample (HS) nmbers. Flexible throghpt: 24- and 96-sample workflow configrations accommodate a range of experiments. Spport for non-indexed seqencing and low-plexity pooling. Optimized shearing for whole-genome reseqencing with insert sizes of 350 bp or 550 bp. Inclsive components: Library Prep components inclde library prep reagents exclding index adapters. Index adapter components mst be prchased separately. See Spporting Information on page 21 for more details. DNA Inpt Recommendations Qantify the inpt gdna and assess the qality before starting library preparation. For best reslts, se the following inpt amonts. Insert Size 350 bp 1 µg 550 bp 2 µg Inpt gdna Lower inpt amonts reslt in low yield and increased dplicates. Qantify Inpt DNA Qantify inpt DNA per the following recommendations: Sccessfl library prep depends on accrate qantification of inpt DNA. Use florometric-based methods for qantification, sch as Qbit or PicoGreen to provide accrate qantification for dsdna. UV spectrophotometric based methods, sch as the Nanodrop, measres any ncleotides present in the sample inclding RNA, dsdna, ssdna, and free ncleotides, which can give an inaccrate measrement of gdna. Qantification methods depend on accrate pipetting methods. Do not se pipettes at the extremes of volme specifications. Make sre that pipettes are calibrated. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 1

5 TrSeq DNA PCR-Free Reference Gide Assess DNA Qality Absorbance measrements at 260 nm are commonly sed to assess DNA qality: The ratio of absorbance at 260 nm to absorbance at 280 nm is sed as an indication of sample prity. Vales from 1.8 throgh 2.0 indicate relatively pre DNA. The presence of RNA or small ncleic acid fragments, sch as ncleotides, can compromise both absorbance measrements. Make sre that samples are free of contaminants. Additional Resorces The following docmentation is available for download from the Illmina website. Resorce Cstom Protocol Selector TrSeq DNA PCR-Free Checklist (docment # ) Index Adapter Pooling Gide (docment # ) Illmina Experiment Manager Gide (docment # ) and IEM TrSeq DNA, RNA, or ChIP Qick Reference Card (docment # ) BaseSpace Seqence Hb help Local Rn Manager Software Gide (docment # ) 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 a checklist of the protocol steps, and is intended for experienced sers. Provides pooling gidelines for preparing libraries for seqencing systems that reqire balanced index combinations. Review this gide before beginning library preparation. Provides information abot creating and editing sample sheets. Provides information abot BaseSpace Seqence Hb, a data analysis tool. Provides an overview of the Local Rn Manager (LRM) software, instrctions for sing software featres, and instrctions for installing analysis modles on the instrment compter. Visit the TrSeq DNA PCR-Free workflow spport page on the Illmina website for access to reqirements and compatibility, additional docmentation, software downloads, online training, freqently asked qestions, and best practices. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 2

6 Chapter 2 Protocol Introdction 3 Tips and Techniqes 3 Library Prep Workflow 5 Prepare for Pooling 5 Fragment DNA 5 Repair Ends and Select Library Size 8 Adenylate 3 Ends 11 Ligate Adapters 12 Check Libraries 15 Normalize and Pool Libraries 18 Introdction This chapter describes the TrSeq DNA PCR-Free Library Prep workflow protocol. Follow the steps in the order shown, sing the specified volmes and incbation parameters. Before proceeding, confirm the delivered contents and make sre that yo have the reqired eqipment and consmables. Review Best Practices from the TrSeq DNA PCR-Free Library Prep workflow spport page on the Illmina website. This protocol provides one workflow with variations for differences in sample nmbers. [HS] and [LS] identify the appropriate option for yor nmber of samples. Expect eqivalent reslts from either option, bt the HS option can yield more consistent reslts between samples. Table 1 Workflow Variable HS LS 24-Sample Workflow 96-Sample Workflow Plate Type Incbation Eqipment Workflow Variations Process > 24 samples with index adapter tbes* Process > 24 samples with index adapter plate 96-well Hard-Shell PCR plate 96-well midi plate Microheating systems Mixing Method Microplate shaker Pipetting Process 24 samples with index adapter tbes* Process 24 samples with index adapter plate 96-well 0.3 ml PCR plate 96-well midi plate 96-well thermal cycler * Combine the Set A and Set B indexes to pool p to 24 libraries. 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. Remove nsed index adapter tbes from the working area. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 3

7 TrSeq DNA PCR-Free Reference Gide 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. Use Microseal 'B' for shaking, centrifging, and long-term storage. Microseal 'A' adhesive film is sed for thermal cycling steps to prevent evaporation. 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 Do not freeze beads. Pipette bead sspensions slowly. Before se, allow the beads to come to room temperatre. Immediately before se, vortex the beads ntil they are well dispersed. The color of the liqid mst appear homogeneos. Vortex throghot protocol as necessary to keep homogenos. 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). When washing beads: Use the specified magnetic stand for the plate. Dispense liqid so that beads on the side of the wells are wetted. 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 # v00 For Research Use Only. Not for se in diagnostic procedres. 4

8 TrSeq DNA PCR-Free Reference Gide Library Prep Workflow The following diagram illstrates the workflow sing a TrSeq DNA PCR-Free Library Prep workflow. Safe stopping points are marked between steps. Figre 1 TrSeq DNA PCR-Free Workflow Prepare for Pooling When pooling samples for seqencing, se IEM, LRM, or BaseSpace Prep Tab to record information abot yor samples before beginning library preparation. Use IEM to create and edit sample sheets for Illmina seqencing systems and analysis software. Use LRM and BaseSpace Prep Tab to organize samples, libraries, pools, and a rn for Illmina seqencing systems and analysis software. Review the planning steps in the Index Adapter Pooling Gide (docment # ) when preparing libraries that reqire balanced index combinations. Fragment DNA This step fragments to an insert size of 350 bp or 550 bp. Covaris shearing generates doble-stranded DNA (dsdna) fragments with 3 or 5 overhangs. Consmables gdna samples [350 bp insert size] 1 µg per sample [550 bp insert size] 2 µg per sample RSB (Resspension Bffer) Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 5

9 TrSeq DNA PCR-Free Reference Gide SPB (Sample Prification Beads) Barcode labels CFP (Covaris Fragmentation Plate) CSP (Clean Up Sheared DNA Plate) DNA (DNA Plate) IMP (Insert Modification Plate) Freshly prepared 80% ethanol (EtOH) Plates [HS] 96-well midi plates (3) [HS] 96-well Hard-Shell 0.3 ml PCR plate (1) [LS] 96-well 0.3 ml PCR plates, semiskirted or skirtless (4) Covaris tbes (1 per sample) Microseal 'B' adhesive seal Abot Reagents Vortex SPB before each se. Vortex SPB freqently to make sre that beads are evenly distribted. Aspirate and dispense SPB slowly de to the viscosity of the soltion. Preparation 1 Prepare the following consmables. Item Storage Instrctions RSB -25 C to -15 C Thaw at room temperatre. After the initial thaw, store at 2 C to 8 C. SPB 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. 2 Trn on and set p the Covaris instrment per manfactrer gidelines. 3 [HS] Calibrate the microplate shaker with a stroboscope and set to 1800 rpm. 4 Apply barcode labels to plates. Barcode Label Plate for HS Plate for LS DNA Midi PCR CFP Hard-Shell PCR PCR CSP Midi PCR IMP Midi PCR Procedre Normalize gdna 1 Qantify gdna sing a florometric-based method. 2 Normalize gdna samples with RSB to a final volme of 55 µl in the DNA plate. 1 g for a 350 bp insert size 2 g for a 550 bp insert size Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 6

10 TrSeq DNA PCR-Free Reference Gide 3 [HS] Mix and centrifge as follows. a b Shake at 1800 rpm for 2 mintes. Centrifge at 280 g for 1 minte. 4 [LS] Pipette to mix, and then centrifge briefly. Fragment DNA 1 Transfer 52.5 µl DNA samples to separate Covaris tbes. Use the wells of the CFP plate to hold the tbes pright. 2 Centrifge at 280 g for 5 seconds. 3 Fragment sing the appropriate Covaris settings: Table bp Insert Setting M220 S220 S2 E210 Dty Cycle (%) Intensity 5.0 Peak/Displayed Power (W) Cycles/Brst 200 Dration (seconds) Mode Freqency sweeping Temperatre ( C) Table bp Insert Setting M220 S220 S2 E210 Dty Cycle (%) Intensity 2.0 Peak/Displayed Power (W) Cycles/Brst 200 Dration (seconds) Mode Freqency sweeping Temperatre ( C) Centrifge at 280 g for 5 seconds. 5 Transfer 50 µl sample from each Covaris tbe to the corresponding well of the CSP plate. Clean Up Fragmented DNA 1 Vortex SPB ntil well-dispersed. 2 Add 80 µl SPB to each well. 3 Mix thoroghly as follows. [HS] Shake at 1800 rpm for 2 mintes. [LS] Pipette p and down. 4 Incbate at room temperatre for 5 mintes. 5 Centrifge at 280 g for 1 minte. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 7

11 TrSeq DNA PCR-Free Reference Gide 6 Place on a magnetic stand and wait ntil the liqid is clear (~8 mintes). 7 Remove and discard all spernatant from each well. 8 Wash two times as follows. a b c Add 200 µl fresh 80% EtOH to each well. Incbate on the magnetic stand for 30 seconds. Remove and discard all spernatant from each well. 9 Use a 20 µl pipette to remove residal EtOH from each well. 10 Air dry on the magnetic stand for 5 mintes. 11 Add RSB to each well, and then remove from the magnetic stand. 12 Mix thoroghly as follows. [HS] Shake at 1800 rpm for 2 mintes. [LS] Pipette p and down. 13 Incbate at room temperatre for 2 mintes. 14 Centrifge at 280 g for 1 minte. 15 Place on a magnetic stand and wait ntil the liqid is clear (2 5 mintes). 16 Transfer 50 µl spernatant to the corresponding well of the IMP plate. Repair Ends and Select Library Size This step ses End Repair Mix 2 to convert the overhangs reslting from fragmentation into blnt ends. A 3 to 5 exonclease activity removes the 3 overhangs. A 5 to 3 polymerase activity completes the 5 overhangs. After end repair, different ratios of Sample Prification Beads are sed to select the appropriate library size. Consmables ERP 2 or ERP 3 (End Repair Mix) RSB (Resspension Bffer) SPB (Sample Prification Beads) Barcode labels ALP (Adapter Ligation Plate) CEP (Clean Up End Repair Plate) Freshly prepared 80% ethanol (EtOH) PCR-grade water Tbe [ 6 samples] 1.7 ml microcentrifge tbe [> 6 samples] 15 ml conical tbe Plates [HS] 96-well midi plates (2) [LS] 96-well 0.3 ml PCR plates, semiskirted or skirtless (2) Microseal 'B' adhesive seals Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 8

12 TrSeq DNA PCR-Free Reference Gide Abot Reagents Vortex SPB before each se. Vortex SPB freqently to make sre that beads are evenly distribted. Aspirate and dispense SPB slowly de to the viscosity of the soltion. Preparation 1 Prepare the following consmables. Item Storage Instrctions ERP 2 or ERP 3-25 C to -15 C Thaw at room temperatre, and then set aside on ice. Retrn to storage after se. RSB 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. SPB 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. 2 [HS] Preheat the microheating system to 30 C. 3 [LS] Save the following ERP program on the thermal cycler: Choose the preheat lid option and set to 100 C 30 C for 30 mintes Hold at 4 C 4 Label plates as follows. Apply an ALP barcode label to a midi or PCR plate. Apply a CEP barcode label to a midi or PCR plate. Procedre Convert Overhangs 1 Centrifge CTE at 600 g for 5 seconds. 2 Add 10 µl CTE or RSB to each well. 3 Add 40 µl ERP 2 or ERP 3 to each well. 4 [HS] Mix, centrifge, and incbate as follows. a b c d Shake at 1800 rpm for 2 mintes. Centrifge at 280 g for 1 minte. Place on the 30 C microheating system, lid closed, for 30 mintes. Place on ice. 5 [LS] Pipette to mix, centrifge, and then place on the thermal cycler and rn the ERP program. Each well contains 100 µl. Remove Large DNA Fragments 1 Vortex SPB ntil well-dispersed. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 9

13 TrSeq DNA PCR-Free Reference Gide 2 Using the following formlas, determine the appropriate volmes of SPB and PCR-grade water for dilting SPB. The formlas inclde 15% excess for mltiple samples. Table 4 Dilted SPB for a 350 bp Insert Size Reagent Formla Example Volme for 12 Samples SPB # of samples µl 1311 µl PCR-grade water # of samples µl 897 µl Yor Calclation Table 5 Dilted SPB for a 550 bp Insert Size Reagent Formla Example Volme for 12 Samples SPB # of samples 92 µl 1104 µl Yor Calclation PCR-grade water # of samples 92 µl 1104 µl 3 Using yor calclations from the previos step, dilte SPB with PCR-grade water. For 6 samples, dilte in a new 1.7 ml microcentrifge tbe. For > 6 samples, dilte in a new 15 ml conical tbe. 4 Vortex dilted SPB ntil well-dispersed. 5 Add 160 µl dilted SPB to each well. 6 Mix thoroghly as follows. [HS] Shake at 1800 rpm for 2 mintes. [LS] Pipette p and down. 7 Incbate at room temperatre for 5 mintes. 8 Centrifge at 280 g for 1 minte. 9 Place on a magnetic stand and wait ntil the liqid is clear (~5 mintes). 10 Transfer 250 µl spernatant to the corresponding well of the CEP plate. 11 Discard remaining dilted SPB. Remove Small DNA Fragments 1 Vortex ndilted SPB ntil well-dispersed. 2 Add 30 µl ndilted SPB to each well. 3 Mix thoroghly as follows. [HS] Shake at 1800 rpm for 2 mintes. [LS] Pipette p and down. 4 Incbate at room temperatre for 5 mintes. 5 Centrifge at 280 g for 1 minte. 6 Place on a magnetic stand and wait ntil the liqid is clear (~5 mintes). 7 Remove and discard all spernatant from each well. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 10

14 TrSeq DNA PCR-Free Reference Gide 8 Wash two times as follows. a b c Add 200 µl fresh 80% EtOH to each well. Incbate on the magnetic stand for 30 seconds. Remove and discard all spernatant from each well. 9 Use a 20 µl pipette to remove residal EtOH from each well. 10 Air dry on the magnetic stand for 5 mintes. 11 Add 17.5 µl RSB to each well, and then remove from the magnetic stand. 12 Mix thoroghly as follows. [HS] Shake at 1800 rpm for 2 mintes. [LS] Pipette p and down. 13 Incbate at room temperatre for 2 mintes. 14 Centrifge at 280 g for 1 minte. 15 Place on a magnetic stand and wait ntil the liqid is clear (~5 mintes). 16 Transfer 15 µl spernatant to the corresponding well of the ALP plate. SAFE STOPPING POINT If yo are stopping, seal the plate and store at -25 C to -15 C for p to 7 days. Adenylate 3 Ends One adenine (A) ncleotide is added to the 3ʹ ends of the blnt fragments to prevent them from ligating to each other dring adapter ligation reaction. One corresponding thymine (T) ncleotide on the 3ʹ end of the adapter provides a complementary overhang for ligating the adapter to the fragment. This strategy ensres a low rate of chimera (concatenated template) formation. Consmables RSB (Resspension Bffer) [Optional] CTA (A-Tailing Control) Microseal 'B' adhesive seals Abot Reagents Using CTA is optional. Use eqal volme of RSB as a sbstitte. Preparation 1 Prepare the following consmables. Item Storage Instrctions ATL or ATL 2-25 C to -15 C Thaw at room temperatre. Retrn to storage after se. CTA -25 C to -15 C Thaw at room temperatre, and then place on ice. RSB 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. 2 [HS] Preheat two microheating systems, one to 37 C and the other to 70 C. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 11

15 TrSeq DNA PCR-Free Reference Gide 3 [LS] Save the following ATAIL70 program on the thermal cycler: Choose the preheat lid option and set to 100 C 37 C for 30 mintes 70 C for 5 mintes Hold at 4 C Procedre 1 Centrifge CTA at 600 g for 5 seconds. 2 Add 2.5 µl CTA to each well. 3 Centrifge ATL or ATL 2 at 600 g for 5 seconds. 4 Add 12.5 µl ATL or ATL 2 to each well. 5 [HS] Mix and incbate as follows. a b c d Shake at 1800 rpm for 2 mintes. Place on the 37 C microheating system, lid closed, for 30 mintes. Move to the 70 C microheating system, lid closed, for 5 mintes. Place on ice for 5 mintes. 6 [LS] Pipette to mix, and then place on the thermal cycler and rn the ATAIL70 program. Each well contains 30 µl. Ligate Adapters This step ligates index adapters to the DNA fragment ends, preparing them for hybridization to a flow cell. Index adapters mst be ordered separately from the Library Prep components. For information on compatible index adapters, see Spporting Information on page 21. Consmables DNA Adapters (tbes or index adapter plate) LIG 2 (Ligation Mix 2) RSB (Resspension Bffer) SPB (Sample Prification Beads) STL (Stop Ligation Bffer) [Optional] CTL (Ligation Control) Barcode labels CAP (Clean Up ALP Plate) Index Adapter Plate TSP1 (Target Sample Plate) Freshly prepared 80% ethanol (EtOH) Plates [HS] 96-well midi plate and 96-well Hard-Shell 0.3 ml PCR plate (1) [LS] 96-well 0.3 ml PCR plates, semiskirted or skirtless (2) Microseal 'B' adhesive seals Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 12

16 TrSeq DNA PCR-Free Reference Gide Abot Reagents Using CTL is optional. Use RSB as a sbstitte. Do not remove LIG 2 from storage ntil instrcted to do so in the procedre. Retrn LIG 2 to storage immediately after se. Vortex SPB before each se. Vortex SPB freqently to make sre that beads are evenly distribted. Aspirate and dispense SPB slowly de to the viscosity of the soltion. Preparation 1 Prepare the following consmables. Item Storage Instrctions CTL -25 C to -15 C Thaw at room temperatre, and then place on ice. DNA Adapters -25 C to -15 C Thaw at room temperatre for 10 mintes. Retrn to storage after se. RSB 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. SPB 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. STL -25 C to -15 C Thaw at room temperatre. Retrn to storage after se. 2 [HS] Preheat a microheating system to 30 C. 3 [LS] Save the following LIG program on the thermal cycler: Choose the preheat lid option and set to 100 C 30 C for 10 mintes Hold at 4 C 4 Label plates as follows. Apply a CAP barcode label to a midi or PCR plate. Apply a barcode label to a Hard-Shell PCR or PCR plate. Procedre Add Index Adapters 1 [HS] Prepare the appropriate Index Adapter Plate as follows. a b c d Remove the tape seal. Centrifge at 280 g for 1 minte. Remove the plastic cover. If yo are not processing the entire plate, save the cover. Apply the index adapter plate barcode label. 2 [LS] Centrifge the adapter tbes at 600 g for 5 seconds. 3 Remove LIG 2 from -25 C to -15 C storage. 4 In the order listed, add the following reagents to each well: CTL (2.5 µl) LIG 2 (2.5 µl) Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 13

17 TrSeq DNA PCR-Free Reference Gide DNA adapters (2.5 µl) 5 Mix thoroghly as follows. [HS] Shake at 1800 rpm for 2 mintes. [LS] Pipette p and down. 6 Centrifge at 280 g for 1 minte. 7 Incbate as follows. [HS] Place on the 30 C microheating system, lid closed, for 10 mintes. Set aside on ice. [LS] Place on the thermal cycler and rn the LIG program. Each well contains 37.5 µl. 8 Centrifge the STL at 600 g for 5 seconds. 9 Add 5 µl STL to each well. 10 Mix thoroghly as follows. [HS] Shake at 1800 rpm for 2 mintes. [LS] Pipette p and down. 11 Centrifge at 280 g for 1 minte. Clean Up Ligated Fragments Steps 1 throgh 14 are performed one time sing the Rond 1 volmes, then repeated sing the Rond 2 volmes. 1 Add the appropriate volme of SPB to each well. Rond µl Rond 2 50 µl 2 Mix thoroghly as follows. [HS] Shake at 1800 rpm for 2 mintes. [LS] Pipette p and down. 3 Incbate at room temperatre for 5 mintes. 4 Centrifge at 280 g for 1 minte. 5 Place on a magnetic stand and wait ntil the liqid is clear (2 5 mintes). 6 Remove and discard all spernatant from each well. 7 Wash two times as follows. a b c Add 200 µl fresh 80% EtOH to each well. Incbate on the magnetic stand for 30 seconds. Remove and discard all spernatant from each well. 8 Use a 20 µl pipette to remove residal EtOH from each well. 9 Air dry on the magnetic stand for 5 mintes. 10 Add the appropriate volme of RSB to each well. Rond µl Rond µl 11 Remove from the magnetic stand, and then mix thoroghly as follows. [HS] Shake at 1800 rpm for 2 mintes. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 14

18 TrSeq DNA PCR-Free Reference Gide [LS] Pipette p and down. 12 Incbate at room temperatre for 2 mintes. 13 Centrifge at 280 g for 1 minte. 14 Place on a magnetic stand and wait ntil the liqid is clear (2 5 mintes). 15 Transfer 50 µl spernatant to the corresponding well of the CAP plate. 16 Repeat steps 1 throgh 14 sing the new plate and the Rond 2 volmes. 17 Transfer 20 µl spernatant to the corresponding well of the TSP1 plate. SAFE STOPPING POINT If yo are stopping, seal the plate and store at -25 C to -15 C for p to 7 days. Check Libraries Qantify Libraries Achieving high-qality data on Illmina seqencing systems reqires optimm clster density across every lane of the flow cell. Optimizing clster densities reqires accrate qantification of DNA libraries sing qpcr. Qantification of TrSeq DNA PCR-Free libraries has been validated with the KAPA Library Qantification Kit Illmina/Universal. Non-qPCR methods qantify molecles that do not have adapters on both ends and do not form clsters. More of these nonclstering molecles can be present de to the absence of PCR enrichment. Ths, qantification by methods other than qpcr might be inaccrate. NOTE For information on handling small liqid volmes, see Best Practices on the spport page for yor workflow. 1 Follow qpcr instrctions for the KAPA kit, with the following modifications: For the library diltion step, se at least 2 µl of the original library stock to ensre accrate and reprodcible qantification. Using at least 2 µl of initial dilted libraries, perform two additional independent (nonserial) 1:10,000 and 1:20,000 diltions to evalate qantification precision. The concentration of each library is calclated as shown in the following tables. Table 6 Diltion Factor Calclated by qpcr Instrment (pm)* Average Dilted Library (pm) Size-Adjsted Dilted library (pm) Undilted Library (pm)* 1:10,000 A1 A2 A = (A1 + A2)/2 W1 = A (452/470) C1 = W1 x 10,000 1:20,000 B1 B2 B = (B1 + B2)/2 W2 = B (452/470) C2 = W2 20,000 Table 7 Diltion Factor 350 bp Library Concentration Calclation 550 bp Library Concentration Calclation Calclated by qpcr Instrment (pm)* Average Dilted Library (pm) Size-Adjsted Dilted Library (pm) Undilted Library (pm)* 1:10,000 C1 C2 C = (C1 + C2)/2 W3 = C (452/670) C3 = W3 10,000 1:20,000 D1 D2 D = (D1 + D2)/2 W4 = D (452/670) C4 = W4 20,000 Undilted Library (pm) (C1 + C2)/2 Undilted Library (pm) (C3 + C4)/2 Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 15

19 TrSeq DNA PCR-Free Reference Gide Obtain the calclated concentration of the 1:10,000 and 1:20,000 library diltions, as determined by qpcr, in relation to the concentrations of the annotated KAPA DNA Standards 1 6. Use the average of the replicate data points to determine the concentration of the dilted library. Make a size adjstment calclation to accont for the size difference between the average fragment length of the library and the KAPA DNA Standard (452 bp). NOTE Do not se the average fragment length of the library insert size based on the Bioanalyzer reslts. Fragment sizes of TrSeq DNA PCR-Free libraries measred on the Bioanalyzer are mch larger than seqencing data can indicate. Calclate the concentration of the ndilted library by acconting for the relevant diltion factor (1:10,000 and 1:20,000). To calclate the concentration of the ndilted library, se the average of the replicate data points corresponding to each library DNA diltion. If a replicate is an otlier, it can be omitted from the calclation. If mltiple replicates are otliers, repeat the assay. Qality Control 1 Verify fragment size by checking the library size distribtion. Rn on an Advanced Analytical Fragment Analyzer with the HS-NGS High Sensitivity 474 kit or an Agilent Technology 2100 Bioanalyzer sing a High Sensitivity DNA kit for qalitative prposes only. a b Dilte the DNA library 1:5 with water. Rn 1 µl dilted DNA library on a High Sensitivity DNA chip or NGS kit. Figre 2 350bp Library Rn on Fragment Analyzer Using High Sensitivity NGS Kit Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 16

20 TrSeq DNA PCR-Free Reference Gide Figre 3 550bp Library Rn on Fragment Analyzer Using High Sensitivity NGS Kit Library fragments measred on the Bioanalyzer are nexpectedly large de to the presence of certain strctral featres, which are normally removed dring sbseqent PCR enrichment. The following figres show example comparisons between library fragment sizes from a Bioanalyzer and the corresponding insert sizes from the alignment of paired-end reads to a reference seqence. Figre 4 350bp Library Rn on Bioanalyzer Using High Sensitivity DNA Kit A B Bioanalyzer Paired-End Alignment Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 17

21 TrSeq DNA PCR-Free Reference Gide Figre bp Library Rn on Bioanalyzer Using High Sensitivity DNA Kit A B Bioanalyzer Paired-End Alignment Normalize and Pool Libraries This step prepares DNA template for clster generation. Non-indexed DNA libraries are normalized to 4 nm in the DCT plate. Indexed DNA libraries are normalized to 4 nm in the DCT plate and then pooled in eqal volmes in the PDP plate. NOTE For best practice, perform normalization and pooling directly prior to seqencing. To minimize index hopping, do not store libraries in the pooled form. For more information, see Minimize index hopping in mltiplexed rns on the Illmina website. Consmables Barcode labels DCT (Dilted Clster Template) PDP (Pooled DCT Plate) (for pooling only) Tris-HCl 10 mm, ph8.5 with 0.1% Tween 20 Plates [LS] 96-well 0.3 ml PCR plates, semiskirted or skirtless (2) (second plate for pooling 40 samples) Microseal 'B' adhesive seals Preparation 1 Label plates as follows. Apply a DCT barcode label to a Hard-Shell PCR or PCR plate. For pooling only, apply a PDP barcode label to the appropriate plate: [> 40 samples] Midi plate Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 18

22 TrSeq DNA PCR-Free Reference Gide [ 40 samples] Hard-Shell PCR or PCR plate Procedre Normalize Libraries 1 Transfer 5 µl library to the corresponding well of the DCT plate. 2 Normalize the library concentration to 4 nm sing Tris-HCl 10 mm, ph 8.5 with 0.1% Tween Mix thoroghly as follows. [HS] Shake at 1800 rpm for 2 mintes. [LS] Pipette p and down. Depending on the yield qantification data of each library, the final volme of each well can vary from µl. 4 Centrifge at 280 g for 1 minte. 5 Proceed to pooling or clstering: To pool libraries, proceed to Pool Libraries. To leave libraries npooled, skip the remaining library prep steps and proceed to clster generation. For instrctions, see the system gide for yor instrment. Pool Libraries The pooling procedre depends on the nmber of libraries being pooled2 24 or Pool 2 24 Libraries 1 Transfer 5 µl of each normalized library to one well of the PDP plate. 2 Mix thoroghly as follows. [HS] Shake at 1800 rpm for 2 mintes. [LS] Pipette p and down. 3 Centrifge at 280 g for 1 minte. 4 Proceed to clster generation. For instrctions, see the system gide for yor Illmina instrment. Pool Libraries 1 Transfer 5 µl of each colmn of normalized library to colmn 1 of the PDP plate. 2 Mix thoroghly as follows. [HS] Shake at 1800 rpm for 2 mintes. [LS] Pipette p and down. 3 Centrifge at 280 g for 1 minte. 4 Transfer the contents from each well of colmn 1 to well A2. 5 Mix thoroghly as follows. [HS] Shake at 1800 rpm for 2 mintes. [LS] Pipette p and down. 6 Centrifge at 280 g for 1 minte. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 19

23 TrSeq DNA PCR-Free Reference Gide 7 Proceed to clster generation. For instrctions, see the system gide for yor Illmina instrment. SAFE STOPPING POINT If yo are stopping, seal the plate and store at -25 C to -15 C. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 20

24 Appendix A Spporting Information Spporting Information Prodct Contents 21 Inline Control DNA [Optional] 22 Consmables and Eqipment 23 Index Adapter Seqences 25 Acronyms 26 Prodct Contents Make sre that yo have all reagents identified in this section before starting the protocol. The following library prep and index adapter components are available to order throgh Illmina to spport the TrSeq DNA PCR-Free Library Prep workflow. From Illmina, order one catalog nmber for the library prep component and one catalog nmber for the index adapter component depending on the nmber of samples for yor experiment. Library Prep Component Catalog # TrSeq DNA PCR-Free Library Prep (24 Samples) TrSeq DNA PCR-Free Library Prep (96 Samples) Index Adapter Component Catalog # IDT for Illmina-TrSeq DNA UD Indexes (24 indexes, 96 samples) IDT for Illmina-TrSeq DNA UD Indexes (96 indexes, 96 samples) TrSeq DNA Combinatorial Dal Indexes (96 indexes, 96 samples) TrSeq DNA Single Indexes (12 indexes, 24 samples) Set A TrSeq DNA Single Indexes (12 indexes, 24 samples) Set B TrSeq DNA PCR-Free Library Prep (24 Samples) This workflow contains two boxes: Box 1 and an SPB (Sample Prification Beads) box. Box 1, Store at -25 C to -15 C Qantity Reagent Description 1 RSB Resspension Bffer 1 ERP 2 or ERP 3 End Repair Mix 1 ATL or ATL 2 A-Tailing Mix 1 LIG 2 Ligation Mix 2 1 CTE End Repair Control 1 CTA A Tailing Control 1 CTL Ligation Control 1 STL Stop Ligation Bffer Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 21

25 TrSeq DNA PCR-Free Reference Gide SPB Box, Store at 2 C to 8 C Qantity Reagent Description 1 SPB Sample Prification Beads TrSeq DNA PCR-Free Library Prep (96 Samples) This workflow contains two boxes: Box 1 and an SPB (Sample Prification Beads) box. Box 1, Store at -25 C to -15 C This box also contains plate barcode labels. Qantity Reagent Description 2 RSB Resspension Bffer 2 ERP 2 or ERP 3 End Repair Mix 2 ATL or ATL 2 A-Tailing Mix 2 LIG 2 Ligation Mix 2 2 CTE End Repair Control 2 CTA A Tailing Control 2 CTL Ligation Control 2 STL Stop Ligation Bffer SPB Box, Store at 2 C to 8 C Qantity Reagent Description 4 SPB Sample Prification Beads Inline Control DNA [Optional] The se of Inline Control DNA provided with this workflow is optional, and only recommended if a cstom analysis pipeline is available. End Repair Control (CTE), A-Tailing Control (CTA), and Ligation Control (CTL) contain fragments sed as controls for the enzymatic activities of End Repair Mix (ERP 2 or ERP 3), A-Tailing Mix (ATL or ATL 2), and Ligation Mix 2 (LIG 2). The inline controls contain dsdna fragments that report the sccess or failre of a specific enzymatic activity in a cstom analysis pipeline. If no sch pipeline is available, it is recommended to omit these controls from the prep. Controls are added to reactions before the corresponding protocol step. The end strctres of the controls match the end strctres of a DNA molecle that has not ndergone the protocol step. If the step is sccessfl, the control molecle is modified to participate in downstream reactions of library generation and reslt in seqencing data. If the step fails, the control molecle does not advance and seqencing data are not generated. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 22

26 TrSeq DNA PCR-Free Reference Gide Table 8 Reagent Fnction Control Reagent ERP 2 or ERP 3 ERP 2 or ERP 3 ATL or ATL 2 Inline Control Fnctions End repair: Generate blnt ended fragments by 3 > 5 exonclease and 5 > 3 polymerase activities. End repair: Add 5 -phosphate grops needed for downstream ligation. A-tailing: Make fragments compatible with adapters and prevent self-ligation by adding a 3 -A overhang LIG 2 Ligation: Join 3 -T overhang adapters to 3 - A overhang inserts CTE 1* CTE 2* CTA CTL Strctre of Control DNA Ends 5 overhang at one end, 3 overhang at the other end Blnt with 5 -OH grop Blnt with 5 -phosphate grop Single-base 3 A base overhang *CTE 1 and CTE 2 are separate controls inclded in the CTE reagent. Inline controls can be sed for varios insert sizes. Each control is provided in ladders ranging from abot 150 bp throgh 850 bp in 100 bp increments. Each control molecle has a niqe DNA seqence that indicates fnction and size. Becase the size selection step precedes A-tailing, CTE 1 and CTE 2 show a narrow size distribtion while CTA and CTL show a broad size distribtion. Inline controls are optional for identifying a specific mode of failre and other trobleshooting. Yo can replace inline controls with eqal volmes of RSB. When seqencing data are not generated from a library, inline controls are ninformative. Consmables and Eqipment Make sre that yo have the reqired ser-spplied consmables and eqipment before starting the protocol. Items that are niqe to the HS or LS workflow are indicated. 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 1.7 ml microcentrifge tbes General lab spplier 15 ml conical tbes General lab spplier 10 µl barrier pipette tips General lab spplier 10 µl mltichannel pipettes General lab spplier 10 µl single channel pipettes 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 Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 23

27 TrSeq DNA PCR-Free Reference Gide Consmable Spplier 1000 µl mltichannel pipettes General lab spplier 1000 µl single channel pipettes General lab spplier 96-well storage plates, rond well, 0.8 ml (midi plate) Adhesive seal roller Ethanol 200 proof (absolte) for moleclar biology (500 ml) Ice bcket KAPA Library Qantification Kit - Illmina/Universal Microseal 'B' adhesive seals microtube AFA Fiber 6x16mm with: Crimp-Cap or Pre-Slit Snap-Cap (for se with Covaris M220) PCR-grade water RNase/DNase-free 8-tbe strips and caps Thermo Fisher Scientific, part # AB-0859 General lab spplier Sigma-Aldrich, part E7023 General lab spplier KAPA Biosystems, part # KK4824 Bio-Rad, part # MSB-1001 Covaris, part # or General lab spplier General lab spplier RNase/DNase-free mltichannel reagent reservoirs, disposable VWR, part # Tris-HCl 10 mm, ph 8.5 Tween 20 [Optional] High Sensitivity NGS Fragment Analysis Kit General lab spplier Sigma-Aldrich, part # P7949 Advanced Analytical, catalog # DNF-474 [Optional] High Sensitivity DNA Kit Agilent Technologies, part # Additional Consmables for HS Workflow Consmable 96-well Hard-Shell 0.3 ml PCR plate Spplier Bio-Rad, part # HSP-9601 Additional Consmables for LS Workflow Consmable 96-well 0.3 ml skirtless PCR plates or Twin.tec 96 well PCR plates Spplier E&K Scientific, part # or Eppendorf, part # Eqipment Eqipment [Optional] Fragment Analyzer [Optional] 2100 Bioanalyzer Desktop System One of the following Covaris systems: S2 S220 E210 M220 Magnetic stand-96 Microplate centrifge Vortexer qpcr system Spplier Advanced Analytical, catalog # FSV2CE2F Agilent Technologies, part # G2940CA Covaris M220, part # * Thermo Fisher Scientific, catalog # AM10027 General lab spplier General lab spplier General lab spplier Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 24

28 TrSeq DNA PCR-Free Reference Gide * Contact Covaris for all other models. Additional Eqipment for HS Workflow Eqipment Spplier High-Speed Microplate Shaker VWR, catalog # (110 V/120 V) or (230 V) SciGene TrTemp Heating System¹ Illmina, catalog # SC (110 V) or SC (220 V) Midi plate insert for heating system² Stroboscope Illmina, catalog # BD General lab spplier ¹ Two systems are recommended to spport sccessive heating procedres. ² Two inserts are recommended to spport sccessive heating procedres. Additional Eqipment for LS Workflow Eqipment 96-well thermal cycler with heated lid Spplier General lab spplier Thermal Cyclers The following table lists the recommended specifications for the thermal cycler. If yor lab has a thermal cycler that is not listed, validate it before starting the protocol. Thermal Cycler Temp Mode Lid Temp Vessel Type Bio-Rad DNA Engine Tetrad 2 Calclated Heated, constant at 100 C Plate MJ Research PTC-225 DNA Engine Tetrad Calclated Heated, constant at 100 C Plate Bio-Rad S1000 N/A Heated, constant at 100 C Plate qpcr Systems The following table lists the validated qpcr systems for the TrSeq DNA PCR-Free protocol. Eqipment CFX96 Toch Real-Time PCR Detection System* Spplier Bio-Rad, part # Mx3000P qpcr System Agilent, part # * Use CFX Manager software version 3.0 with Cq Determination mode: Single Threshold; Baseline Setting:Baseline Sbtracted Crve Fit and Apply Florescent Drift Correction for data analysis. This setting can correct for abnormalities in florescence intensity of the standard crve cased by the instrment. For software installation, contact Bio-Rad. Index Adapter Seqences For information on index adapter seqences, see Illmina Adapter Seqences (docment # ) which provides information regarding the ncleotide seqences that comprise Illmina oligoncleotides sed in Illmina seqencing technologies. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 25

29 TrSeq DNA PCR-Free Reference Gide Acronyms Acronym Definition ALP Adapter Ligation Plate ATL A-Tailing Mix CAP Clean Up ALP Plate CEP Clean Up End Repair Plate CFP Covaris Fragmentation Plate CSP Clean Up Sheared DNA Plate CTA A-Tailing Control CTE End Repair Control CTL Ligation Control DCT Dilted Clster Template Plate DNA Cstomer Sample DNA Plate ERP End Repair Mix HS High Sample IEM Illmina Experiment Manager IMP Insert Modification Plate LIG Ligation Mix LRM Local Rn Manager LS Low Sample PDP Pooled Diltion Plate RSB Resspension Bffer SPB Sample Prification Beads STL Stop Ligation Bffer TSP1 Target Sample Plate 1 Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 26

30 TrSeq DNA PCR-Free Reference Gide 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 # v00 For Research Use Only. Not for se in diagnostic procedres. 27

31 Docment # v00 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.