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TruSeq Nano DNA Sample Preparation Guide 15041110 B

1. 13 Perform End Repair and Size Selection 2 2 2 2 eee cece ccc eceececccceccceceeceeeeeeeceeeees 19 Adenylate Sh En c RT 26 MEV ALS Hielo ice euros no rt a a ea 28 Enrich DNA Fragments ooooccccccccccccccccccccccccoc nn 36 Validate Library cc 41 Nomalize and Pool Libraries cesse tete sc c o eibi s c pnis od y i fuc 43 sorar ans e wt e Qu SEA TOC GG Ck ry vegas tci 1C4 U TruSeq Nano DNA Sample Preparation Guide O c Je1deuo Low Sample LS Protocol Introduction 10 This chapter describes the TruSeq Nano DNA Sample Preparation LS protocol This protocol is intended for preparing up to 24 samples at one time using either the LT or HT kit Follow the protocol in the order described using the specified volumes and incubation parameters Review Best Practices before proceeding See Additional Resources on page 6 for information on how to access TruSeq Nano DNA Sample Preparation Best Practices on the Illumina website Review the TruSeg Sample Preparation Pooling Guide part 15042173 before proceeding See Additional Resources on page 6 for information on how to download the guide from the Illumina website Review Appendix A Supporting Information before proceeding to confirm your kit contents and make sure that you have obtained all of the requisite equipment and consumables for the LS protocol Part 15041110 Rev B Sample Prep Workflow The following figure illustrates the proces
2. ILLUMINA PROPRIETARY Catalog FC 121 9010DOC Part 15041110 Rev B November 2013 This document and its contents are proprietary to Illumina Inc and its affiliates Illumina and are intended solely for the contractual use of its customer in connection with the use of the product s described herein and for no other purpose This document and its contents shall not be used or distributed for any other purpose and or otherwise communicated disclosed or reproduced in any way whatsoever without the prior written consent of Illumina Illumina does not convey any license under its patent trademark copyright or common law rights nor similar rights of any third parties by this document The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order to ensure the proper and safe use of the product s described herein All of the contents of this document must be fully read and understood prior to using such product 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 OR ANY USE OF SUCH PRODUCT S OUTSIDE THE SCOPE OF THE EXPRESS WRITTEN LICENSES OR PERMISSIONS GRANTED BY ILLUMINA IN
3. LT kit 1 tube per 24 15 C to 25 C reactions or HT kit 1 tube per 48 reactions 1 tube 2 C to 8 C 1 tube per 24 ZF C tio GC reactions LT kit 1 tube per 24 15 C to 25 C reactions or HT kit 1 tube per 48 reactions This process ligates multiple indexing adapters to the ends of the DNA fragments Supplied By Illumina Illumina Illumina Illumina Illumina Part 15041110 Rev B Item Quantity Storage Supplied By Barcode labels for 1 label per plate IBC 0 SAC Illumina CAP Clean Up ALP Plate DAP DNA Adapter Plate if using the HT kit PCR Polymerase Chain Reaction Plate 96 well 0 3 ml PCR plates 2 15 C to 30 C User Freshly prepared 80 ethanol 800 ul per sample TIC O BOE User EtOH Microseal B adhesive seals 2 15 C to 30 C User RNase DNase free eight tube Sy TIS C tio STU CC User strips and caps if using multichannel pipettes RNase DNase free reagent 3 27 15 C to 30 C User reservoirs if using multichannel pipettes Preparation Remove the following from 15 C to 25 C storage and thaw them at room temperature Appropriate DNA Adapter tubes depending on the DNA Adapter Indices being used or the DAP NOTE Review the TruSeq Sample Preparation Pooling Guide part 15042173 See Additional Resources on page 6 for information on how to download the guide from the Illumina website e When indexing libraries using adapter index tubes Ilumina recomme
4. TruSeq Nano DNA Sample Preparation Guide 8 3 SeueJqr OOd pue ezieuuoN If pooling 49 96 samples Using a multichannel pipette transfer 5 ul of each normalized sample library in column 1 of the DCT plate to column 1 of the new MIDI plate labeled with the PDP barcode Transfer 5 ul of each normalized sample library in column 2 of the DCT plate to column 1 of the PDP plate Repeat the transfer for as many times as there are remaining columns in the DCT plate The result is a PDP plate with pooled samples in column 1 Mix the PDP plate as follows Seal the PDP plate with a Microseal B adhesive seal Shake the PDP plate on a microplate shaker at 1800 rpm for 2 minutes Centrifuge the PDP plate at 280 x g for 1 minute Remove the adhesive seal from the PDP plate Combine the contents of each well of column 1 into a 1 7 ml microcentrifuge tube for the final pool High Sample HS Protocol 3 Mix as follows depending on the item that contains the final pool For the PDP plate Seal the PDP plate with a Microseal B adhesive seal Shake the PDP plate on a microplate shaker at 1800 rpm for 2 minutes Centrifuge the PDP plate at 280 x g for 1 minute For the 1 7 ml microcentrifuge tube Cap the tube Vortex the tube to mix thoroughly 4 Do one of the following Proceed to cluster generation For more information see the cluster generation section of the user guide for your Illumina platform Store the sealed PDP plate or capped 1 7 m
5. c cece cece cece cccceeeceeeccceceeeeceeececeeceeeereeseee 3 Insert Size Options 2 22 e cece ccceeeeeeeeeeeees 3 Insert Size Options sss cece 0000 a001 a 222r 2222 13 Covaris S220 Settings esa reene ren eE ie a e NEE 15 Covans M220 Settings lesson aaa E irte SEEE lin 16 Covaris S2 and E210 Settings 2 0 0 0 0 cece cece ccc e eee cee eceeeeeees 16 Diluted Bead Mixture for a 350 bp Insert Size sse 22 Diluted Bead Mixture for a 550 bp Insert Size 2 22 Insert Size ODHODnS cortar tue cU I Duiei te EDD hat e NL ewes 51 Covaris S220 Settings 2 0 0 2 cee cece cece cece cece cece Re erre rere 53 Covans M220 Settings soi ccesdeaseced teas torear ltda 54 Covaris S2 and E210 Settings 0 0002 ee 54 Diluted Bead Mixture for a 350 bp Insert Size lle 60 Diluted Bead Mixture for a 550 bp Insert Size 2 2 60 TruSeq Nano DNA Sample Preparation Acronyms 22222222 87 TruSeq Nano DNA Sample Prep Kits 0 oooocccccccccccccccccccccccccccccccccoos 89 User Supplied Consumables sssssssss sensns ssi 95 User Supplied Consumables Additional Items for LS Processing 97 User Supplied Consumables Additional Items for HS Processing 97 User Supplied Equipment 0 0 000000 sese 97 User Supplied Equipment Additional Items for LS Processing 98 User Supplied Equipment Additional I
6. MIDI plate insert for heating system Illumina catalog BD 60 601 Note Two inserts are recommended to support successive heating procedures Stroboscope General lab supplier SciGene TruTemp Heating System Illumina catalog SC 60 503 115 V Note Two systems are recommended Illumina catalog SC 60 504 220 V to support successive heating procedures Part 15041110 Rev B Thermal Cyclers The following table lists the recommended settings for the Illumina recommended thermal cycler as well as other comparable models If your lab has a thermal cycler that is not listed validate the thermal cycler before performing the TruSeq Nano DNA Sample Preparation protocol Thermal Cycler Bio Rad DNA Engine Tetrad 2 MJ Research PTC 225 DNA Engine Tetrad Bio Rad S1000 TruSeq Nano DNA Sample Preparation Guide Temp Mode Calculated Calculated N A Lid Temp Heated constant at 100 C Heated constant at 100 C Heated constant at 100 C Vessel Type Plate Plate Plate 99 juaudinby pue sejqeuunsuoy Supporting Information Indexed Adapter Sequences This section details the indexed adapter sequences TruSeq Nano DNA LT Sample Prep Kit Indexed Adapter Sequences The TruSeq Nano DNA LT Sample Prep Kit contains the following indexed adapter sequences NOTE E The index numbering is not contiguous There is no Index 17 24 or 26 The base in parentheses indicates the base for the seve
7. TruSeq Nano DNA Sample Preparation Guide 91 SJU9 UOD 1M Supporting Information Figure 11 TruSeq Nano DNA LT Sample Prep Kit 24 Samples SP Beads Box 2 of 2 part 15041758 Slot Reagent Part Description 1 SPB 15041032 Sample Purification Beads TruSeq Nano DNA HT Sample Prep Kit The TruSeq Nano DNA HT Sample Prep Kit contains three boxes a core reagent box an Adapter Plate box and an SP Beads box 96 Samples Core Reagents Box Store at 15 C to 25 C This box is shipped on dry ice As soon as you receive it store the following components at 15 C to 25 C This box also contains plate barcode labels Figure 12 TruSeq Nano DNA HT Sample Prep Kit 96 Samples Box 1 of 2 part 15041877 92 Part 15041110 Rev B Slot Reagent Part Description 1 2 RSB 15026770 Resuspension Buffer 34 End Repair Mix 2 56 A Tailing Mix 7 8 LIG2 15036184 Ligation Mix 2 9 10 15012546 Stop Ligation Buffer 11 12 PPC 15031748 PCR Primer Cocktail 13 14 15041027 Enhanced PCR Mix 96 Samples Adapter Plate Box Store at 15 C to 25 C This box is shipped on dry ice As soon as you receive it store the contents at 15 C to 25 C Figure 13 TruSeq Nano DNA HT Sample Prep Kit 96 Adapter Plate Box part 15032317 Slot Reagent Part Description 1 DAP 15016426 DNA Adapter Plate 96plex 96 Samples SP Beads Box Store at 2 C to 8 C This box is shipped at 2 C to 8 C As soon as you recei
8. 25 C storage Incubate IMP 1 Place the sealed IMP plate on the pre heated microheating system Close the lid and incubate at 30 C for 30 minutes 2 Remove the IMP plate from the microheating system and place the plate on ice until you are ready for the next step Clean Up IMP and Size Selection 1 Remove the adhesive seal from the IMP plate Remove Large DNA Fragments 1 Vortex the Sample Purification Beads for at least 1 minute or until they are well dispersed TruSeq Nano DNA Sample Preparation Guide 5 9 uonoejegs ezig pue Jreday pug wIou d High Sample HS Protocol Add the Sample Purification Beads and PCR grade water to one of the following tubes to create a diluted bead mixture of 160 ul per 100 ul of end repaired sample New 15 ml conical tube when processing gt 6 samples at a time New 1 7 ml microcentrifuge tube when processing lt 6 samples at a time Determine the volumes using the following formulas which include 15 excess for multiple samples Table 13 Diluted Bead Mixture for a 350 bp Insert Size Formula Example Amount Your per 12 Calculation samples Sample Purification Beads of samples X 10923 ul BI PCR grade water of samples X 897 ul 74 75 ul Table 14 Diluted Bead Mixture for a 550 bp Insert Size Formula Example Amount Your per 12 Calculation samples Sample Purification Beads of samples X 1104 ul 92 ul PCR grade water of samples X 1104 ul 92 ul Vortex the diluted bead mi
9. ul then gently pipette the entire volume up and down 10 times to mix thoroughly 5 Incubate the PCR plate at room temperature for 5 minutes 6 Place the PCR plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear 7 Remove and discard 95 ul of the supernatant from each well of the PCR plate NOTE k Leave the PCR plate on the magnetic stand while performing the following 80 EtOH wash steps 8 10 TruSeq Nano DNA Sample Preparation Guide 3 9 s juauBe14 vNG u9uu3 Low Sample LS Protocol 10 11 12 13 14 15 16 17 40 With the PCR plate on the magnetic stand add 200 ul freshly prepared 80 EtOH to each well without disturbing the beads Incubate the PCR plate at room temperature for 30 seconds and then remove and discard all of the supernatant from each well Repeat steps 8 and 9 one time for a total of two 8076 EtOH washes With the PCR plate on the magnetic stand let the samples air dry at room temperature for 5 minutes Remove and discard any remaining EtOH from each well of the PCR plate with a 10 ul pipette With the PCR plate on the magnetic stand add 32 5 ul Resuspension Buffer to each well of the PCR plate Remove the PCR plate from the magnetic stand Resuspend the beads in each well of the PCR plate by repeatedly dispensing the Resuspension Buffer over the bead pellet until it is immersed in the solution Gently pipette the entire volume up and down 10 times t
10. 240 ul If pooling 25 48 samples Using a multichannel pipette transfer 5 ul of each normalized sample library in column 1 of the DCT plate to column 1 of the new 0 3 ml PCR or MIDI plate labeled with the PDP barcode Transfer 5 ul of each normalized sample library in column 2 of the DCT plate to column 1 of the PDP plate Repeat the transfer for as many times as there are remaining columns in the DCT plate The result is a PDP plate with pooled samples in column 1 Gently pipette the entire volume of each well of column 1 up and down 10 times to mix thoroughly Combine the contents of each well of column 1 into well A2 of the PDP plate for the final pool If pooling 49 96 samples Using a multichannel pipette transfer 5 ul of each normalized sample library in column 1 of the DCT plate to column 1 of the new MIDI plate labeled with the PDP barcode Transfer 5 ul of each normalized sample library in column 2 of the DCT plate to column 1 of the PDP plate Repeat the transfer for as many times as there are remaining columns in the DCT plate The result is a PDP plate with pooled samples in column 1 Gently pipette the entire volume of each well of column 1 up and down 10 times to mix thoroughly Combine the contents of each well of column 1 into a 1 7 ml microcentrifuge tube for the final pool TruSeq Nano DNA Sample Preparation Guide A 5 SeueJqr OOd pue SZI EULUON Low Sample LS Protocol 3 Gently pipette the entire volume up and down 10
11. CSP plate Take care not to disturb the beads E NOTE Leave the CSP plate on the magnetic stand while performing the following steps 8 12 8 With the CSP plate on the magnetic stand add 200 ul freshly prepared 80 EtOH to each well without disturbing the beads 9 Incubate the CSP plate at room temperature for 30 seconds and then remove and discard all of the supernatant from each well Take care not to disturb the beads TruSeq Nano DNA Sample Preparation Guide 5 5 VNG Juauwbely High Sample HS Protocol 96 10 11 12 13 14 15 16 17 18 19 20 Repeat steps 8 and 9 one time for a total of two 80 EtOH washes With the CSP plate on the magnetic stand let the samples air dry at room temperature for 5 minutes Remove and discard any remaining EtOH with a 10 ul pipette With the CSP plate on the magnetic stand add 62 5 ul Resuspension Buffer to each well of the plate Remove the CSP plate from the magnetic stand Mix thoroughly as follows a Seal the CSP plate with a Microseal B adhesive seal b Shake the CSP plate on a microplate shaker at 1800 rpm for 2 minutes Incubate the CSP plate at room temperature for 2 minutes Centrifuge the CSP plate at 280 x g for 1 minute Remove the adhesive seal from the CSP plate Place the CSP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear Transfer 60 ul of the clear supernatant from each well of the CSP plate to the co
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13. Illumina User 19 VNG 1ueuuDeJJ Low Sample LS Protocol Item Covaris tubes DNA samples Freshly prepared 8076 ethanol EtOH Microseal B adhesive seal Preparation Review DNA Input Recommendations on page 4 Quantity 1 per sample 100 ng per sample for a 350 bp insert size Or 200 ng per sample for a 550 bp insert size 400 ul per sample Storage 15 C to 30 C 15 C to 25 C 15 C to 30 C ISS to SUE Supplied By User User User User Review Best Practices for Handling Magnetic Beads See Additional Resources on page 6 for information on how to access TruSeq Nano DNA Sample Preparation Best Practices on the lumina website Remove the Sample Purification Beads from 2 C to 8 C storage and let stand for at least 30 minutes to bring them to room temperature Remove one tube of Resuspension Buffer from 15 C to 25 C storage and thaw it at room temperature E NOTE The Resuspension Buffer can be stored at 2 C to 8 C after the initial thaw Turn on the Covaris instrument and follow the manufacturer s guidelines to set up your instrument Apply a CFP barcode label to a new 96 well 0 3 ml PCR plate Apply a CSP barcode label to a new 96 well 0 3 ml PCR plate Apply a DNA barcode label to a new 96 well 0 3 ml PCR plate Apply an IMP barcode label to a new 96 well 0 3 ml PCR plate 14 Part 15041110 Rev B Make CFP 1 Illumina recommends quantifying gDNA s
14. PDP plate if you are not pooling samples 1 Determine the number of samples to be combined together for each pool NOTE Make a note of which sample goes into which well to avoid pooling two samples with the same index 2 Doone of the following If pooling 2 24 samples Transfer 10 ul of each normalized sample library to be pooled from the DCT plate to one well of the new MIDI plate labeled with the PDP barcode The total volume in each well of the PDP plate is 10X the number of combined sample libraries and 20 240 ul 2 24 libraries For example the volume for 2 samples is 20 ul the volume for 12 samples is 120 ul or the volume for 24 samples is 240 ul If pooling 25 48 samples Using a multichannel pipette transfer 5 ul of each normalized sample library in column 1 from the DCT plate to column 1 of the new MIDI plate labeled with the PDP barcode Transfer 5 ul of each normalized sample library in column 2 of the DCT plate to column 1 of the PDP plate Repeat the transfer for as many times as there are remaining columns in the DCT plate The result is a PDP plate with pooled samples in column 1 Mix the PDP plate as follows Seal the PDP plate with a Microseal B adhesive seal Shake the PDP plate on a microplate shaker at 1800 rpm for 2 minutes Centrifuge the PDP plate at 280 x g for 1 minute Remove the adhesive seal from the PDP plate Combine the contents of each well of column 1 into well A2 of the PDP plate for the final pool
15. Part 4 Revision Date 15041110 November 2013 15041110 May 2013 TruSeq Nano DNA Sample Preparation Guide Description of Change Renamed Incubate 1 IMP to Incubate IMP Added recommended thermal cycler settings to Consumables and Equipment Initial Release Part 15041110 Rev B Table of Contents Revision History ooccccccccccccccccccccccccccccnnccccnncnn cnn Table of Contents o oooccccccccccccccccccccccccccccccncnnnncnccco List of Tables eee a a ea e e araea ni Chapter 1 Overview oc ccc ccoo coo n nn ncccccccccccccccno Introduction 2 2 2 oe ese cece c cece cee ce cceecsceeeeseeeeees Protocol Features 22 ccc c eee ccccce cece ceecccceeee DNA Input Recommendations sseeueuuse Positive Control 2 2 2 0 eco eee cece cece cece cceeeeeceeees Chapter 2 Low Sample LS Protocol INTFOGUCTION ERR Seeks talco ais asses Sample Prep Workflow 2 222 2222222 e eee eeeeee eee Prepare Adapter Setup 22 22 e eee ee Fragment DNA 0022 0022222 e cece cece cece ccc cece cece cece eeees Perform End Repair and Size Selection Adenylate 3 Ends o ooocooccccccccccccccccccccocccccccnnnncccco Ligate Adapters 22 2 0 ooo eee cece cece cece sese sese Enrich DNA Fragments 20 222 cece eee ee eee eee ee Validate Library 20 2 22 22 eee Chapter 3 High Sample HS Protoco
16. Per Sample 100 ng 200 ng Recommended Read Length lt 2x 101 bp lt 2 x 151 bp Read lengths greater than 2 x 151 bp produce a significantly higher percentage of overlapping read pairs TruSeq Nano DNA Sample Preparation Guide 3 soJnjee 0901014 Overview DNA Input Recommendations It is important to quantitate the input DNA and assess the DNA quality before performing TruSeq Nano DNA Sample Preparation Input DNA Quantitation Follow these DNA input recommendations Correct quantification of gDNA is essential 100 ng input DNA is recommended for the 350 bp insert size workflow and 200 ng for the 550 bp insert size workflow The ultimate success or failure of library preparation strongly depends on using an accurately quantified amount of input DNA Illumina recommends using fluorometric based methods for quantification including Qubit or PicoGreen to provide accurate quantification of dsDNA UV spectrophotometric based methods such as the Nanodrop measure any nucleotides present in the sample including RNA dsDNA ssDNA and free nucleotides which can give an inaccurate measurement of gDNA Use multiple methods of quantification to verify results DNA quantification methods that rely on intercalating fluorescent dyes measure only double stranded DNA and are less subject to the presence of excess nucleic acids These methods require the preparation of calibration curves and are highly sensitive to pipetting error Make sure tha
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18. and let stand for at least 30 minutes to bring them to room temperature Pre program the thermal cycler with the following program and save as LIG Choose the thermal cycler pre heat lid option and set to 100 C 30 C for 10 minutes Hold at 4 C Apply a CAP barcode label to a new 96 well 0 3 ml PCR plate Apply a PCR barcode label to a new 96 well 0 3 ml PCR plate Add LIG 1 Do one of the following If using DNA Adapter tubes centrifuge the thawed tubes at 600 x g for 5 seconds 30 Part 15041110 Rev B fusing a DAP Thaw the plate for 10 minutes at room temperature on the benchtop Visually inspect the wells to make sure that they all are thawed Remove the adapter plate tape seal Centrifuge the plate at 280 x g for 1 minute to collect all of the adapter to the bottom of the well Remove the plastic cover Save the cover if you are not processing the entire plate at one time If it is the first time using this DAP apply the DAP barcode label to the plate Centrifuge the Stop Ligation Buffer tube at 600 x g for 5 seconds Immediately before use remove the Ligation Mix 2 tube from 15 C to 25 C storage Remove the adhesive seal from the ALP plate Add 2 5 ul Resuspension Buffer to each well of the ALP plate Add 2 5 ul Ligation Mix 2 to each well of the ALP plate Return the Ligation Mix 2 tube to 15 C to 25 C storage immediately after use oo N A 0 A C N Do one of the following If using DNA Adapter
19. are by products of inefficiencies in the ligation reaction Neither species can be used to make clusters Fragments without any adapters cannot hybridize to surface bound primers in the flow cell Fragments with an adapter on only one end can hybridize to surface bound primers but cannot form clusters Consumables Item Quantity Storage Supplied By Enhanced PCR Mix EPM LT kit 1 tube per 24 15 C to 25 C Ilumina reactions or HT kit 1 tube per 48 reactions PCR Primer Cocktail PPC LT kit 1 tube per 24 15 C to 25 C Ilumina reactions or HT kit 1 tube per 48 reactions Sample Purification Beads SPB 1 tube per 24 2 C to 8 C Illumina reactions Resuspension Buffer RSB 1 tube PTO E Ilumina Barcode labels for 1 label per plate 15 C to 30 C Illumina e CPP Clean Up PCR Plate e TSP1 Target Sample Plate 96 well HSP plate il lot oS 029 User Part 15041110 Rev B Item Quantity Storage Supplied By 96 well MIDI plate 1 15 C to 30 C User Freshly prepared 80 ethanol 400 ul per sample TIC O SE User EtOH Microseal A film 1 15 C to 30 C User Microseal B adhesive seals 3 IAC 0 HOKE User RNase DNase free eight tube 5 15 C to 30 C User strips and caps if using multichannel pipettes RNase DNase free reagent 5 15 C to 30 C User reservoirs if using multichannel pipettes Preparation Remove the Enhanced PCR Mix and PCR Primer Cocktail from 15 C to 25 C storage and thaw them at room temper
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21. generates dsDNA fragments with 3 or 5 overhangs The fragmentation process is optimized to obtain final libraries with the following average insert sizes Table 9 Insert Size Options Insert Size Input DNA Per Sample Recommended Read Length 350 bp 100 ng lt 2x101 bp 550 bp 200 ng lt 2x151 bp Read lengths greater than 2 x 151 bp produce a significantly higher percentage of overlapping read pairs Consumables Item Resuspension Buffer RSB Sample Purification Beads SPB Barcode labels for CFP Covaris Fragmentation Plate CSP Clean Up Sheared DNA Plate DNA DNA Plate MP Insert Modification Plate 96 well HSP plate TruSeq Nano DNA Sample Preparation Guide Quantity 1 tube 1 tube per 24 reactions 1 label per plate Storage 15 C to 25 C 2 C to 8 C after initial thaw 2C io BAC 15 C to 30 C ISS tro SUE Supplied By Illumina Illumina Illumina User 51 VNG Juauwbely High Sample HS Protocol Item Quantity Storage Supplied By 96 well MIDI plates 3 15 C to 30 C User Covaris tubes 1 per sample ISAC SAC User DNA samples 100 ng per sample 15 C to 25 C User for a 350 bp insert size or 200 ng per sample for a 550 bp insert size Freshly prepared 80 ethanol 400 ul per sample 15 to 30 C User EtOH Microseal B adhesive seal 1 15 C to 30 C User Preparation Review DNA Input Recommendations on page 4 Review
22. high library yields 100 ng for a 350 bp insert size 200 ng for a 550 bp insert size 1 Add 5 ul thawed PCR Primer Cocktail to each well of the PCR plate 2 Add 20 ul thawed Enhanced PCR Mix to each well of the PCR plate a Seal the PCR plate with a Microseal A film WARNING Follow vendor instructions for applying Microseal A sealing films Improper use could zi lead to inefficient sealing evaporation of sample or cross contamination or too efficient sealing parts of the seal remain in the well after removing the whole seal b Shake the PCR plate on a microplate shaker at 1600 rpm for 20 seconds 3 Centrifuge the PCR plate at 280 x g for 1 minute 76 Part 15041110 Rev B Amp PCR Place the sealed PCR plate containing 50 ul of each sample on the pre programmed thermal cycler Close the lid then select and run PCRNano to amplify the plate a Choose the pre heat lid option and set to 100 C b 95 C for 3 minutes c 8 cycles of 98 C for 20 seconds 60 C for 15 seconds 72 C for 30 seconds d 72 C for 5 minutes e Hold at 4 C Clean Up PCR e O Mon Centrifuge the PCR plate at 280 x g for 1 minute Remove the adhesive seal from the PCR plate Vortex the Sample Purification Beads until they are well dispersed Do one of the following depending on the adapter type used If using the DNA Adapter tubes add 50 ul mixed Sample Purification Beads to each well of the new MIDI plate labeled with the CPP barcode If usin
23. nM using Tris HCl 10 mM pH 8 5 with 0 176 Tween 20 i NOTE Depending on the yield quantification data of each sample library the final volume in the DCT plate can vary from 10 400 pl Gently pipette the entire normalized sample library volume up and down 10 times to mix thoroughly Depending on the type of library you want to generate do one of the following For non pooled libraries the protocol stops here Do one of the following Proceed to cluster generation For more information see the cluster generation section of the user guide for your Illumina platform Seal the DCT plate with a Microseal B adhesive seal and store at 15 C to 25 C For pooled libraries proceed to Make PDP for pooling only Make PDP for pooling only 44 D NOTE Do not make a PDP plate if you are not pooling samples Part 15041110 Rev B 1 Determine the number of samples to be combined together for each pool NOTE j Note the sample that is in each well to avoid pooling two samples with the same index 2 Doone of the following If pooling 2 24 samples Transfer 10 ul of each normalized sample library to be pooled from the DCT plate to one well of the new 0 3 ml PCR plate labeled with the PDP barcode The total volume in each well of the PDP plate is 10 X the number of combined sample libraries and 20 240 ul 2 24 libraries For example the volume for 2 samples is 20 ul the volume for 12 samples is 120 ul or the volume for 24 samples is
24. preparing them for hybridization onto a flow cell Supplied By Illumina Illumina Illumina Illumina Illumina Item Quantity Storage Supplied By Barcode labels for 1 label per plate ISAC o SAC Illumina CAP Clean Up ALP Plate DAP DNA Adapter Plate if using the HT kit PCR Polymerase Chain Reaction Plate 96 well HSP plate 1 15 C to 30 C User 96 well MIDI plate il ISC o BOC User Freshly prepared 80 800 ul per sample 15 C to 30 C User ethanol EtOH Ice bucket As needed 15 to 25 User Microseal B adhesive seals 7 15 C to 30 C User RNase DNase free eight 9 27 15 to 30 User tube strips and caps if using multichannel pipettes RNase DNase free reagent 3 27 15 C to 30 C User reservoirs if using multichannel pipettes Preparation Prepare an ice bucket Remove the following from 15 C to 25 C storage and thaw them at room temperature Appropriate DNA Adapter tubes depending on the DNA Adapter Indices being used or the DAP TruSeq Nano DNA Sample Preparation Guide 67 sJejdepy oe1e6r High Sample HS Protocol 68 P NOTE Review the TruSeq Sample Preparation Pooling Guide part 15042173 See Additional Resources on page 6 for information on how to download the guide from the Illumina website e When indexing libraries using adapter index tubes Ilumina recommends arranging samples that are going to be combined into a common pool in the
25. room temperature for 5 minutes Remove and discard any remaining EtOH with a 10 ul pipette With the CEP plate on the magnetic stand add 20 ul Resuspension Buffer to each well of the plate Remove the CEP plate from the magnetic stand Resuspend the beads in each well of the CEP plate by repeatedly dispensing the Resuspension Buffer over the bead pellet until it is immersed in the solution Gently pipette the entire volume up and down 10 times to mix thoroughly Part 15041110 Rev B 14 Incubate the CEP plate at room temperature for 2 minutes 15 Place the CEP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear 16 Transfer 17 5 ul of the clear supernatant from each well of the CEP plate to the corresponding well of the new 0 3 ml PCR plate labeled with the ALP barcode SAFESTOPPING POINT If you do not plan to proceed immediately to Adenylate 3 Ends on page 26 the protocol can 1 be safely stopped here If you are stopping seal the ALP plate with a Microseal B adhesive seal and store at 15 C to 25 C for up to 7 days TruSeq Nano DNA Sample Preparation Guide 2 5 uonoejegs ezig pue Jreday pug wIou d Low Sample LS Protocol Adenylate 3 Ends A single A nucleotide is added to the 3 ends of the blunt fragments to prevent them from ligating to one another during the adapter ligation reaction A corresponding single T nucleotide on the 3 end of the adapter
26. samples that will be combined into a common pool in the same row Include a common index in each column This arrangement facilitates pipetting operations when dispensing indexed adapters and pooling indexed libraries later in the protocol If you are pooling with the DAP arrange samples that will be pooled together in the same orientation as the indices in the DAP Part 15041110 Rev B Fragment DNA This process describes how to optimally fragment the gDNA depending on the downstream application Covaris shearing generates dsDNA fragments with 3 or 5 overhangs The fragmentation process is optimized to obtain final libraries with the following average insert sizes Table 3 Insert Size Options Insert Size Input DNA Per Sample Recommended Read Length 350 bp 100 ng lt 2x101 bp 550 bp 200 ng lt 2x151 bp Read lengths greater than 2 x 151 bp produce a significantly higher percentage of overlapping read pairs Consumables Item Quantity Resuspension Buffer RSB 1 tube Sample Purification Beads SPB 1 tube per 24 reactions Barcode labels for 1 label per plate CFP Covaris Fragmentation Plate CSP Clean Up Sheared DNA Plate DNA DNA Plate MP Insert Modification Plate 96 well 0 3 ml PCR plates 4 TruSeq Nano DNA Sample Preparation Guide Storage 15 C to 25 C 2 C to 8 C after initial thaw 2C io BAC 15 C to 30 C T O SUE Supplied By Illumina Illumina
27. this Products Specifications or Documentation or iv any Excluded Claim Conditions to Indemnification Obligations The parties indemnification obligations are conditioned upon the party seeking indemnification i promptly notifying the other party in writing of such claim or action ii giving the other party exclusive control and authority over the defense and settlement of such claim or action iii not admitting infringement of any intellectual property right without prior written consent of the other party iv not entering into any settlement or compromise of any such claim or action without the other party s prior written consent and v providing reasonable assistance to the other party in the defense of the claim or action provided that the party reimburses the indemnified party for its reasonable out of pocket expenses incurred in providing such assistance Third Party Goods and Indemnification Illumina has no indemnification obligations with respect to any goods originating from a third party and supplied to Purchaser Third party goods are those that are labeled or branded TruSeq Nano DNA Sample Preparation Guide V with a third party s name Purchaser s indemnification rights if any with respect to third party goods shall be pursuant to the original manufacturer s or licensor s indemnity Upon written request Illumina will attempt to pass through such indemnity if any to Purchaser Part 15041110 Rev B Revision History
28. times to mix thoroughly 4 Do one of the following Proceed to cluster generation For more information see the user guide for your Illumina sequencer Do one of the following depending on the item that contains the final pool Seal the PDP plate with a Microseal B adhesive seal and store at 15 C to 25 C Cap the 1 7 ml microcentrifuge tube and store at 15 C to 25 C 46 Part 15041110 Rev B High Sample HS Protocol Introduction A ean oa Ob den an 48 Sample Prep Workflow sssssssssssssssII III cece cece ee eeeeeeeeeeeeeeteteetttteteees 49 Prepare Adapter Setup sone 2 oci dar it ile 50 denaib 51 Perform End Repair and Size Selection ssuuuuuuuuuuuesseeeeessssssssssss sess 57 Adenylate 3 Ends tese aeree acer ene Ded DD be rne ism D rU D nh becodad 64 MENG ALS givelo r ice a sn hs sn a deere eft laa 66 Enrich DNA Fragments 00 00002000 c cece ccc nn 74 Validate Library cc 79 Nomalize and Pool Libraries csse sh eiea anere Eipre tear SAd G aee Er ERSS 81 j n m amd acq dn SSeS ETT e gt af 4 6 re m a m Ar recae TruSeq Nano DNA Sample Preparation Guide AT e Jejdeuo High Sample HS Protocol Introduction 48 This chapter describes the TruSeq Nano DNA Sample Preparation HS protocol This protocol is intended for preparing more than 24 samples at one time using e
29. tube ZIXC O BAC Ice bucket As needed 15 C to 25 C Microseal B adhesive seal 1 15 C to 30 C RNase DNase free eight tube 2 15 C to 30 C strips and caps if using multichannel pipettes RNase DNase free reagent 2 ISC to 30 E reservoirs if using multichannel pipettes Preparation Prepare an ice bucket Supplied By Illumina Illumina User User User User Remove the A Tailing Mix from 15 C to 25 C storage and thaw it at room temperature Remove the Resuspension Buffer from 2 C to 8 C storage and bring it to room temperature 64 Part 15041110 Rev B Add ATL Remove the ALP plate from 15 C to 25 C storage if it was stored at the conclusion of Clean Up IMP and Size Selection on page 59 Let it thaw at room temperature Centrifuge the thawed ALP plate at 280 x g for 1 minute Remove the adhesive seal from the ALP plate Pre heat two microheating systems system 1 to 37 C and system 2 to 70 C Centrifuge the thawed A Tailing Mix tube at 600 x g for 5 seconds Add 12 5 ul thawed A Tailing Mix to each well of the ALP plate Mix thoroughly as follows a Seal the ALP plate with a Microseal B adhesive seal b Shake the ALP plate on a microplate shaker at 1800 rpm for 2 minutes Centrifuge the ALP plate at 280 x g for 1 minute Return the A Tailing Mix tube to 15 to 25 C storage Incubate 1 ALP 1 Place the sealed ALP plate on the pre heated microheating system 1 Close
30. tubes add 2 5 ul thawed DNA Adapter Index to each well of the ALP plate Set a 200 ul pipette to 35 ul then gently pipette the entire volume up and down 10 times to mix thoroughly Ifusing a DAP Place the DAP on the benchtop so that the part number barcode on the long side of the plate is facing you and the clipped corner is on the lower left Figure 2 Correct DAP Orientation e gt gt gt gt gt gt gt TruSeq Nano DNA Sample Preparation Guide 31 sJo1depy o1e6r Low Sample LS Protocol Do one of the following to pierce the foil seal If using the entire plate at one time use the bottom of a clean 96 well semi skirted PCR plate to pierce a hole in all of the well seals simultaneously Gently but firmly press the clean plate over the foil seal If using only part of the plate use the bottom of a clean eight tube strip with caps attached to pierce holes in the seals of the wells that will be used for ligation Repeat with a new clean eight tube strip with caps attached for each row or column of adapters that will be used for ligation Using an eight tip multichannel pipette transfer 2 5 ul thawed DNA Adapter from the DAP well to each well of the ALP plate Set a 200 ul pipette to 35 ul then gently pipette the entire volume up and down 10 times to mix thoroughly 9 Seal the ALP plate with a Microseal B adhesive seal 10 Centrifuge the ALP plate at 280 x g for 1 minute Incu
31. 3 24 25 26 27 28 29 Transfer 50 ul of the clear supernatant from each well of the ALP plate to the corresponding well of the new 0 3 ml PCR plate labeled with the CAP barcode Take care not to disturb the beads Vortex the Sample Purification Beads until they are well dispersed Add 50 ul mixed Sample Purification Beads to each well of the CAP plate for a second cleanup Set a 200 ul pipette to 90 ul and then gently pipette the entire volume up and down 10 times to mix thoroughly Incubate the CAP plate at room temperature for 5 minutes Place the CAP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear Remove and discard 95 ul of the supernatant from each well of the CAP plate Take care not to disturb the beads NOTE j Leave the CAP plate on the magnetic stand while performing the following steps 21 25 With the CAP plate on the magnetic stand add 200 ul freshly prepared 8076 EtOH to each well Take care not to disturb the beads Incubate the CAP plate at room temperature for 30 seconds and then remove and discard all of the supernatant from each well Take care not to disturb the beads Repeat steps 21 and 22 one time for a total of two 80 EtOH washes With the CAP plate on the magnetic stand let the samples air dry at room temperature for 5 minutes Remove and discard any remaining EtOH with a 10 ul pipette With the CAP plate on the magnetic stand add 27 5 ul Resusp
32. ACK IAAGALIACT IGATSCACTGA CAACGTA CO VEA S CL LM posa ie M E ACCGTAACGAACG TATCAATTGAGACTAAATAT TAACG TACCAT TAAGAGC TACCGTGCAACGACGAAAAGAATGATAACAG TAACACACT TCTGTTAACCTT ACGAAAAGAATGATAACAG TAACACACT TCTGT TAACCT TAAGAT TACTT GAT CCACTGATTCAACGTACCGTAAAGAT TACT TGATCCACTGAT I CAACGTACCGTAACGAACG TATCAAT TGAGACTAAATAT TAACG TACCATTAAGAGOTACCE O ESOO ARTO TT AECA AATGATAACAGTAACACACT TCTGTTAACCTTAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAGC TACCGTCTTCTGTTAACCTTAAGAT TACTTGATCCACTGATTCAACGTA ITACTTGATCCACTGAT TCAACGTTIAAGAT TACTT GAT CCACT GATT CAACGTACCGTAACGAACGTATCAATT GAGCT TCTGT TAACCT TAAGAT TACTT GATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT I GAGACTAGCAACGACGAA ACC C G AACGACGAAAAGAATGAT ACACTTCTGTTAACC MAC TTOMCCACTEATTCAACGTTAAGALTACTTGATCCACT GATTCAAGGTACCGTAACGAACGTATCAATIGAGCHICTOL AACCTTAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAGCAACGACGAA Mee ee ee a EL TT T AT ee ATTE ee eee er A LT a doe TMA Ea ATT TRAC GAL LANA AATGATAACAGTAACACACTTCTGTTAACCT TAAGAT TA SABE a aE o ee E LP e CTAAATAT TAACGTACCAT TAAGAGCTACCGTCTTCTGTTAACCTTAAGAT TACT TGATCCACTGAT TCAACGTACCGTAA NTCCACTGATTCAACGTACCAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TGAGAC TAAATAT TAACGTACCAT TAAGAGC TACCGTCTTCTGT TAACCT TAAGAT TACTTGAT CCACTGAT TCAACGTACCGTAACGAAC eg CA aR Toy ee ELTE ATA OT NEA AINT EMT Tea RACE TTA ASTRA LCG inl TAA DAE AT LARA GUI AATGATAACAG TAACACACT TCTGTTAACCTTAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAA
33. Best Practices for Handling Magnetic Beads See Additional Resources on page 6 for information on how to access TruSeq Nano DNA Sample Preparation Best Practices on the Illumina website Remove the Sample Purification Beads from 2 C to 8 C storage and let stand for at least 30 minutes to bring them to room temperature Remove one tube of Resuspension Buffer from 15 C to 25 C storage and thaw it at room temperature 1 NOTE The Resuspension Buffer can be stored at 2 C to 8 C after the initial thaw Turn on the Covaris instrument and follow the manufacturer s guidelines to set up your instrument Calibrate the microplate shaker with a stroboscope and set it to 1800 rpm Apply a CFP barcode label to a new 96 well HSP plate Apply a CSP barcode label to a new 96 well MIDI plate Apply a DNA barcode label to a new 96 well MIDI plate Apply an IMP barcode label to a new 96 well MIDI plate Part 15041110 Rev B Make CFP 1 Illumina recommends quantifying gDNA samples using a fluorometric quantification method that uses dsDNA binding dyes Normalize the gDNA samples with Resuspension Buffer to one of the following in each well of the new 0 3 ml PCR plate labeled with the DNA barcode 100 ng in a final volume of 52 5 ul for a 350 bp insert size 200 ng in a final volume of 52 5 ul for a 550 bp insert size Fragment DNA 1 Shear one of the following amounts of gDNA sample by transferring 52 5 ul of each DNA sample from the DNA plate to a se
34. CAACGACGAAAAGAAT GATAACAGTAACACA CCA CCTTAAGATTACTT C A VT TGAGACTAAATAT TAACG A AA CGAACTTCTGTTAA 3CIACCGTGCAACGAAAATAACCT TAAGATTACT TGATCCACTGAT TCAACGTACTTCTGTTAACCTTAAGAT TACTTGATCCACTGATTCAACGTACCG TAACGAACG TATCAAT TGAGACTAAGCTACCGTGCAACGACGAAAAGAATGAT A ae wise O MADE e A die EQUI USE M AOV Ate E PIE A YE a Sg VER AREIS TS S EUIS 3ATAACAGTAACACACT TCTGTTAACCT T Su AES E S pie p RH SEES TCAATTGAGACT m Uu CGACGAAAAGAATGATAACAGTAACACAC TTC TGT I CCAT TAAGAGC TACCGTGCAACAGTAACACACT TCTGTTAACCTTAAGAT TACT TGATCCACTGAT TCAACGTACCG TAACGAACGTAT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTGCAACGACGAAAAGAAT GATAA 3aATAACAGTAACACACT TCTGTTAACCT TAAGAT TACTTGATCCACTGAT TCAACGTACCG TAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCG TCTTCTGT TAACCTTAAGAT TACTTGATCCACTGAT TCAACG STACCGT eU RE ee og OE A E vr M aces ATCAATTG O CUPS TTAAGAGCTACCGTGCAACGACGAAAAGAATGATAACAGTAACACACTTCTGTTAACCT TA JAUNES A LA CGTTAAGATTACTTGATCCACTGATTCAACG TACCGTAACGAACG TATCAAT TGAGCTICTGTIAACCTTY RAGATTAOT TE EAT TAL TET RON TCAATTGAGACTAGCAACGACQG NIN GAG TAACACACTICIGTIAACGT MARA ee ee eee ew UN roe NAG gla AN Moyle Near GTAACGAACGTATCAAT TGAGACTAAATATTAACGTACCAT TAAGAGC TACC SATAA ACTRAC ACACTTCTGTTAACCTTAAGAT TACTTGATCCACTGATTCAACGTACCG TAACGAACG TATCAAT TGAGACTAAA AN KAGAGC TACCGTCTTCTGTIAACCTIAAGATIACTTGA YER EAT a m TCAT TAAGAT TACTTGATCCACTGATTCAACGTACCGTAACGAACG TAT CAAT TGAGACTAAATAT TAACG TACCAT TAAGAGC TACCGTGCAACGACGAAAAGAATGATAACAG TAACACACT TCTG
35. CG TATCAAT TGAGACTAAATATTAACGTACCAT TAAGAGC TACCGTCT TCTGT TAACCT TAAGATTACTTGATCCACTGAT TCAACGTA AAT TGAGACTAAATAT TAACGTTGTTAACCTTAAGATTACTTGATCCACTGAT TCAACG TACCGTAACGAACG TATCAATTGAGACTAAATATTAACGTACCAT TAAGAGCT TCTGT TAACCT TAAGAT TACTTGATCCACTGAT TCAACGTACCGTAAC ERA CERE DM E MAT HA A LATTE EUM AA TE TCAA M Ad GAGAC TAAATAT TAACGTACCAT TAAGAGCTACAACCTTAAGA BEC SE SEES DO ne TCAAT TGAGACTAAATAT TAACG TACCAT TAAGAGCTACCGTGCAACGACGAAAAGAAT GA NEA AATGATAACAGTAACACACT TCTGTTAACCTTAAGAT TACTTGATCCACTGAT T CAACGTACCGTAACGAACGTA ARA CIC TACCOTOTIOTO TAAGO TTAAGATTACITGATCOACIGA AACG GTACCATTAAGAGC TACCGTGCAACTTAACCTTAAGAT TACTTGATCCACTGATTCAACGTACCGTAACGAACGTAT CAAT TGAGACTAAATAT ATTA AC AAGATIACTIGATCC ES ee TE AACCTT EE CTGTTAACCTTAAGATTACTTGATCCAC GAAAAGAATGATAA ACGAAAAGAATGATAACAG TAACACACTTCTGT TAACCT T TTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGAT C ITTAAGAGCTACCGT AATGATAACAGTAACACACTICTGTTAACCTTAAG KIA TIOATOGACT GATT GAACGTACCOTAACGAACG TAT CANT GAGAG TA ICACACTTCTGTTAZ p M M REIS STS ae CU eee A GAATGATAACA AATGATAACAGTAACACACTICTGTTAACCTT TTGATCCACTGATTCAACGTACCGTAACGAA TCAATTGAGACTA CTGATTCAACGT CO COCA GAACGATOATIAAGATTACT AT GOAGTGATTOAACQTA AA C AA AT CARTTQACA CTAAATAT T TGTTAACCTTAAC I TACTTGATCCACTGATTCAACGTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACG TATCAAT TGAGCT TCTGT TAACI AGCAACGACGAA ACGAAAAGAATGATAACAGTAACACACT CTGTTAACCT TAAGATTACTTGATCCACTGAT TCAACGTACCGTAAAGAT TACTTGATC AAGAGCTACCGT FOR RESEARCH USE ONLY
36. CONNECTION WITH CUSTOMER S ACQUISITION OF SUCH PRODUCT S FOR RESEARCH USE ONLY O 2013 Illumina Inc All rights reserved Illumina IlluminaDx BaseSpace BeadArray BeadXpress cBot CSPro DASL DesignStudio Eco GAIIx Genetic Energy Genome Analyzer GenomeStudio GoldenGate HiScan HiSeq Infinium iSelect MiSeq Nextera NuPCR SeqMonitor Solexa TruSeq TruSight VeraCode the pumpkin orange color and the Genetic Energy streaming bases design are trademarks or registered trademarks of Illumina Inc All other brands and names contained herein are the property of their respective owners Read Before Using this Product This Product and its use and disposition is subject to the following terms and conditions If Purchaser does not agree to these terms and conditions then Purchaser is not authorized by Illumina to use this Product and Purchaser must not use this Product 1 Definitions Application Specific IP means Illumina owned or controlled intellectual property rights that pertain to this Product and use thereof only with regard to specific field s or specific application s Application Specific IP excludes all Illumina owned or controlled intellectual property that cover aspects or features of this Product or use thereof that are common to this Product in all possible applications and all possible fields of use the Core IP Application Specific IP and Core IP are separate non overlapping subsets of all Illumina owned or
37. E Transfer do not discard the supernatant It contains the DNA of interest 10 Repeat step 9 one time Each CEP plate well now contains a total of 250 ul of DNA of interest 11 Discard the IMP plate containing the beads 12 Discard any remaining diluted bead mixture Remove Small DNA Fragments lg NOTE In the following steps use undiluted Sample Purification Beads 1 Vortex the Sample Purification Beads for at least 1 minute or until they are well dispersed 2 Add30 ul undiluted Sample Purification Beads to each well of the CEP plate containing 250 ul of supernatant with the DNA of interest Mix thoroughly as follows a Seal the CEP plate with a Microseal B adhesive seal b Shake the CEP plate on a microplate shaker at 1800 rpm for 2 minutes TruSeq Nano DNA Sample Preparation Guide 61 uonoejes ezig pue Jreday pug uuopegd High Sample HS Protocol 62 ON 0 m Q 10 11 12 13 14 NOTE Aspirate the Sample Purification Beads slowly and dispense them slowly due to the viscosity of the solution Changes in the volume of the bead mixture affect the insert size of your library NOTE Vortex the Sample Purification Beads frequently to make sure that they are evenly distributed Illumina recommends the following If using a single channel pipette vortex the beads after processing four samples If using a multichannel pipette vortex the beads after processing four columns If the beads are in a reagent
38. GTAACACACT TCTGT TAACCT TAAGAT TACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGC TACCGTCTTCT AAGAT T BOT GAT RG an O as GTACCGT AA ANC ATCATTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGAC TAAATAT TAACG TACCATTAAGAGC TACCGTGCAACGA CORABACAA AGAT TAACAGTAACACACTTCTGTTAACCT T STTGATOCAGT GAIT GAACG TTAAGATTACTTGATCCACTGATT GAACGTACCGTAACGAACGTATCAATT GAGCTTCTG HACC AAGAT TACT IGATCCAG TGA CAAGOTACCGTAACGAACG TA CARTTGAGACTAGGAACGACG AAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATATTAACGTACCATTAAGAGCTACC lllumina San Diego California U S A 1 800 809 ILMN 4566 1 858 202 4566 outside North America techsupport illumina com www illumina com
39. IGTGY IAACCLTAAGATIACT TGATCGACT GATT GAACGTAGCG AAAGAT AC EGATOCAC GAT ICAAGGTACCGTAACGAACGIATCAAI TGAGAC TAAATATTAACGTAGCATTAAGAGC AGO e a GTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACG TATCAA LO E ARR eiue GATTACTTGATCCACTGATTCAACG JGAGACTAAATAT TAACGTTGTTAACCTTAAGAT TACT TGATCCACTGATTCAACGTACCGTAACGAACGTAT CAATT GAGACTAAATAT TAACGTACCAT T GCTTCTGTTAACCT TAAGAT TACTTGATCCACTGAT TCAACGTACCGTA ATCAATTGAGA CTAANTALT AACGTACTTAACCTIAAGAT TACTTGATCCACTGATTCAACGTACCGTAACGAACG TCTTCTGT TAACCT TAAGAT T KETIGMCCACIGATTCAACG TACCGTAACGAACGTATCAATTGAGACTAACGACGA E AE 3ATAACAGTAACACACTT AACCTTAAGATTACTTGATCCACTGATTCAACG TACCG TAACGAACG T SS MN TRATTE TET CTGATTCAACG CCA I TANG AGCIACCOTECAACTIAACCTY AAGA HAGTIGATCCACIGATTCAACCTACECTARCOAACOTA TCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTGCAACGACGAACTICTGTT RACCTIANGATTAGTIGAT TACOS TGCAACGAAAATAACC TTAAGATTACT IGATCCACTGAT ICAACGTAGTICTG TAAGGTTAAGATTACT IGATCCACT GAL CAACGACOGTAACGAACG TAI GAAT TGAGAG TAAGGAGCGIGCAACGACGAAAAGAATAT AAAAGAATGA e de UL R AACCTIAAGATTACTT EE AE GMT S M AAAGATTACTTGAT VER ACTGATICAACGTACCGTAACGAACG TA TTGAGACTAAATAT TAACGTACCAT TAAGAGCTACC 3ATAACAGTAACACACT TCTGTTAACCT TAAGAT TACT T PORT OCACTGALICAACGT ACCGTAACGAA ACTA TCAAT TGAGACTAAATAT TAACGT AC CATAAGAGCTACCGTOCAA AOGA OINA GA TAACAGTAACACACTTCTGTI CCATTAAGAGCTACEGT SC AACAGTAACACAC E TGTOT IAACCTTAAGATTACT GAT GGAGTGATTCAA OTACOG IAACGAACG IAT CAAT GAGAGTAAATAT TAACGTACGAI TAAGAGCTACCG I GCAACGACGARAAGAAT SATAN 3ATAACA
40. IN CONNECTION WITH THESE TERMS AND CONDITIONS INCLUDING WITHOUT LIMITATION THIS PRODUCT INCLUDING USE THEREOF AND ILLUMINA S PERFORMANCE HEREUNDER WHETHER IN CONTRACT TORT INCLUDING NEGLIGENCE STRICT LIABILITY OR OTHERWISE SHALL IN NO EVENT EXCEED THE AMOUNT PAID TO ILLUMINA FOR THIS PRODUCT TruSeq Nano DNA Sample Preparation Guide Limitations on Illumina Provided Warranties TO THE EXTENT PERMITTED BY LAW AND SUBJECT TO THE EXPRESS PRODUCT WARRANTY MADE HEREIN ILLUMINA MAKES NO AND EXPRESSLY DISCLAIMS ALL WARRANTIES EXPRESS IMPLIED OR STATUTORY WITH RESPECT TO THIS PRODUCT INCLUDING WITHOUT LIMITATION ANY IMPLIED WARRANTY OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE NONINFRINGEMENT OR ARISING FROM COURSE OF PERFORMANCE DEALING USAGE OR TRADE WITHOUT LIMITING THE GENERALITY OF THE FOREGOING ILLUMINA MAKES NO CLAIM REPRESENTATION OR WARRANTY OF ANY KIND AS TO THE UTILITY OF THIS PRODUCT FOR PURCHASER S INTENDED USES Product Warranty All warranties are personal to the Purchaser and may not be transferred or assigned to a third party including an affiliate of Purchaser All warranties are facility specific and do not transfer if the Productis moved to another facility of Purchaser unless Illumina conducts such move a Warranty for Consumables Illumina warrants that Consumables other than custom Consumables will conform to their Specifications until the later of i 3 months from the date of shipment from Illumina
41. PPC pad RSB Wer SPB Plato ea E zu TSP1 1 4 Validate Library Adenylate 3 Ends Consumables ATL 4 RSB Y Plato Normalize and Pool ALP Libraries Consumables Tris HCl 10 mM w Tween 20 Plates DCT PDP pooling only TruSeq Nano DNA Sample Preparation Guide A 9 High Sample HS Protocol Prepare Adapter Setup o0 If you are pooling record information about your samples before beginning library preparation for later use in data analysis Use IEM to create and edit a sample sheet for Illumina sequencers and analysis software See Additional Resources on page 6 for information on how to download IEM software and documentation from the Illumina website Review planning steps in the TruSeq Sample Preparation Pooling Guide part 15042173 See Additional Resources on page 6 for information on how to download the guide from the Illumina website If you are pooling using adapter index tubes Illumina recommends arranging samples that will be combined into a common pool in the same row Include a common index in each column This arrangement facilitates pipetting operations when dispensing indexed adapters and pooling indexed libraries later in the protocol If you are pooling with the DAP arrange samples that will be pooled together in the same orientation as the indices in the DAP Part 15041110 Rev B Fragment DNA This process describes how to optimally fragment the gDNA depending on the downstream application Covaris shearing
42. Preparation Guide 6 9 SJo1depy b High Sample HS Protocol Do one of the following to pierce the foil seal If using the entire plate at one time use the bottom of a clean 96 well semi skirted PCR plate to pierce a hole in all of the well seals simultaneously Gently but firmly press the clean plate over the foil seal If using only part of the plate use the bottom of a clean eight tube strip with caps attached to pierce holes in the seals of the wells that will be used for ligation Repeat with a new clean eight tube strip with caps attached for each row or column of adapters that will be used for ligation Using an eight tip multichannel pipette transfer 2 5 ul thawed DNA Adapter from the DAP well to each well of the ALP plate 9 Mix thoroughly as follows a Seal the ALP plate with a Microseal B adhesive seal b Shake the ALP plate on a microplate shaker at 1800 rpm for 2 minutes 10 Centrifuge the ALP plate at 280 x g for 1 minute Incubate 2 ALP 1 Incubate the ALP plate on the pre heated microheating system with the lid closed at 30 C for 10 minutes 2 Remove the ALP plate from the microheating system and place the plate on ice until you are ready for the next step Add STL 1 Remove the adhesive seal from the ALP plate 2 Add5 ul Stop Ligation Buffer to each well of the ALP plate to inactivate the ligation mix Mix thoroughly as follows a Seal the ALP plate with a Microseal B adhesive seal b Shake
43. T TAACC T T IAAAAGAATGATAACAGTAACACACT TCTGTTAACC T TAAGAT TACTTGAT CCACTGAT TCAACGTACCGTAAAGAT TACTT AT CACTGATICAACGTAGCOTAACGAACETATOAATTGAGAC TANAAN TAA C GIA OATTARGAGU ACG CCAT TAAGAGCTACCGTGCAACAGTAACACACT TCTGTTAACCTTAAGATTACT TGATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGT GCAACGACGAAAAGAAT GATAA 3ATAACAGTAACACACTTCTGTTAACCT TAAGATTACTTGATCCACTGATTCAACGTACCG TAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGC TACCG TCTTCTGTTAACCT TAAGAT TACTTGATCCACTGAT TCAACG Z2LTGATCCACTGATTCAACGTTAAGAT TACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGCT TCTGT TAACCTTAAGAT TACT TGATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TGAGAC TAGCAACGACQ A a O DARET C ACA AA ON Galt ore aL GTACCGTAACGAACGTATCATTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACG TATCAAT TGAGA P pe E ACCGTGCAACGACGAAAAGAATGATAACAG TAACACACT TCTGT TAACCT A ZTTGATCCACTGATTCAACGTTIAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACG TATCAAT TGAGCTTCTGT TAACCT TAAGAT TACT TGAT CCACTGAT TCAACG TACCGTAACGAACGTAT CAAT TGAGACTAGCAACGACG IAAAAGAATGATAACAGTAACACAC T TCTGT TAACCT TAAGAT TACTT GATCCACTGAT TCAACGTACCGTAAAGATTACT TGATCCACTGAT TCAACGTACCGTAACGAACG TATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACC 3ATAACAGTAACACACT TCTGTTAACCT TAAGAT TA quU A di eie SIL RE I Real AR SCIRE MAUS ARES ATTACTT OE Oe cU E ZACTGATTCAACGTACCAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTCTTCTGT TAACCT TAAGAT TACT TGATCCACTGATTCAACGTACCG TAACGA AAAAGANTGATAACAGTAAGAGAGT
44. TGATCCACT GAT TCAACGTACCGTAACGAACGTATCAATT GAGACTAAATAT TAACG TACCA WD I e CGACG TICTGTT Se ET GAGCTACCGTGCAACGAAAATAACCTTAAGATTACT TGATCCACTGAT TCAACGTACTTCTGTTAACCTTAAGAT TACTTGATCCACTGAT TCAACGTACCGTAACGAACGTA TRATIGAGACTAARCIACCOTECAACGA CGAAAAGAATGAT A GRAAAGAATQATARCAGTARCACAG CTO IAACCTAAGATTACT TGATCCACT GAT TORACOTACOS AAAGATTAC TGATCCAGTGAT I CAAGG ACTAS GAAGG IATGAALI GAGACIAAATATTAACGIAGCAT TAA GAG TACCA AATGATAACAGTAACACACTICTGTTAACCTTAAGA Quodam aes CCGTAACGAACG TATCAAT TGAGACT Ree er A e E CI E CGACGAAAAGAA UES a para Eve pees TE Ala GTACCAT TAAGAGC TACCGTGCAACAGTAACACACT TCTGT TAACC TTAAGAT TACTT GATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TGAGAC TAAATAT TAACGTACCAT TAAGAGCTACCGTGCAACGACGAAAAGAATGATAACA AATOGATAACAGTAACACACTIOTOTTAACOTIAAGATTACTIGATOGACTGA MT OAACOTACCGIAACOAACOTATCAATI GAGA TAAATATTAAGGTACCATAAGHOCTASCOTCTTOTC MAACO TIAAGATIACH GATGCACTGATICANCGTA MACGTACCGTAACGAACGTATCATTAAGATIA DEM CENA eU UAI ED S ally ig STG re a S afe CGAAAAGAATGA ae ed CTTCTGTTAACCTTAAC I TACTTGATCCACTGATTCAACGTTAAGATTACTTGATCCACTGATTICAACGTACCGTAACGAACG TAT CAAT TGAGCTTCTGTTAACCT TAAGAT TACT TGATCCACT ES EAR E CES VA TCAAT TGAGACTAGCAACGACGAA ACGAAAAGAATGATAACAGTAACACAC T TCTGTTAACCT TAAGAT TACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGAT TCAACG TACCGTAACGAACG TATCAAT TGAGAC TAAATATTAACGTACCAT TAAGAGC TACCGT AATGATAACAG TAACACACT TC TGT TAACCT TAAGAT TACT TGATCCACTGAT TCAACG TACCG TAACGAACG TATCAAT TGAGA ET TATTAACGTA SCANAAGAGCIAGCGTCTTCT GI T
45. adhesive seal b Shake the CEP plate on a microplate shaker at 1800 rpm for 2 minutes Incubate the CEP plate at room temperature for 2 minutes Centrifuge the CEP plate at 280 x g for 1 minute Remove the adhesive seal from the CEP plate Place the CEP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear Transfer 17 5 ul of the clear supernatant from each well of the CEP plate to the corresponding well of the new MIDI plate labeled with the ALP barcode SAFESTOPPING POINT If you do not plan to proceed to Adenylate 3 Ends on page 64 immediately the protocol can 1 be safely stopped here If you are stopping seal the ALP plate with a Microseal B adhesive seal and store at 15 C to 25 C for up to 7 days TruSeq Nano DNA Sample Preparation Guide 6 3 uonoejegs ezig pue Jreday pug wIou d High Sample HS Protocol Adenylate 3 Ends A single A nucleotide is added to the 3 ends of the blunt fragments to prevent them from ligating to one another during the adapter ligation reaction A corresponding single T nucleotide on the 3 end of the adapter provides a complementary overhang for ligating the adapter to the fragment This strategy ensures a low rate of chimera concatenated template formation Consumables Item Quantity Storage A Tailing Mix ATL LT kit 1 tube per 24 15 C to 25 C reactions or HT kit 1 tube per 48 reactions Resuspension Buffer RSB 1
46. amples using a fluorometric quantification method that uses dsDNA binding dyes Normalize the gDNA samples with Resuspension Buffer to one of the following in each well of the new 0 3 ml PCR plate labeled with the DNA barcode 100 ng in a final volume of 52 5 ul for a 350 bp insert size 200 ng in a final volume of 52 5 ul for a 550 bp insert size Fragment DNA 1 Shear one of the following amounts of gDNA sample by transferring 52 5 ul of each DNA sample from the DNA plate to a separate new Covaris tube 100 ng for a 350 bp insert size 200 ng for a 550 bp insert size Use the wells of the new 0 3 ml PCR plate labeled with CFP barcode or another device to hold the Covaris tubes upright L NOTE Load the DNA sample into the Covaris tube slowly to avoid creating air bubbles However air bubbles might not be preventable Centrifuge the CFP plate at 600 x g for 5 seconds Fragment the DNA using the following settings Table 4 Covaris S220 Settings Setting 350 bp Insert 550 bp Insert Duty factor 5 Peak Incident Power 175 W Cycles per burst 200 Duration 50 seconds 25 seconds Mode Frequency sweeping Temperature 55 HD OC TruSeq Nano DNA Sample Preparation Guide 1 5 VNG JUaubely Low Sample LS Protocol 16 Table 5 Covaris M220 Settings Setting 350 bp Insert 550 bp Insert Duty factor 20 Peak Incident Power 50 W Cycles per burst 200 Duration 65 seconds 45 seconds Temperature 20 C 4 NOTE The Covaris M220 se
47. and ii any expiration date or the end of the shelf life pre printed on such Consumable by Illumina but in no event later than 12 months from the date of shipment With respect to custom Consumables i e Consumables made to specifications or designs made by Purchaser or provided to Illumina by or on behalf of Purchaser Illumina only warrants that the custom Consumables will be made and tested in accordance with Illumina s standard manufacturing and quality control processes Illumina makes no warranty that custom Consumables will work as intended by Purchaser or for Purchaser s intended uses b Warranty for Hardware Illumina warrants that Hardware other than Upgraded Components will conform to its Specifications for a period of 12 months after its shipment date from Illumina unless the Hard ware includes Illumina provided installation in which case the warranty period begins on the date of installation or 30 days after the date it was delivered whichever occurs first Base Hardware Warranty Upgraded Components means Illumina provided components modifications or enhancements to Hardware that was previously acquired by Purchaser Illumina warrants that Upgraded Components will conform to their Specifications for a period of 90 days from the date the Upgraded Components are installed Upgraded Components do not extend the warranty for the Hardware unless the upgrade was conducted by Illumina at Illumina s facilities in which case the upgraded
48. ared 80 ethanol EtOH TruSeq Nano DNA Sample Preparation Guide Quantity LT kit 1 tube per 24 reactions or HT kit 1 tube per 48 reactions 1 tube 1 tube per 24 reactions 1 label per plate p 400 ul per sample Storage 15 C to 25 C LC O BAC 2 C to 8 C ISS to SUE 15 C to 30 C IBC to SUE 15 C to 30 C Supplied By Ilumina Ilumina Ilumina Ilumina User User User 19 uonoejes ezig pue Jreday pug uuopegd Low Sample LS Protocol Item Quantity Storage Supplied By Ice bucket As needed AAC 1 2529 User Microseal B adhesive seals 2 15 C to 30 C User PCR grade water 1 bottle 15 C to 302 C User RNase DNase free eight tube 6 15 C to 30 C User strips and caps if using multichannel pipettes RNase DNase free reagent 6 15 to 30 C User reservoirs if using multichannel pipettes Preparation Make IMP 1 20 Prepare an ice bucket Remove the End Repair Mix 2 from 15 C to 25 C storage and thaw it at room temperature Place the tube on ice Remove the Sample Purification Beads and Resuspension Buffer from 2 C to 8 C storage and bring them to room temperature Review Best Practices for Handling Magnetic Beads See Additional Resources on page 6 for information on how to access TruSeq Nano DNA Sample Preparation Best Practices on the Illumina website Pre program the thermal cycler with the following program and save as ERP Choose the thermal
49. ature Centrifuge the thawed Enhanced PCR Mix and PCR Primer Cocktail tubes to 600 x g for 5 seconds Remove the Resuspension Buffer from 2 C to 8 C storage and bring it to room temperature Remove the Sample Purification Beads from 2 C to 8 C storage and let stand for at least 30 minutes to bring them to room temperature Review Best Practices for Handling Magnetic Beads See Additional Resources on page 6 for information on how to access TruSeq Nano DNA Sample Preparation Best Practices on the Illumina website Remove the PCR plate from 15 C to 25 C storage if it was stored at the conclusion of Clean Up ALP on page 70 Let it thaw at room temperature Centrifuge the thawed PCR plate at 280 x g for 1 minute Remove the adhesive seal from the thawed PCR plate TruSeq Nano DNA Sample Preparation Guide v 5 sjuauBe14 vNG u9uu3 High Sample HS Protocol Pre program the thermal cycler with the following program and save as PCRNano Choose the pre heat lid option and set to 100 C 95 C for 3 minutes 8 cycles of 98 C for 20 seconds 60 C for 15 seconds 72 C for 30 seconds 72 C for 5 minutes Hold at 4 C Igy NOTE Ilumina recommends 8 cycles of PCR for robust protocol performance Apply a CPP barcode label to a new 96 well MIDI plate Apply a TSP1 barcode label to a new 96 well HSP plate Make PCR The following procedure assumes the following amount of input DNA sample for library preparation and is designed to result in
50. bate 2 ALP 1 Place the sealed ALP plate containing 37 5 ul of each sample on the pre programmed thermal cycler Close the lid then select and run the LIG program a Choose the thermal cycler pre heat lid option and set to 100 C b 30 C for 10 minutes c Hold at 4 C 2 Remove the ALP plate from the thermal cycler when the program reaches 4 C Add STL 1 Remove the adhesive seal from the ALP plate 2 Add 5 ul Stop Ligation Buffer to each well of the ALP plate to inactivate the ligation Set a 200 ul pipette to 40 ul then gently pipette the entire volume up and down 10 times to mix thoroughly Clean Up ALP 1 Vortex the Sample Purification Beads for at least 1 minute or until they are well 32 dispersed Part 15041110 Rev B 10 11 12 13 14 Add 42 5 ul well mixed Sample Purification Beads to each well of the ALP plate Set a 200 ul pipette to 75 ul and then gently pipette the entire volume up and down 10 times to mix thoroughly NOTE f Vortex the Sample Purification Beads frequently to make sure that they are evenly distributed Illumina recommends the following If using a single channel pipette vortex the beads after processing four samples If using a multichannel pipette vortex the beads after processing four columns sJo1depy e1e6r f the beads are in a reagent reservoir mix with a 1000 ul pipette Incubate the ALP plate at room temperature for 5 minutes Place the ALP plate on the magnetic stan
51. controlled intellectual property By way of non limiting example Illumina intellectual property rights for specific diagnostic methods for specific forensic methods or for specific nucleic acid biomarkers sequences or combinations of biomarkers or sequences are examples of Application Specific IP Consumable s means Illumina branded reagents and consumable items that are intended by Illumina for use with and are to be consumed through the use of Hardware Documentation means Illumina s user manual for this Product including without limitation package inserts and any other documentation that accompany this Product or that are referenced by the Product or in the packaging for the Product in effect on the date of shipment from Illumina Documentation includes this document Hardware means Illumina branded instruments accessories or peripherals Illumina means Illumina Inc or an Illumina affiliate as applicable Product means the product that this document accompanies e g Hardware Consumables or Software Purchaser is the person or entity that rightfully and legally acquires this Product from Illumina or an Illumina authorized dealer Software means Illumina branded software e g Hardware operating software data analysis software All Software is licensed and not sold and may be subject to additional terms found in the Software s end user license agreement Specifications means Illumina s written specifications for this Produc
52. cycler pre heat lid option and set to 100 C 30 C for 30 minutes Hold at 4 C Apply an ALP barcode label to a new 96 well 0 3 ml PCR plate Apply a CEP barcode label to a new 96 well 0 3 ml PCR plate Centrifuge the thawed End Repair Mix 2 tube at 600 x g for 5 seconds Part 15041110 Rev B 2 Add 40 ul End Repair Mix 2 to each well of the IMP plate containing the fragmented DNA Set a 200 ul pipette to 95 ul and then gently pipette the entire volume up and down 10 times to mix thoroughly 3 Seal the IMP plate with a Microseal B adhesive seal 4 Return the End Repair Mix 2 tube to 15 C to 25 C storage Incubate IMP 1 Place the sealed IMP plate on the pre programmed thermal cycler Close the lid then select and run the ERP program a Choose the thermal cycler pre heat lid option and set to 100 C b 30 C for 30 minutes c Hold at 4 C 2 Remove the IMP plate from the thermal cycler when the program reaches 4 C Clean Up IMP and Size Selection 1 Remove the adhesive seal from the IMP plate Remove Large DNA Fragments 1 Vortex the Sample Purification Beads for at least 1 minute or until they are well dispersed TruSeq Nano DNA Sample Preparation Guide 2 1 uonoejes ezig pue Jreday pug uuonegd Low Sample LS Protocol Add the Sample Purification Beads and PCR grade water to one of the following tubes to create a diluted bead mixture of 160 ul per 100 ul of end repaired sample New 15 ml conical tube when processi
53. d at room temperature for 5 minutes or until the liquid is clear Remove and discard 80 ul of the supernatant from each well of the ALP plate Take care not to disturb the beads NOTE j Leave the ALP plate on the magnetic stand while performing the following steps 6 10 With the ALP plate on the magnetic stand add 200 ul freshly prepared 80 EtOH to each well without disturbing the beads Incubate the ALP plate at room temperature for 30 seconds and then remove and discard all of the supernatant from each well Take care not to disturb the beads Repeat steps 6 and 7 one time for a total of two 8076 EtOH washes With the ALP plate on the magnetic stand let the samples air dry at room temperature for 5 minutes Remove and discard any remaining EtOH with a 10 ul pipette With the ALP plate on the magnetic stand add 52 5 ul Resuspension Buffer to each well of the plate Remove the ALP plate from the magnetic stand Resuspend the beads in each well of the ALP plate by repeatedly dispensing the Resuspension Buffer over the bead pellet until it is immersed in the solution Gently pipette the entire volume up and down 10 times to mix thoroughly Incubate the ALP plate at room temperature for 2 minutes Place the ALP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear TruSeq Nano DNA Sample Preparation Guide 3 3 Low Sample LS Protocol 34 15 16 17 18 19 20 21 22 2
54. due to the viscosity of the solution Changes in the volume of the bead mixture affect the insert size of your library j NOTE Vortex the Sample Purification Beads frequently to make sure that they are evenly distributed Illumina recommends the following If using a single channel pipette vortex the beads after processing four samples If using a multichannel pipette vortex the beads after processing four columns If the beads are in a reagent reservoir mix with a 1000 ul pipette Incubate the CEP plate at room temperature for 5 minutes Place the CEP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear Using a 200 ul single channel or multichannel pipette set to 138 ul remove and discard 138 ul of the supernatant from each well of the CEP plate Take care not to disturb the beads Repeat step 5 one time removing and discarding a total of 276 ul of the supernatant from each well NOTE E Leave the CEP plate on the magnetic stand while performing the following steps 7 11 With the CEP plate on the magnetic stand add 200 ul freshly prepared 80 EtOH to each well without disturbing the beads Incubate the CEP plate at room temperature for 30 seconds and then remove and discard all of the supernatant from each well Take care not to disturb the beads Repeat steps 7 and 8 one time for a total of two 8076 EtOH washes With the CEP plate on the magnetic stand let the samples air dry at
55. e Preparation 350 bp Insert Library Distribution 700 10380 Parts 15041110 Rev B Normalize and Pool Libraries This process describes how to prepare DNA templates for cluster generation Indexed DNA libraries are normalized to 10 nM in the DCT plate and then pooled in equal volumes in the PDP plate DNA libraries not intended for pooling are normalized to 10 nM in the DCT plate Consumables Item Barcode labels for DCT Diluted Cluster Template PDP Pooled DCT Plate for pooling only 1 7 ml microcentrifuge tube when processing gt 48 samples ata time 96 well MIDI plates 96 well 0 3 ml PCR plate for pooling only if pooling lt 40 samples Microseal B adhesive seals Tris HCl 10 mM pH8 5 with 0 196 Tween 20 TruSeq Nano DNA Sample Preparation Guide Quantity 1 label per plate 2 second plate for pooling only if pooling gt 40 samples il 2 Enough to normalize the concentration of each sample library to 10nM Storage 15 C to 30 C LAS 0 SAS 15 C to 30 C TIS 9 ENE 15 C to 30 C ISS to XU Supplied By Ilumina User User User User User 81 SeueJqr OOd pue ZIjewIoN High Sample HS Protocol Preparation Remove the TSP1 plate from 15 C to 25 C storage if it was stored at the conclusion of Clean Up PCR on page 39 Let it thaw at room temperature Centrifuge the thawed TSP1 plate at 280 x g for 1 minute Remove the adhes
56. egd High Sample HS Protocol Item Quantity Storage Supplied By Ice bucket As needed AAC 1 2529 User Microseal B adhesive seals 5 15 C to 30 C User PCR grade water 1 bottle 15 C to 302 C User RNase DNase free eight tube 5 15 C to 30 C User strips and caps if using multichannel pipettes RNase DNase free reagent 5 15 to 30 C User reservoirs if using multichannel pipettes Preparation Make IMP 58 Prepare an ice bucket Remove the End Repair Mix 2 from 15 C to 25 C storage and thaw it at room temperature Place the tube on ice Review Best Practices for Handling Magnetic Beads See Additional Resources on page 6 for information on how to access TruSeq Nano DNA Sample Preparation Best Practices on the Illumina website Make sure that the Sample Purification Beads and Resuspension Buffer are at room temperature Pre heat the microheating system to 30 C Apply an ALP barcode label to a new 96 well MIDI plate Apply a CEP barcode label to a new 96 well MIDI plate Centrifuge the thawed End Repair Mix 2 tube at 600 x g for 5 seconds Add 40 pl End Repair Mix 2 to each well of the IMP plate containing the fragmented DNA Mix thoroughly as follows a Seal the IMP plate with a Microseal B adhesive seal b Shake the IMP plate on a microplate shaker at 1800 rpm for 2 minutes Centrifuge the IMP plate at 280 x g for 1 minute Part 15041110 Rev B 4 Return the End Repair Mix 2 tube to 15 C to
57. enced users before starting sample preparation Click Training on TruSeq Nano DNA LT Sample Prep Kit Support or Click Training on TruSeq Nano DNA HT Sample Prep Kit Support Provides best practices specific to this protocol Review these best practices before starting sample preparation Topics include Handling Liquids Handling Master Mix Reagents Handling Magnetic Beads Avoiding Cross Contamination Potential DNA Contaminants Temperature Considerations Equipment Click Best Practices on TruSeq Nano DNA LT Sample Prep Kit Support or Click Best Practices on TruSeq Nano DNA HT Sample Prep Kit Support Part 15041110 Rev B Resource TruSeq Nano DNA Sample Preparation LS Protocol Experienced User Card and Lab Tracking Form part 15041111 TruSeq Nano DNA Sample Preparation HS Protocol Experienced User Card and Lab Tracking Form part 15041112 TruSeq Sample Preparation Pooling Guide part 15042173 Illumina Experiment Manager IEM TruSeq Nano DNA Sample Preparation Guide Description Provides LS protocol instructions but with less detail than what is provided in this user guide New or less experienced users are advised to follow this user guide and not the EUC and LTF Click Documentation amp Literature on TruSeq Nano DNA LT Sample Prep Kit Support or Click Documentation amp Literature on TruSeq Nano DNA HT Sample Prep Kit Support Provides HS protocol ins
58. ension Buffer to each well of the plate Remove the CAP plate from the magnetic stand Resuspend the beads in each well of the CAP plate by repeatedly dispensing the Resuspension Buffer over the bead pellet until it is immersed in the solution Gently pipette the entire volume up and down 10 times to mix thoroughly Incubate the CAP plate at room temperature for 2 minutes Place the CAP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear Part 15041110 Rev B 30 Transfer 25 ul of the clear supernatant from each well of the CAP plate to the corresponding well of the new 0 3 ml PCR plate labeled with the PCR barcode Take care not to disturb the beads SAFESTOPPING POINT If you do not plan to proceed immediately to Enrich DNA Fragments on page 36 you can 1 safely stop the protocol here If you are stopping seal the PCR plate with a Microseal B adhesive seal and store at 15 C to 25 C for up to 7 days TruSeq Nano DNA Sample Preparation Guide 3 5 sJo1depy e1e6r Low Sample LS Protocol Enrich DNA Fragments 36 This process uses PCR to selectively enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library The PCR is performed with a PCR Primer Cocktail that anneals to the ends of the adapters Minimize the number of PCR cycles to avoid skewing the representation of the library lg NOTE PCR enriches for fragments tha
59. er 200 ul barrier pipette tips General lab supplier 200 ul multichannel pipettes General lab supplier 200 ul single channel pipettes General lab supplier TruSeq Nano DNA Sample Preparation Guide 95 jueuudinb4 pue se qeuunsuo Supporting Information Consumable 96 well storage plates round well 0 8 ml MIDI plate Distilled water Ethanol 200 proof absolute for molecular biology 500 ml Optional Fluorometric quantitation with dsDNA binding dye reagents Ice bucket Optional KAPA Library Quantification Kit Illumina Universal Microseal B adhesive seals microTUBE AFA Fiber 6x16mm with Crimp Cap or Pre Slit Snap Cap for use with Covaris M220 PCR grade water RNaseZap to decontaminate surfaces RNase DNase free eight tubes strips and caps RNase DNase free multichannel reagent reservoirs disposable Tris HCl 10 mM pH 8 5 Tween 20 Supplier Fisher Scientific part AB 0859 General lab supplier Sigma Aldrich part E7023 General lab supplier General lab supplier KAPA Biosystems part KK4824 Bio Rad part MSB 1001 Covaris part 520052 or part 520045 General lab supplier General lab supplier General lab supplier VWR part 4 89094 658 General lab supplier Sigma Aldrich part P7949 Part 15041110 Rev B Table 18 User Supplied Consumables Additional Items for LS Processing Consumable Supplier 96 well 0 3 ml skirtless PCR
60. esult in high library yields 100 ng for a 350 bp insert size 200 ng for a 550 bp insert size 1 Add5 ul thawed PCR Primer Cocktail to each well of the PCR plate 2 Add 20 ul thawed Enhanced PCR Mix to each well of the PCR plate Set a 200 ul pipette to 40 ul then gently pipette the entire volume up and down 10 times to mix thoroughly 3 Seal the PCR plate with a Microseal B adhesive seal 38 Part 15041110 Rev B Amp PCR 1 Place the sealed PCR plate containing 50 ul of each sample on the pre programmed thermal cycler Close the lid then select and run PCRNano to amplify the plate a Choose the pre heat lid option and set to 100 C b 95 C for 3 minutes c 8 cycles of 98 C for 20 seconds 60 C for 15 seconds 72 C for 30 seconds d 72 C for 5 minutes e Hold at 4C Clean Up PCR 1 Centrifuge the PCR plate at 280 x g for 1 minute 2 Remove the adhesive seal from the PCR plate 3 Vortex the Sample Purification Beads until they are well dispersed 4 Do one ofthe following depending on the adapter type used If using the DNA Adapter tubes add 50 ul mixed Sample Purification Beads to each well of the PCR plate containing 50 ul of the PCR amplified library Set a 200 ul pipette to 90 ul then gently pipette the entire volume up and down 10 times to mix thoroughly If using the DAP add 47 5 ul mixed Sample Purification Beads to each well of the PCR plate containing 50 ul of the PCR amplified library Set a 200 ul pipette to 90
61. ev B Kit Contents Check to make sure that you have all of the reagents identified in this section before starting the TruSeq Nano DNA Sample Preparation protocol The TruSeq Nano DNA LT Sample Prep Kits are available as Set A and B Each TruSeq Nano DNA LT Sample Prep Kit contains enough reagents to prepare up to 24 samples When used together TruSeq Nano DNA LT Sample Prep Kits A and B allow for pooling up to 24 samples using the 12 different indices in each kit Table 16 TruSeq Nano DNA Sample Prep Kits Kit Name Catalog Number Number of of Samples Indices Supported TruSeq Nano DNA LT Sample FC 121 4001 24 12 Prep Kit Set A TruSeq Nano DNA LT Sample FC 121 4002 24 12 Prep Kit Set B TruSeq Nano DNA HT Sample FC 121 4003 96 96 Prep Kit TruSeq Nano DNA LT Sample Prep Kit The TruSeq Nano DNA LT Sample Prep Kit contains two boxes a Set A or Set B box and an SP Beads box 24 Samples Set A or Set B Box You receive either box A or B with the kit depending on the set you ordered These boxes also contain plate barcode labels Store at 15 C to 25 C These boxes are shipped on dry ice As soon as you receive them store the following components at 15 C to 25 C TruSeq Nano DNA Sample Preparation Guide 8 9 S USJUO YM Supporting Information 90 Set A Figure 9 TruSeq Nano DNA LT Sample Prep Kit 24 Samples Set A Box 1 of 2 part 15041757 Slot Reagent Part 4 Description 1 RSB 15026770 Resuspensi
62. for 5 minutes Remove and discard any remaining EtOH from each well of the CPP plate with a 10 ul pipette With the CPP plate on the magnetic stand add 32 5 ul Resuspension Buffer to each well of the CPP plate Remove the CPP plate from the magnetic stand Mix thoroughly as follows a Seal the CPP plate with a Microseal B adhesive seal b Shake the CPP plate on a microplate shaker at 1800 rpm for 2 minutes Incubate the CPP plate at room temperature for 2 minutes Centrifuge the CPP plate at 280 x g for 1 minute Remove the adhesive seal from the CPP plate Place the CPP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear Transfer 30 ul of the clear supernatant from each well of the CPP plate to the corresponding well of the new HSP plate labeled with the TSP1 barcode SAFESTOPPING POINT If you do not plan to proceed immediately to Validate Library on page 79 you can safely stop j the protocol here If you are stopping seal the TSP1 plate with a Microseal B adhesive seal and store at 15 C to 25 C for up to 7 days Part 15041110 Rev B Validate Library Illumina recommends performing the following procedures for quality control analysis on your sample library and quantification of the DNA library templates Quantify Libraries To achieve the highest data quality on Illumina sequencing platforms it is important to create optimum cluster densities across every lane of a f
63. from 15 C to 25 C storage and thaw them at room temperature Centrifuge the thawed Enhanced PCR Mix and PCR Primer Cocktail tubes to 600 x g for 5 seconds Remove the Resuspension Buffer from 2 C to 8 C storage and bring it to room temperature Remove the Sample Purification Beads from 2 C to 8 C storage and let stand for at least 30 minutes to bring them to room temperature Review Best Practices for Handling Magnetic Beads See Additional Resources on page 6 for information on how to access TruSeq Nano DNA Sample Preparation Best Practices on the Illumina website Remove the PCR plate from 15 C to 25 C storage if it was stored at the conclusion of Clean Up ALP on page 32 Let it thaw at room temperature Centrifuge the thawed PCR plate at 280 x g for 1 minute Remove the adhesive seal from the thawed PCR plate TruSeq Nano DNA Sample Preparation Guide 37 s juauBe14 vNG u9uu3 Low Sample LS Protocol Pre program the thermal cycler with the following program and save as PCRNano Choose the pre heat lid option and set to 100 C 95 C for 3 minutes 8 cycles of 98 C for 20 seconds 60 C for 15 seconds 72 C for 30 seconds 72 C for 5 minutes Hold at 4 C Igy NOTE Ilumina recommends 8 cycles of PCR for robust protocol performance Apply a TSP1 barcode label to a new 96 well 0 3 ml PCR plate Make PCR The following procedure assumes the following amount of input DNA sample for library preparation and is designed to r
64. g the DAP add 47 5 ul mixed Sample Purification Beads to each well of the new MIDI plate labeled with the CPP barcode Transfer the entire contents from each well of the PCR plate to the corresponding well of the CPP plate containing 50 ul mixed Sample Purification Beads Mix thoroughly as follows a Seal the CPP plate with a Microseal B adhesive seal b Shake the CPP plate on a microplate shaker at 1800 rpm for 2 minutes Incubate the CPP plate at room temperature for 5 minutes Centrifuge the CPP plate at 280 x g for 1 minute Remove the adhesive seal from the CPP plate TruSeq Nano DNA Sample Preparation Guide T1 s juauBe14 vNG u9uu3 High Sample HS Protocol 78 10 11 12 13 14 15 16 17 18 19 20 21 22 Place the CPP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear Remove and discard 95 ul of the supernatant from each well of the CPP plate NOTE j Leave the CPP plate on the magnetic stand while performing the following 8076 EtOH wash steps 11 13 With the CPP plate on the magnetic stand add 200 ul freshly prepared 80 EtOH to each well without disturbing the beads Incubate the CPP plate at room temperature for 30 seconds and then remove and discard all of the supernatant from each well Repeat steps 11 and 12 one time for a total of two 80 EtOH washes With the CPP plate on the magnetic stand let the samples air dry at room temperature
65. illumina TruSeq Nano DNA Sample Preparation Guide ACGAAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATAT TAACGTACCATTAAGAGCTACCGT AATGATAACAGTAACACACTTCTGTTAACCT TAAGATTACTTGT TGATCCACTGAT TCAACGTACCG TATCAAT TGAGAC TAAATAT TAACGTACCAT TAAGAGCTACCGTCTTCTGTTAACCTTAAGAT TACTTGATCCACTGATTCAACGTACCGTAA ete MM RGA TAGES ME Ta En TAER TAA RA CGT GR RAGAT Tied USC T ACCATTAAGAGCTACCGTCTTCTGT T e PS al Y ACCGTAACGAAC AAAAGAATGATAACAGTAACACACTTCTGT TAACCTTAAGAT T ATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAA TTGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGT AAIGATAACAGAACACASTICTGITAACLT IAAGATTAGT I GATGCAGT GAT TCAACGTACOGTAACGAACGIATGAAT GAGAC TAAATAT TAACGTAGCAT TAAGAGCTACCGTC TCTGT IARC IAAGATIACT TGATCCACTGATTCAAGGTA AAT TGAGAC TAAATAT TAACGTTGTTAACCTTAAGATTACTTGATCCACTGATTCAACG T CU NAD GW ae TRACT AMTATTAG UAR TTAAGAGCT TCTGT TAACCT TAAGAT TACTT GATCCACTGAT TCAACGTACCGTAAC CGTATCAATTGAGACTAAATAT TAACGTACT TAACCT TAAGAT TACT TGATCCACTGATTCAACGTACCGTAACGAACG TCTTC TGT TAACC T TAAGAT TACTTGATCCACTGAT TCAACGTACCGIAACGAACGTATCAAT TGAGAC TAACGACGAAA GAGAC TAAATAT TAACGTACCAT TAAGAGCTACAACCT TAAGAT TACT TGATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TGAGACTAAATAT TAACG TACCAT TAAGAGC TACCGTGCAACGACGAAAAGAAT GATAACAGTAACACACT MALADES TARDA TTR TOT TIARO I o AGER ARA ATE A TASA AMATEURS UNE Ee red eg TAG TI a Leg PARIS TRACTA GTACCATTAAGAGC TACCGTGCAACTTAACCTTAAGAT TACT
66. ion Beads tube at room temperature for later use in the protocol Incubate the CSP plate at room temperature for 5 minutes Place the CSP plate on the magnetic stand at room temperature for 8 minutes or until the liquid is clear Using a 200 ul single channel or multichannel pipette set to 125 ul remove and discard 125 ul of the supernatant from each well of the CSP plate NOTE j Leave the CSP plate on the magnetic stand while performing the following steps 6 10 With the CSP plate on the magnetic stand add 200 ul freshly prepared 80 EtOH to each well without disturbing the beads Incubate the CSP plate at room temperature for 30 seconds and then remove and discard all of the supernatant from each well Take care not to disturb the beads Repeat steps 6 and 7 one time for a total of two 8076 EtOH washes With the CSP plate on the magnetic stand let the samples air dry at room temperature for 5 minutes Remove and discard any remaining EtOH with a 10 ul pipette TruSeq Nano DNA Sample Preparation Guide 1 rj VNG 1ueuuDeJJ Low Sample LS Protocol 18 10 11 12 13 14 15 16 With the CSP plate on the magnetic stand add 62 5 ul Resuspension Buffer to each well of the plate Remove the CSP plate from the magnetic stand Resuspend the beads in each well of the CSP plate by repeatedly dispensing the Resuspension Buffer over the bead pellet until it is immersed in the solution Gently pipette the entire vol
67. ion in nM perform the following insert size adjustment For 350 bp libraries use 470 bp for the average fragment length For 550 bp libraries use 670 bp for the average fragment length Igy NOTE You can download the KAPA Library Quantification Kits for Illumina sequencing platforms Technical Data Sheet from the Kapa Biosystems website www kapabiosystems com Optional Quality Control To verify the size of your fragments check the template size distribution Run samples on a Bioanalyzer for qualitative purposes only Do one of the following If using a High Sensitivity DNA chip Prepare a 1 100 dilution of the DNA library with water Run 1 ul of the diluted DNA library on an Agilent Technologies 2100 Bioanalyzer If using a DNA 7500 chip run 1 ul of undiluted DNA library on an Agilent Technologies 2100 Bioanalyzer TruSeq Nano DNA Sample Preparation Guide 41 Heq e3epi eA Low Sample LS Protocol 42 Figure 3 Example TruSeq Nano DNA Sample Preparation 350 bp Insert Library Distribution 700 10380 Parts 15041110 Rev B Normalize and Pool Libraries This process describes how to prepare DNA templates for cluster generation Indexed DNA libraries are normalized to 10 nM in the DCT plate and then pooled in equal volumes in the PDP plate DNA libraries not intended for pooling are normalized to 10 nM in the DCT plate Consumables Item Barcode labels for DCT Diluted Cluster Template PDP Pooled DCT P
68. ither the LT or HT kit Follow the protocols in the order described using the specified volumes and incubation parameters Review Best Practices before proceeding See Additional Resources on page 6 for information on how to access TruSeq Nano DNA Sample Preparation Best Practices on the Illumina website Review the TruSeq Sample Preparation Pooling Guide part 15042173 before proceeding See Additional Resources on page 6 for information on how to download the guide from the Illumina website Review Appendix A Supporting Information before proceeding to confirm your kit contents and make sure that you have obtained all of the requisite equipment and consumables for the HS protocol This HS protocol requires shaking and heating equipment to mix reagents and for incubation see User Supplied Equipment Additional Items for HS Processing on page 98 Part 15041110 Rev B Sample Prep Workflow The following figure illustrates the processes of the TruSeq Nano DNA Sample Preparation HS protocol to prepare templates using indexed adapter tubes or a DAP Figure 5 TruSeq Nano DNA Sample Preparation HS Workflow MOJ P440M day ajduwes Prepare Adapter Setup Ligate Indexed Paired End Adapters Purified Genomic DNA Consumables DNA Adapters or DAP EtOH uG2 Fragment RSB Genomic DNA e Consumable RSB Plato SPB CAP PCR Plate CFP CSP DNA IMP 1 Repair Ends and Mai Size Selection POR Ampificition Consumables er EtOH R2 EPM EtOH
69. ive seal from the thawed TSP1 plate Apply a DCT barcode label to a new 96 well MIDI plate For pooling only Apply a PDP barcode label to a new 96 well 0 3 ml PCR plate if pooling x 40 samples or a 96 well MIDI plate if pooling gt 40 samples Make DCT 1 Transfer 10 ul of sample library from each well of the TSP1 plate to the corresponding well of the new MIDI plate labeled with the DCT barcode 2 Normalize the concentration of sample library in each well of the DCT plate to 10 nM using Tris HCl 10 mM pH 8 5 with 0 1 Tween 20 i NOTE Depending on the yield quantification data of each sample library the final volume in the DCT plate can vary from 10 400 pl 3 Mix the DCT plate as follows a Seal the DCT plate with a Microseal B adhesive seal b Shake the DCT plate on a microplate shaker at 1000 rpm for 2 minutes 4 Centrifuge the DCT plate at 280 x g for 1 minute 5 Remove the adhesive seal from the DCT plate 6 Depending on the type of library you want to generate do one of the following For non pooled libraries the protocol stops here Do one of the following Proceed to cluster generation For more information see the cluster generation section of the user guide for your Illumina platform Seal the DCT plate with a Microseal B adhesive seal and store at 15 C to 25 C For pooled libraries proceed to Make PDP for pooling only Q2 Part 15041110 Rev B Make PDP for pooling only ley NOTE Do not make a
70. l Introduction o occccccccccccccccccccccnnnnncccc cc ccnnnnnnnnncco Sample Prep Workflow ssec Prepare Adapter Setup 2 2 0222 e eee ee ee Fragment DNA 2 222 000 2 2 c cece cece cc ccccccceceeceeeeeeeeeeeeeeees Perform End Repair and Size Selection Adenylate 3 Ends o oooooococcccccccccccccccccnoccccccnnnccccoo Ligate Adapters o oooocooccccccccccccccccccccocccccccnnnnccccccio Enrich DNA Fragments 2002 eee eee cece e eee ee Validate Library iouis Normalize and Pool Libraries ssuuuuuuuseususuus Appendix A Supporting Information ooo TruSeq Nano DNA Sample Preparation Guide Introduction s eret tte ee A e e OS Ni 86 AGFODVITIS A iA bmi te ae neo O 87 Kit Cotriterils act oo oe ecd oUm A DLE eU Ut A A UD e DOR FLU ds 89 Consumables and Equipment 0 00 0 0 cece eee eeeeeeeeeeees 95 Indexed Adapter Sequences 2 2 2 2 22 2 c cee eee cccceeeeeecececceeeees 100 o AA A AR ANA 103 Technical ASSISTANCE s reise t EE tr eese Do ERO ER i ERR beh 105 Part 15041110 Rev B List of Tables Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 Table 7 Table 8 Table 9 Table 10 Table 11 Table 12 Table 13 Table 14 Table 15 Table 16 Table 17 Table 18 Table 19 Table 20 Table 21 Table 22 Table 23 Table 24 Table 25 Table 26 Table 27 Table 28 Protocol Features 22 2 0 2
71. l microcentrifuge tube at 15 C to 25 C 834 Part 15041110 Rev B Supporting Information Introduction io acie io copitio idiota ein Dd oa Needles FP iN Ou eU 86 ACLONYIMS NER 87 eed Ero 410 58 i TREE ERROR dto aras 89 Consumables and Equipment 2 0 0 e cece cc eee cece eee cece eeeeeeeeeeeeeeeesees 95 Indexed Adapter Sequences 2 2 eee cece cee cccccccccecccccccceecccccceceeeccccceeeeeeeeees 100 E gil Cani SI TOC GC riy vegas ici tCU UA TruSeq Nano DNA Sample Preparation Guide 8 5 v xipueddw Supporting Information Introduction The protocols described in this guide assume that you have reviewed the contents of this appendix confirmed your kit contents and obtained all of the requisite consumables and equipment ao Part 15041110 Rev B Acronyms Table 15 TruSeq Nano DNA Sample Preparation Acronyms Acronym Definition ALP Adapter Ligation Plate Clean Up ALP Plate Covaris Fragmentation Plate Clean Up Sheared DNA Plate DCT Diluted Cluster Template dsDNA double stranded DNA End Repair Mix 2 genomic DNA High Sample TruSeqNano DNA Sample Preparation Guide 87 sw uoIoy Supporting Information 88 Acronym TEM LIG2 LT PCR PPC SPB TSP Definition Illumina Experiment Manager Ligation Mix 2 Low Throughput Polymerase Chain Reaction PCR Primer Cocktail Sample Purification Beads Target Sample Plate Part 15041110 R
72. late for pooling only 1 7 ml microcentrifuge tube when processing gt 48 samples ata time 96 well MIDI plates 96 well 0 3 ml PCR plate for pooling only if pooling lt 40 samples Microseal B adhesive seals Tris HCl 10 mM pH8 5 with 0 196 Tween 20 TruSeq Nano DNA Sample Preparation Guide Quantity 1 label per plate 2 second plate for pooling only if pooling gt 40 samples il 2 Enough to normalize the concentration of each sample library to 10nM Storage 15 C to 30 C LAS 0 SAS 15 C to 30 C TIS 9 ENE 15 C to 30 C ISS to XU Supplied By Ilumina User User User User User 43 SeueJqr OOd pue ZIjewIoN Make DCT 1 Low Sample LS Protocol Preparation Remove the TSP1 plate from 15 C to 25 C storage if it was stored at the conclusion of Clean Up PCR on page 39 Let it thaw at room temperature Centrifuge the thawed TSP1 plate at 280 x g for 1 minute Remove the adhesive seal from the thawed TSP1 plate Apply a DCT barcode label to a new 96 well MIDI plate For pooling only Apply a PDP barcode label to a new 96 well 0 3 ml PCR plate if pooling x 40 samples or a 96 well MIDI plate if pooling gt 40 samples Transfer 10 ul of sample library from each well of the TSP1 plate to the corresponding well of the new MIDI plate labeled with the DCT barcode Normalize the concentration of sample library in each well of the DCT plate to 10
73. low cell Optimizing cluster densities requires accurate quantitation of DNA library templates Quantify your libraries using a fluorometric quantification method that uses dsDNA binding dyes or qPCR ly NOTE TruSeq Nano DNA Sample Prep library quantitation has been validated using the Eco Real Time PCR System and KAPA Library Quantification Kit specified in the Consumables and Equipment on page 95 Follow the KAPA instructions with the KAPA standard To calculate the library concentration in nM perform the following insert size adjustment For 350 bp libraries use 470 bp for the average fragment length For 550 bp libraries use 670 bp for the average fragment length Igy NOTE You can download the KAPA Library Quantification Kits for Illumina sequencing platforms Technical Data Sheet from the Kapa Biosystems website www kapabiosystems com Optional Quality Control To verify the size of your fragments check the template size distribution Run samples on a Bioanalyzer for qualitative purposes only Do one of the following If using a High Sensitivity DNA chip Prepare a 1 100 dilution of the DNA library with water Run 1 ul of the diluted DNA library on an Agilent Technologies 2100 Bioanalyzer If using a DNA 7500 chip run 1 ul of undiluted DNA library on an Agilent Technologies 2100 Bioanalyzer TruSeq Nano DNA Sample Preparation Guide F Q Heq e3epi eA High Sample HS Protocol 80 Figure 7 Example TruSeq Nano DNA Sampl
74. ly a PCR barcode label to a new 96 well HSP plate Part 15041110 Rev B Add LIG 1 Do one of the following If using DNA Adapter tubes centrifuge the thawed tubes at 600 x g for 5 seconds Ifusing a DAP Thaw the plate for 10 minutes at room temperature on the benchtop Visually inspect the wells to make sure that they all are thawed Remove the adapter plate tape seal Centrifuge the plate at 280 x g for 1 minute to collect all of the adapter to the bottom of the well Remove the plastic cover Save the cover if you are not processing the entire plate at one time Fitis the first time using this DAP apply the DAP barcode label to the plate Centrifuge the Stop Ligation Buffer tube at 600 x g for 5 seconds Immediately before use remove the Ligation Mix 2 tube from 15 C to 25 C storage Remove the adhesive seal from the ALP plate Add 2 5 ul Resuspension Buffer to each well of the ALP plate Add 2 5 ul Ligation Mix 2 to each well of the ALP plate Return the Ligation Mix 2 tube to 15 C to 25 C storage immediately after use oo N A a A CQ N Do one of the following Ifusing DNA Adapter tubes add 2 5 ul thawed DNA Adapter Index to each well of the ALP plate If using a DAP Place the DAP on the benchtop so that the part number barcode on the long side of the plate is facing you and the clipped corner is on the lower left Figure 6 Correct DAP Orientation Bey TruSeq Nano DNA Sample
75. ly equivalent reconditioned or new Hardware or components if only a component of Hardware is non conforming If the Hardware is replaced in its entirety the warranty period for the replacement is 90 days from the date of shipment or the remaining period on the original Hardware warranty whichever is shorter If only a component is being repaired or replaced the warranty period for such component is 90 days from the date of shipment or the remaining period on the original Hardware warranty whichever ends later IV Part 15041110 Rev B The preceding states Purchaser s sole remedy and Illumina s sole obligations under the warranty provided hereunder Third Party Goods and Warranty Illumina has no warranty obligations with respect to any goods originating from a third party and supplied to Purchaser hereunder Third party goods are those that are labeled or branded with a third party s name The warranty for third party goods if any is provided by the original manufacturer Upon written request Illumina will attempt to pass through any such warranty to Purchaser 9 Indemnification a Infringement Indemnification by Illumina Subject to these terms and conditions including without limitation the Exclusions to Illumina s Indemnification Obligations Section 9 b below the Conditions to Indemnification Obligations Section 9 d below Illumina shall i defend indemnify and hold harmless Purchaser against any third party claim or action
76. mple preparation protocol offers Streamlined Workflow Master mixed reagents to reduce reagent containers and pipetting Universal adapter for preparation of single read paired end and indexing Optimized shearing for whole genome resequencing with 350 bp and 550 bp insert size workflows Bead based size selection reagents included in each kit Optimized workflows for processing low sample LS and high sample HS numbers in parallel Compatibility with LT and HT kit configurations High Throughput Adapter plate allows for simultaneous preparation of 96 dual indexed DNA samples Volumes optimized for standard 96 well plate Index Adapter Tags All Samples Additional adapters and primers not necessary Each TruSeq Nano DNA LT Sample Prep Kit contains adapter index tubes recommended for preparing up to 24 samples for sequencing Together kits A and B allow for pooling up to 24 samples The TruSeq Nano DNA HT Sample Prep Kit contains a 96 well plate with 96 uniquely indexed adapter combinations designed for manual or automated preparation of 96 uniquely indexed samples The protocol is compatible with single sample sequencing or lower indexing pooling levels Part 15041110 Rev B Protocol Features This guide documents the TruSeq Nano DNA Sample Preparation protocol using a TruSeq Nano DNA LT Sample Prep Kit or TruSeq Nano DNA HT Sample Prep Kit Chapter 2 Low Sample LS Protocol explains how to perform the TruSeq Nano DNA Sample Preparation
77. nds arranging samples that are going to be combined into a common pool in the same row Also include a common index in each column This arrangement facilitates pipetting operations when dispensing indexed adapters and pooling indexed libraries later in the protocol e When indexing libraries with the DAP arrange samples that will be pooled together in the same orientation as the indices in the DAP TruSeq Nano DNA Sample Preparation Guide A Q sJe1depy oe1e6r Low Sample LS Protocol i NOTE When indexing libraries with the DAP Review Handling Adapter Plate in the TruSeq Sample Preparation Pooling Guide part 15042173 See Additional Resources on page 6 for information on how to download the guide from the Illumina website Illumina recommends that the DAP does not undergo more than four freeze thaw cycles To maximize the use of the DAP process more than 24 samples at a time These samples can then be pooled in any supported configuration Stop Ligation Buffer NOTE 4 Do not remove the Ligation Mix 2 tube from 15 C to 25 C storage until instructed to do so in the procedures Remove the Resuspension Buffer from 2 C to 8 C storage and bring it to room temperature Review Best Practices for Handling Magnetic Beads See Additional Resources on page 6 for information on how to access TruSeq Nano DNA Sample Preparation Best Practices on the Illumina website Remove the Sample Purification Beads from 2 C to 8 C storage
78. ng gt 6 samples at a time New 1 7 ml microcentrifuge tube when processing lt 6 samples at a time Determine the volumes using the following formulas which include 15 excess for multiple samples Table 7 Diluted Bead Mixture for a 350 bp Insert Size Formula Example Amount Your per 12 Calculation samples Sample Purification Beads of samples X 10923 ul BI PCR grade water of samples X 897 ul 74 75 ul Table 8 Diluted Bead Mixture for a 550 bp Insert Size Formula Example Amount Your per 12 Calculation samples Sample Purification Beads of samples X 1104 ul 92 ul PCR grade water of samples X 1104 ul 92 ul Vortex the diluted bead mixture for 5 seconds to make sure that the beads are evenly dispersed Add 160 ul of the diluted bead mixture to each well of the IMP plate containing 100 ul of the end repaired sample Set a 200 ul pipette to 200 ul and then gently pipette the entire volume up and down 10 times to mix thoroughly y NOTE Aspirate the diluted bead mixture slowly and dispense it slowly due to the viscosity of the solution Changes in the volume of the diluted bead mixture affect the insert size of your library Part 15041110 Rev B 9 NOTE j Vortex the diluted bead mixture frequently Illumina recommends the following If using a single channel pipette vortex the mixture after processing four samples If using a multichannel pipette vortex the mixture after processing four columns If the mix
79. nth cycle and is not considered as part of the index sequence Record the index in the sample sheet as only six bases For indices 13 and above the seventh base in parentheses might not be A which is seen in the seventh cycle of the index read For more information on the number of cycles used to sequence the index read reference your instrument user guide Table 23 TruSeq Nano DNA LT Sample Prep Kit Set A Indexed Adapter Sequences Adapter Sequence Adapter Sequence AD002 CGATGT A ADO013 AGTCAA C AD004 TGACCA A ADO014 AGTICC G AD005 ACAGTG A ADO015 ATGTCA G AD006 GCCAAT A AD016 EEECTEE C AD007 CAGATC A ADO018 GTCCGC A AD012 CTTGTA A AD019 GTGAAA C 1 OO Part 15041110 Rev B Table 24 TruSeq Nano DNA LT Sample Prep Kit Set B Indexed Adapter Sequences Adapter Sequence Adapter Sequence ADO001 ATCACG A GTGGCC T AD003 GTTICG G AD008 CGTACG T AD009 GAGTGG A ADO010 ACTGAT A ADO11 GGCTAC A AD027 ATICCT T TruSeq Nano DNA HT Sample Prep Kit Indexed Adapter Sequences The DAP in the TruSeq Nano DNA HT Sample Prep Kit contains the following indexed adapter sequences Ww NOTE seouenbes Je1depy pexepu v The Index recorded in the sample sheet is the full 8 bases and 8 bases are sequenced per indexed read Table 25 TruSeq Nano DNA HT Sample Prep Kit Indexed Adapter 1 Sequences Adapter Sequence Adapter Sequence D701 ATTACTCG D707 CTGAAGCT E CE NEL INN NN cas TruSeq Nano DNA Sample Preparation G
80. o each well of the ALP plate Set a 20 ul pipette to 20 ul then gently pipette the entire volume up and down 10 times to mix thoroughly 3 Seal the ALP plate with a Microseal B adhesive seal 4 Return the A Tailing Mix tube to 15 to 25 C storage Incubate 1 ALP 1 Place the sealed ALP plate containing 30 ul of each sample on the pre programmed thermal cycler Close the lid then select and run the ATAIL70 program a Choose the pre heat lid option and set to 100 C b 37 C for 30 minutes c 70 C for 5 minutes d 4 C for 5 minutes e Hold at 4 C 2 When the thermal cycler temperature has been at 4 C for 5 minutes remove the ALP plate from the thermal cycler 3 Centrifuge the ALP plate at 280 x g for 1 minute 4 Proceed immediately to Ligate Adapters on page 28 TruSeq Nano DNA Sample Preparation Guide P T Spu3 e aje huspy Low Sample LS Protocol Ligate Adapters preparing them for hybridization onto a flow cell Consumables Item Choose from the following depending on the kit you are using e TruSeq Nano DNA LT Sample Prep Kit contents DNA Adapter Indices AD001 ADO016 AD018 AD023 AD025 AD027 TruSeq Nano DNA HT Sample Prep Kit contents DAP DNA Adapter Plate Ligation Mix 2 LIG2 Resuspension Buffer RSB Sample Purification Beads SPB Stop Ligation Buffer STL Quantity Storage 1 tube of each index 15 C to 25 C being used per column of 8 reactions or 1 DAP
81. o mix thoroughly Incubate the PCR plate at room temperature for 2 minutes Place the PCR plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear Transfer 30 ul of the clear supernatant from each well of the PCR plate to the corresponding well of the new 0 3 ml PCR plate labeled with the TSP1 barcode SAFESTOPPING POINT If you do not plan to proceed immediately to Validate Library on page 41 you can safely stop 1 the protocol here If you are stopping seal the TSP1 plate with a Microseal B adhesive seal and store at 15 C to 25 C for up to 7 days Part 15041110 Rev B Validate Library Illumina recommends performing the following procedures for quality control analysis on your sample library and quantification of the DNA library templates Quantify Libraries To achieve the highest data quality on Illumina sequencing platforms it is important to create optimum cluster densities across every lane of a flow cell Optimizing cluster densities requires accurate quantitation of DNA library templates Quantify your libraries using a fluorometric quantification method that uses dsDNA binding dyes or qPCR ly NOTE TruSeq Nano DNA Sample Prep library quantitation has been validated using the Eco Real Time PCR System and KAPA Library Quantification Kit specified in the Consumables and Equipment on page 95 Follow the KAPA instructions with the KAPA standard To calculate the library concentrat
82. on Product documentation in PDF is available for download from the Illumina website Go to www illumina com support select a product then click Documentation amp Literature TruSeq Nano DNA Sample Preparation Guide 1 O 5 99UB SISSY e9IUuu2e AAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATATTAACGTACCATTAAGAGCTACC SATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGT TGATCCACTGATTCAACGTACCGTATCAAT TGAGAC TAAATAT TAACGTACCAT TAAGAGCTACCGTCTTCTGTTAACCTTAAGAT TACT TGATCCACTGAT TCAACGTACCGT PATATE CEDAT ECT TOC RAD EAS STA TCRA RASTA AAT A TAM BOCA TANGO IR CELO PACTI TAGAT AC TRAE ABE TARTE TCR TAA IAAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGAT TACTTGATCCACTGAT TCAACGTACCGTAAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACC 3ATAACAGTAACACACT TCTGTTAACCTIAAGAT TACT TGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTCTTCTGTTAACCT TAAGATTACTTGATCCACTGAT TCAACG TGAGACTAAATAT TAACGTTGTTAACCTTAAGATTACTTGATCCACTGATTCAACG TACCGTAACGAACG TATCAAT TGAGACTAAATATTAACGTACCAT TAAGAGCT TCTGT TAACCT TAAGAT TACTTGATCCACTGAT TCAACG TACCG TA ATCAAT TGAGAC TAAATAT TAACGTACT TAACCT TAAGAT TACT TGATCCACTGATTCAACGTACCGTAACGAACG TCTTCTGTTAACCTTAAGAT TACTT GATCCACTGATTCAACGTACCGTAACGAACG TATCAAT TGAGAC TAACGACGA SAC TAAATAT TAACGTACCAT TAAGAGCTACAACCT TAAGAT TACT TGATCCACTGATTCAACGTACCGTAACGAACG AT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAGC TACCGTG
83. on Buffer 2 End Repair Mix 2 3 A Tailing Mix 4 Ligation Mix 2 5 Stop Ligation Buffer 6 PPC PCR Primer Cocktail 7 Enhanced PCR Mix 8 15026621 DNA Adapter Index 2 9 15026623 DNA Adapter Index 4 10 15026624 DNA Adapter Index 5 11 15026625 DNA Adapter Index 6 12 15026627 DNA Adapter Index 7 13 15026632 DNA Adapter Index 12 14 15024641 DNA Adapter Index 13 15 15024642 DNA Adapter Index 14 16 15024643 DNA Adapter Index 15 17 15024644 DNA Adapter Index 16 18 15024646 DNA Adapter Index 18 19 15024647 DNA Adapter Index 19 Part 15041110 Rev B Set B Figure 10 TruSeq Nano DNA LT Sample Prep Kit 24 Samples Set B Box 1 of 2 part 15041759 Slot Reagent Part Description 1 RSB 15026770 Resuspension Buffer 2 End Repair Mix 2 3 A Tailing Mix 4 Ligation Mix 2 5 Stop Ligation Buffer 6 PPC PCR Primer Cocktail 7 Enhanced PCR Mix 8 15026620 DNA Adapter Index 1 9 15026622 DNA Adapter Index 3 10 15026628 DNA Adapter Index 8 11 15026629 DNA Adapter Index 9 12 15026630 DNA Adapter Index 10 13 15026631 DNA Adapter Index 11 14 15024648 DNA Adapter Index 20 15 15024649 DNA Adapter Index 21 16 15024650 DNA Adapter Index 22 17 15024651 DNA Adapter Index 23 18 15024653 DNA Adapter Index 25 19 15024654 DNA Adapter Index 27 24 Samples SP Beads Box Store at 2 C to 8 C This box is shipped at 2 C to 8 C As soon as you receive it store the components at 2 C to 8 C
84. on the magnetic stand add 200 ul freshly prepared 80 EtOH to each well without disturbing the beads Incubate the ALP plate at room temperature for 30 seconds and then remove and discard all of the supernatant from each well Take care not to disturb the beads Repeat steps 9 and 10 one time for a total of two 8076 EtOH washes With the ALP plate on the magnetic stand let the samples air dry at room temperature for 5 minutes Remove and discard any remaining EtOH with a 10 ul pipette With the ALP plate on the magnetic stand add 52 5 ul Resuspension Buffer to each well of the plate Remove the ALP plate from the magnetic stand TruSeq Nano DNA Sample Preparation Guide 11 High Sample HS Protocol Te 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Mix thoroughly as follows a Seal the ALP plate with a Microseal B adhesive seal b Shake the ALP plate on a microplate shaker at 1800 rpm for 2 minutes Incubate the ALP plate at room temperature for 2 minutes Centrifuge the ALP plate at 280 x g for 1 minute Remove the adhesive seal from the ALP plate Place the ALP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear Transfer 50 ul of the clear supernatant from each well of the ALP plate to the corresponding well of the new MIDI plate labeled with the CAP barcode Take care not to disturb the beads Vortex the Sample Purification Beads until they a
85. parate new Covaris tube 100 ng for a 350 bp insert size 200 ng for a 550 bp insert size Use the wells of the new HSP plate labeled with CFP barcode or another device to hold the Covaris tubes upright NOTE i Load the DNA sample into the Covaris tube slowly to avoid creating air bubbles However air bubbles might not be preventable Centrifuge the CFP plate at 600 x g for 5 seconds Fragment the DNA using the following settings Table 10 Covaris S220 Settings Setting 350 bp Insert 550 bp Insert Duty factor 5 Peak Incident Power 175 W Cycles per burst 200 Duration 50 seconds 25 seconds Mode Frequency sweeping Temperature 55 O OC TruSeq Nano DNA Sample Preparation Guide 5 3 VNG JUaubely High Sample HS Protocol o4 Table 11 Covaris M220 Settings Setting 350 bp Insert 550 bp Insert Duty factor 20 Peak Incident Power 50 W Cycles per burst 200 Duration 65 seconds 45 seconds Temperature 20 C i E NOTE The Covaris M220 settings are optimized for use with the Covaris microTUBE AFA Fiber Pre Slit Snap Cap 6x16mm Table 12 Covaris S2 and E210 Settings Setting 350 bp Insert 550 bp Insert Duty cycle 1076 Intensity 5 0 2 0 Cydles per burst 200 Duration 45 seconds Mode Frequency sweeping Displayed Power 92 29 W 92 9 W E210 14W E210 7W Temperature 5 8 1 OE Centrifuge the CFP plate at 600 x g for 5 seconds Transfer 50 ul of fragmented DNA from each Covaris tube in the CFP plate to the cor
86. plates or E amp K Scientific part 480096 Twin tec 96 well PCR plates Eppendorf part 951020303 Table 19 User Supplied Consumables Additional Items for HS Processing Consumable Supplier Hard Shell 96 well PCR Plates HSP plate Bio Rad part HSP 9601 Table 20 User Supplied Equipment Equipment Supplier Optional 2100 Bioanalyzer Desktop System Agilent part G2940CA Optional Agilent DNA 7500 Kit Agilent part 5067 1506 Optional Agilent High Sensitivity DNA Kit Agilent part 4 5067 4626 One of the following Covaris systems Covaris M220 part 500295 e S2 For all other models contact e 5220 Covaris e E210 e M220 Optional Eco Real Time PCR System Ilumina catalog EC 100 1000 110 V EC 100 1001 220 V Optional Fluorometer for quantitation with General lab supplier dsDNA binding dyes Magnetic stand 96 Life Technologies catalog AM10027 Microplate centrifuge General lab supplier Vortexer General lab supplier TruSeq Nano DNA Sample Preparation Guide O7 juaudinby pue se qeuunsuo Supporting Information 98 Table 21 User Supplied Equipment Additional Items for LS Processing Equipment Supplier 96 well thermal cycler General lab supplier with heated lid See Thermal Cyclers on page 99 Table 22 User Supplied Equipment Additional Items for HS Processing Equipment Supplier High Speed Microplate Shaker VWR catalog 13500 890 110 V 120 V VWR catalog 14216 214 230 V
87. provides a complementary overhang for ligating the adapter to the fragment This strategy ensures a low rate of chimera concatenated template formation Consumables Item Quantity Storage A Tailing Mix ATL LT kit 1 tube per 24 15 C to 25 C reactions or HT kit 1 tube per 48 reactions Resuspension Buffer RSB 1 tube ZIXC t9 BAC Microseal B adhesive seal 1 15 C to 30 C RNase DNase free eight tube 2 15 C to 30 C strips and caps if using multichannel pipettes RNase DNase free reagent 2 15 C to 30 C reservoirs if using multichannel pipettes Preparation Supplied By Illumina Illumina User User User Remove the A Tailing Mix from 15 C to 25 C storage and thaw it at room temperature Remove the Resuspension Buffer from 2 C to 8 C storage and bring it to room temperature Remove the ALP plate from 15 C to 25 C storage if it was stored at the conclusion of Clean Up IMP and Size Selection on page 21 Let it thaw at room temperature 26 Part 15041110 Rev B Centrifuge the thawed ALP plate at 280 x g for 1 minute Remove the adhesive seal from the ALP plate Pre program the thermal cycler with the following program and save as ATAIL70 Choose the pre heat lid option and set to 100 C 37 C for 30 minutes 70 C for 5 minutes 4 C for 5 minutes Hold at 4 C Add ATL 1 Centrifuge the thawed A Tailing Mix tube at 600 x g for 5 seconds 2 Add125 ul thawed A Tailing Mix t
88. re well dispersed Add 50 ul mixed Sample Purification Beads to each well of the CAP plate Mix thoroughly as follows a Seal the CAP plate with a Microseal B adhesive seal b Shake the CAP plate on a microplate shaker at 1800 rpm for 2 minutes Incubate the CAP plate at room temperature for 5 minutes Centrifuge the CAP plate at 280 x g for 1 minute Remove the adhesive seal from the CAP plate Place the CAP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear Remove and discard 95 ul of the supernatant from each well of the CAP plate Take care not to disturb the beads NOTE E Leave the CAP plate on the magnetic stand while performing the following steps 28 32 With the CAP plate on the magnetic stand add 200 ul freshly prepared 8076 EtOH to each well Take care not to disturb the beads Incubate the CAP plate at room temperature for 30 seconds and then remove and discard all of the supernatant from each well Take care not to disturb the beads Repeat steps 28 and 29 one time for a total of two 8076 EtOH washes Part 15041110 Rev B 31 32 33 34 35 36 37 38 39 With the CAP plate on the magnetic stand let the samples air dry at room temperature for 5 minutes Remove and discard any remaining EtOH with a 10 ul pipette With the CAP plate on the magnetic stand add 27 5 ul Resuspension Buffer to each well of the plate Remove the CAP plate from the magnetic
89. red end 2 CR 29 67 PCR grade water 20 58 PDP 43 81 pooled oo volumes 45 83 pooling guidelines 7 ositive control 5 PC 36 74 Q qPCR 41 79 quality control 41 79 quantify libraries 41 79 quantitation 4 quantity and quality 4 R Reagent Reservoirs 20 26 29 37 58 64 67 75 RSB 13 19 26 28 36 51 57 64 66 74 S shear gDNA 15 53 shearing 2 single read 2 Size Selection 21 59 SPB 13 19 28 36 51 57 66 74 STL 28 66 strip tubes and caps 20 26 29 37 58 64 67 75 T technical assistance 105 thermal cycler 3 Training 6 Tris HCl 43 81 TSP1 36 44 74 82 W workflow diagram 11 49 Parts 15041110 Rev B Technical Assistance For technical assistance contact Illumina Technical Support Table 27 Illumina General Contact Information Illumina Website www illumina com Email techsupport illumina com Table 28 Illumina Customer Support Telephone Numbers Region Contact Number Region Contact Number North America 1 800 809 4566 Italy 800 874909 Austria 0800 296575 Netherlands 0800 0223859 Belgium 0800 81102 Norway 800 16836 Denmark 80882346 Spain 900 812168 Finland 0800 918363 Sweden 020790181 France 0800 911850 Switzerland 0800 563118 Germany 0800 180 8994 United Kingdom 0800 917 0041 Ireland 1 800 812949 Other countries 44 1799 534000 Safety Data Sheets Safety data sheets SDSs are available on the Illumina website at www illumina com msds Product Documentati
90. reservoir mix with a 1000 ul pipette Incubate the CEP plate at room temperature for 5 minutes Centrifuge the CEP plate at 280 x g for 1 minute Remove the adhesive seal from the CEP plate Place the CEP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear Using a 200 ul single channel or multichannel pipette set to 138 ul remove and discard 138 ul of the supernatant from each well of the CEP plate Take care not to disturb the beads Repeat step 7 one time removing and discarding a total of 276 ul of supernatant from each well NOTE j Leave the CEP plate on the magnetic stand while performing the following steps 9 13 With the CEP plate on the magnetic stand add 200 ul freshly prepared 80 EtOH to each well without disturbing the beads Incubate the CEP plate at room temperature for 30 seconds and then remove and discard all of the supernatant from each well Take care not to disturb the beads Repeat steps 9 and 10 one time for a total of two 80 EtOH washes With the CEP plate on the magnetic stand let the samples air dry at room temperature for 5 minutes Remove and discard any remaining EtOH with a 10 ul pipette With the CEP plate on the magnetic stand add 20 ul Resuspension Buffer to each well of the plate Remove the CEP plate from the magnetic stand Part 15041110 Rev B 15 16 17 18 19 20 Mix thoroughly as follows a Seal the CEP plate with a Microseal B
91. responding well of the new MIDI plate labeled with the CSP barcode using a single channel pipette Part 15041110 Rev B 6 Proceed immediately to Clean Up Fragmented DNA Clean Up Fragmented DNA 1 Vortex the room temperature Sample Purification Beads for at least 1 minute or until they are well dispersed 2 Add 80 ul well mixed Sample Purification Beads to each well of the CSP plate containing 50 ul of fragmented gDNA Mix thoroughly as follows a Seal the CSP plate with a Microseal B adhesive seal b Shake the CSP plate on a microplate shaker at 1800 rpm for 2 minutes E NOTE Vortex the Sample Purification Beads frequently to make sure that they are evenly distributed Illumina recommends the following If using a single channel pipette vortex the beads after processing four samples If using a multichannel pipette vortex the beads after processing four columns If the beads are in a reagent reservoir mix with a 1000 ul pipette 1 NOTE Keep the Sample Purification Beads tube at room temperature for later use in the protocol 3 Incubate the CSP plate at room temperature for 5 minutes 4 Centrifuge the CSP plate at 280 x g for 1 minute 5 Remove the adhesive seal from the CSP plate 6 Place the CSP plate on the magnetic stand at room temperature for 8 minutes or until the liquid is clear 7 Using a 200 ul single channel or multichannel pipette set to 125 ul remove and discard 125 ul of the supernatant from each well of the
92. rresponding well of the new MIDI plate labeled with the IMP barcode Take care not to disturb the beads Proceed immediately to Perform End Repair and Size Selection on page 57 Part 15041110 Rev B Perform End Repair and Size Selection This process converts the overhangs resulting from fragmentation into blunt ends using End Repair Mix 2 The 3 to 5 exonuclease activity of this mix removes the 3 overhangs and the 5 to 3 polymerase activity fills in the 5 overhangs Following end repair the appropriate library size is selected using different ratios of the Sample Purification Beads Consumables Item End Repair Mix 2 ERP2 Resuspension Buffer RSB Sample Purification Beads SPB Barcode labels for ALP Adapter Ligation Plate CEP Clean Up End Repair Plate 15 ml conical tube when processing gt 6 samples ata time or 1 7 ml microcentrifuge tube when processing lt 6 samples ata time 96 well MIDI plates Freshly prepared 80 ethanol EtOH TruSeq Nano DNA Sample Preparation Guide Quantity LT kit 1 tube per 24 reactions or HT kit 1 tube per 48 reactions 1 tube 1 tube per 24 reactions 1 label per plate p 400 ul per sample Storage 15 C to 25 C LC O BAC 2 C to 8 C ISS to SUE 15 C to 30 C IBC to SUE 15 C to 30 C Supplied By Ilumina Ilumina Ilumina Ilumina User User User r4 uonoejes ezig pue Jreday pug uuop
93. same row Also include a common index in each column This arrangement facilitates pipetting operations when dispensing indexed adapters and pooling indexed libraries later in the protocol When indexing libraries with the DAP arrange samples that will be pooled together in the same orientation as the indices in the DAP NOTE When indexing libraries with the DAP Review Handling Adapter Plate in the TruSeq Sample Preparation Pooling Guide part 15042173 See Additional Resources on page 6 for information on how to download the guide from the Illumina website e Illumina recommends that the DAP does not undergo more than four freeze thaw cycles To maximize the use of the DAP process more than 24 samples at a time These samples can then be pooled in any supported configuration Stop Ligation Buffer NOTE Do not remove the Ligation Mix 2 tube from 15 C to 25 C storage until instructed to do so in the procedures Remove the Resuspension Buffer from 2 C to 8 C storage and bring it to room temperature Review Best Practices for Handling Magnetic Beads See Additional Resources on page 6 for information on how to access TruSeq Nano DNA Sample Preparation Best Practices on the Illumina website Remove the Sample Purification Beads from 2 C to 8 C storage and let stand for at least 30 minutes to bring them to room temperature Pre heat the microheating system 1 to 30 C Apply a CAP barcode label to a new 96 well MIDI plate App
94. ses of the TruSeq Nano DNA Sample Preparation LS protocol to prepare templates using indexed adapter tubes or a DAP Figure TruSeq Nano DNA Sample Preparation LS Workflow Prepare Adapter Setup Ligate Indexed Paired End Adapters Purified Genomic DNA Consumables DNA Adapters or DAP EtOH uG2 Fragment RSB Genomic DNA P Consumable RSB Plato SPB CAP PCR Piste CFP CSP DNA IMP 1 Repair Ends and Size Selection PCR Amplification Consumables Consumables ERP2 EtOH EtOH EPM RSB PPC SPB RSB Water SPB Plates Plate ALP TSP1 CEP 1 1 Validate Library Adenylate 3 Ends Consumables ATL 1 RSB Plato Normalize and Pool ALP Libraries Consumables Tris HCl 10 mM w Tween 20 Plates DCT POP pooling only TruSeq Nano DNA Sample Preparation Guide 1 1 MOP JON day ajduwes Low Sample LS Protocol Prepare Adapter Setup 12 If you are pooling record information about your samples before beginning library preparation for later use in data analysis Use IEM to create and edit a sample sheet for Illumina sequencers and analysis software See Additional Resources on page 6 for information on how to download IEM software and documentation from the Illumina website Review planning steps in the TruSeq Sample Preparation Pooling Guide part 15042173 See Additional Resources on page 6 for information on how to download the guide from the Illumina website If you are pooling using adapter index tubes Illumina recommends arranging
95. ssemble the Product ii separate extract or isolate components of this Product or subject this Product or components thereof to any analysis not expressly authorized in this Products Documentation iii gain access to or attempt to determine the methods of operation of this Product or iv transfer to a third party or grant a sublicense to any Software or any third party software Purchaser further agrees that the contents of and methods of operation of this Product are proprietary to lumina and this Product contains or embodies trade secrets of Illumina The conditions and restrictions found in these terms and conditions are bargained for conditions of sale and therefore control the sale of and use of this Product by Purchaser 5 Limited Liability TO THE EXTENT PERMITTED BY LAW IN NO EVENT SHALL ILLUMINA OR ITS SUPPLIERS BE LIABLE TO PURCHASER OR ANY THIRD PARTY FOR COSTS OF PROCUREMENT OF SUBSTITUTE PRODUCTS OR SERVICES LOST PROFITS DATA OR BUSINESS OR FOR ANY INDIRECT SPECIAL INCIDENTAL EXEMPLARY CONSEQUENTIAL OR PUNITIVE DAMAGES OF ANY KIND ARISING OUT OF OR IN CONNECTION WITH WITHOUT LIMITATION THE SALE OF THIS PRODUCT ITS USE ILLUMINA S PERFORMANCE HEREUNDER OR ANY OF THESE TERMS AND CONDITIONS HOWEVER ARISING OR CAUSED AND ON ANY THEORY OF LIABILITY WHETHER IN CONTRACT TORT INCLUDING NEGLIGENCE STRICT LIABILITY OR OTHERWISE 6 ILLUMINA S TOTAL AND CUMULATIVE LIABILITY TO PURCHASER OR ANY THIRD PARTY ARISING OUT OF OR
96. stand Mix thoroughly as follows a Seal the CAP plate with a Microseal B adhesive seal b Shake the CAP plate on a microplate shaker at 1800 rpm for 2 minutes Incubate the CAP plate at room temperature for 2 minutes Centrifuge the CAP plate at 280 x g for 1 minute Remove the adhesive seal from the CAP plate Place the CAP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear Transfer 25 ul of the clear supernatant from each well of the CAP plate to the corresponding well of the new HSP plate labeled with the PCR barcode Take care not to disturb the beads SAFESTOPPING POINT If you do not plan to proceed immediately to Enrich DNA Fragments on page 74 the protocol 1 can be safely stopped here If you are stopping seal the PCR plate with a Microseal B adhesive seal and store at 15 C to 25 C for up to 7 days TruSeq Nano DNA Sample Preparation Guide T 3 sJo1depy o1e6rq High Sample HS Protocol Enrich DNA Fragments 14 This process uses PCR to selectively enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library The PCR is performed with a PCR Primer Cocktail that anneals to the ends of the adapters Minimize the number of PCR cycles to avoid skewing the representation of the library NOTE PCR enriches for fragments that have adapters ligated on both ends Fragments with only one or no adapters on their ends
97. t have adapters ligated on both ends Fragments with only one or no adapters on their ends are by products of inefficiencies in the ligation reaction Neither species can be used to make clusters Fragments without any adapters cannot hybridize to surface bound primers in the flow cell Fragments with an adapter on only one end can hybridize to surface bound primers but cannot form clusters Consumables Item Enhanced PCR Mix EPM PCR Primer Cocktail PPC Sample Purification Beads SPB Resuspension Buffer RSB TSP1 Target Sample Plate barcode label 96 well 0 3 ml PCR plate Quantity LT kit 1 tube per 24 reactions Or HT kit 1 tube per 48 reactions LT kit 1 tube per 24 reactions Or HT kit 1 tube per 48 reactions 1 tube per 24 reactions 1 tube 1 label per plate Storage 15 C to 25 C 15 C to 25 C 2 C to 8 C BE ro E 15 C to 30 C Cto 0S9 Supplied By Mumina Illumina Illumina Illumina Illumina User Part 15041110 Rev B Item Quantity Storage Supplied By Freshly prepared 80 400 ul per sample 15 C to 30 C User ethanol EtOH Microseal B adhesive seals 2 USMC tio SOME User RNase DNase free eight 5 15 C to 30 C User tube strips and caps if using multichannel pipettes RNase DNase free reagent 5 TES o SOURCE User reservoirs if using multichannel pipettes Preparation Remove the Enhanced PCR Mix and PCR Primer Cocktail
98. t in effect on the date that the Product ships from Illumina Part 15041110 Rev B 2 Research Use Only Rights Subject to these terms and conditions and unless otherwise agreed upon in writing by an officer of Illumina Purchaser is granted only a non exclusive non transferable personal non sublicensable right under Illumina s Core IP in existence on the date that this Product ships from Illumina solely to use this Product in Purchaser s facility for Purchaser s internal research purposes which includes research services provided to third parties and solely in accordance with this Product s Documentation but specifically excluding any use that a would require rights or a license from Illumina to Application Specific IP b is a re use of a previously used Consumable c is the disassembling reverse engineering reverse compiling or reverse assembling of this Product d is the separation extraction or isolation of components of this Product or other unauthorized analysis of this Product e gains access to or determines the methods of operation of this Product f is the use of non Illumina reagent consumables with Illumina s Hardware does not apply if the Specifications or Documentation state otherwise or g is the transfer to a third party of or sub licensing of Software or any third party software All Software whether provided separately installed on or embedded in a Product is licensed to Purchaser and not sold Except as
99. t pipettes are correctly calibrated and are not used at the volume extremes of their performance specifications Assessing DNA Quality Absorbance measurements at 260 nm are commonly used to assess DNA quality The ratio of absorbance at 260 nm to absorbance at 280 nm is used as an indication of sample purity and values of 1 8 2 0 are considered indicative of relatively pure DNA The presence of RNA or small nucleic acid fragments such as nucleotides can compromise both absorbance measurements Carefully collect gDNA samples to make sure that they are free of contaminants A Part 15041110 Rev B Positive Control In line controls are not provided in TruSeq Nano DNA Sample Preparation kits Therefore Illumina recommends using Coriell Human 1 DNA NA 18507 or Promega Human Genomic DNA G3041 as a positive control sample for this protocol TruSeq Nano DNA Sample Preparation Guide 5 O43002 SAINSOJ Overview Additional Resources The following resources are available for TruSeq Nano DNA Sample Preparation protocol guidance and sample tracking Access these and other resources on the Illumina website at support illumina com sequencing kits ilmn Then select TruSeq Nano DNA LT Sample Prep Kit Support or TruSeq Nano DNA HT Sample Prep Kit Support Resource Training Best Practices Description Illustrates elements of the TruSeq Nano DNA Sample Preparation process Viewing these videos is recommended for new and less experi
100. tems for HS Processing 98 TruSeq Nano DNA LT Sample Prep Kit Set A Indexed Adapter Sequences 100 TruSeq Nano DNA LT Sample Prep Kit Set B Indexed Adapter Sequences 101 TruSeq Nano DNA HT Sample Prep Kit Indexed Adapter 1 Sequences 101 TruSeq Nano DNA HT Sample Prep Kit Indexed Adapter 2 Sequences 102 Illumina General Contact Information o oooccccccccccccccccccccccccnccccccccoo 105 Illumina Customer Support Telephone Numbers 105 TruSeq Nano DNA Sample Preparation Guide XI Part 15041110 Rev B Overview Introduction a Ls cuneos E tarado pets 2 Protocol Features nn 3 DNA Input Recommendations 2 2 2 2 22 ccc cece cece cece cccccccceececeeeeecscssessseeees 4 Positive Control cout stare ect ct elena LE dda ita 5 Additional Resources 2 2 ieee cece cece cece cccccccececccceccececccceeceeeececceeeeeeeecceeeeees 6 J PRT eaa e P A TruSeq Nano DNA Sample Preparation Guide 1 1ajdeyo Overview Introduction This protocol explains how to prepare up to 96 pooled indexed paired end libraries of genomic DNA gDNA for subsequent cluster generation and DNA sequencing using the reagents provided in the Illumina TruSeq Nano DNA Sample Prep Kits low throughput LT and high throughput HT The goal of this protocol is to add adapter sequences onto the ends of DNA fragments to generate indexed single read or paired end sequencing libraries The sa
101. the ALP plate on a microplate shaker at 1800 rpm for 2 minutes 3 Centrifuge the ALP plate at 280 x g for 1 minute Clean Up ALP 1 Remove the adhesive seal from the ALP plate 70 Part 15041110 Rev B N Oo OF q 10 11 12 15 14 Vortex the Sample Purification Beads for at least 1 minute or until they are well dispersed Add 42 5 ul well mixed Sample Purification Beads to each well of the ALP plate Mix thoroughly as follows a Seal the ALP plate with a Microseal B adhesive seal b Shake the ALP plate on a microplate shaker at 1800 rpm for 2 minutes i NOTE Vortex the Sample Purification Beads frequently to make sure that they are evenly distributed Illumina recommends the following sJo1depy e1e6r If using a single channel pipette vortex the beads after processing four samples If using a multichannel pipette vortex the beads after processing four columns If the beads are in a reagent reservoir mix with a 1000 ul pipette Incubate the ALP plate at room temperature for 5 minutes Centrifuge the ALP plate at 280 x g for 1 minute Remove the adhesive seal from the ALP plate Place the ALP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear Remove and discard 80 ul of the supernatant from each well of the ALP plate Take care not to disturb the beads NOTE E Leave the ALP plate on the magnetic stand while performing the following steps 9 13 With the ALP plate
102. the lid and incubate at 37 C for 30 minutes Immediately after the 37 C incubation remove the ALP plate from system 1 and place the plate on the pre heated microheating system 2 Close the lid and incubate at 70 C for 5 minutes Set the microheating system 1 to 30 C in preparation for Ligate Adapters Immediately remove the ALP plate from the microheating system 2 and place the plate on ice for 5 minutes Proceed immediately to Ligate Adapters on page 66 TruSeq Nano DNA Sample Preparation Guide 6 5 Spu3 e aje huspy High Sample HS Protocol Ligate Adapters 66 Consumables Item Choose from the following depending on the kit you are using e TruSeq Nano DNA LT Sample Prep Kit contents DNA Adapter Indices AD001 AD016 AD018 AD023 AD025 AD027 e TruSeq Nano DNA HT Sample Prep Kit contents DAP DNA Adapter Plate Ligation Mix 2 LIG2 Resuspension Buffer RSB Sample Purification Beads SPB Stop Ligation Buffer STL Quantity 1 tube of each index being used per column of 8 reactions or 1 DAP LT kit 1 tube per 24 reactions or HT kit 1 tube per 48 reactions 1 tube 1 tube per 24 reactions LT kit 1 tube per 24 reactions or HT kit 1 tube per 48 reactions Storage 15 C to 25 C 15 C to 25 C 2 C to 8 C LC O E 15 C to 25 C Part 15041110 Rev B This process ligates indexing adapters to the ends of the DNA fragments
103. tructions but with less detail than what is provided in this user guide New or less experienced users are advised to follow this user guide and not the EUC and LTF Click Documentation amp Literature on TruSeq Nano DNA LT Sample Prep Kit Support or Click Documentation amp Literature on TruSeq Nano DNA HT Sample Prep Kit Support Provides TruSeq pooling guidelines for sample preparation Review this guide before beginning library preparation Click Documentation amp Literature on TruSeq Nano DNA LT Sample Prep Kit Support or Click Documentation amp Literature on TruSeq Nano DNA HT Sample Prep Kit Support Enables you to create and edit appropriate sample sheets for Illumina sequencers and analysis software and record parameters for your sample plate To download the software Click Downloads on TruSeq Nano DNA LT Sample Prep Kit Support or Click Downloads on TruSeq Nano DNA HT Sample Prep Kit Support To download the documentation Click Documentation amp Literature on TruSeq Nano DNA LT Sample Prep Kit Support or Click Documentation amp Literature on TruSeq Nano DNA HT Sample Prep Kit Support S92JnoseH EeuonippV Part 15041110 Rev B Low Sample LS Protocol Introduction A ean oa den P i UON nds 10 Sample Prep Workflow 10 0 02 2222 c cece ccc cc ccc cc cece cece cece cece ee eeeeeeeteeeeeetetettttteesees 11 Prepare Adapter Setup 2 eere tar it il gue 12 denaib
104. ttings are optimized for use with the Covaris micro TUBE AFA Fiber Pre Slit Snap Cap 6x16mm Table 6 Covaris S2 and E210 Settings Setting 350 bp Insert 550 bp Insert Duty cycle 1076 Intensity 5 0 2 0 Cydles per burst 200 Duration 45 seconds Mode Frequency sweeping Displayed Power 92 293 W S2 9 W E210 14 W E210 7 W Temperature 935 ie OC Centrifuge the CFP plate at 600 x g for 5 seconds Transfer 50 ul of fragmented DNA from each Covaris tube in the CFP plate to the corresponding well of the new 0 3 ml PCR plate labeled with the CSP barcode using a single channel pipette Part 15041110 Rev B 6 Proceed immediately to Clean Up Fragmented DNA Clean Up Fragmented DNA 1 Vortex the room temperature Sample Purification Beads for at least 1 minute or until they are well dispersed Add 80 ul well mixed Sample Purification Beads to each well of the CSP plate containing 50 ul of fragmented gDNA Set a 200 ul pipette to 125 ul and then gently pipette the entire volume up and down 10 times to mix thoroughly E NOTE Vortex the Sample Purification Beads frequently to make sure that they are evenly distributed Illumina recommends the following If using a single channel pipette vortex the beads after processing four samples If using a multichannel pipette vortex the beads after processing four columns If the beads are in a reagent reservoir mix with a 1000 ul pipette E NOTE Keep the Sample Purificat
105. ture is in a reagent reservoir mix with a 1000 ul pipette Incubate the IMP plate at room temperature for 5 minutes Place the IMP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear Use a 200 ul single channel or multichannel pipette set to 125 ul to transfer 125 ul of the supernatant containing the DNA of interest from each well of the IMP plate to the corresponding well of the new 0 3 ml PCR plate labeled with the CEP barcode Take care not to disturb the beads NOTE Transfer do not discard the supernatant It contains the DNA of interest Repeat step 7 one time Each CEP plate well now contains a total of 250 ul of DNA of interest Discard the IMP plate containing the beads 10 Discard any remaining diluted bead mixture Remove Small DNA Fragments NOTE In the following steps use undiluted Sample Purification Beads Vortex the Sample Purification Beads for at least 1 minute or until they are well dispersed Add 30 ul undiluted Sample Purification Beads to each well of the CEP plate containing 250 ul of the supernatant with the DNA of interest Set a 200 ul pipette to 200 ul and then gently pipette the entire volume up and down 10 times to mix thoroughly TruSeq Nano DNA Sample Preparation Guide 2 3 UOI 93 9S ezig pue Jreday pug uuopegd Low Sample LS Protocol 24 10 11 12 13 NOTE Aspirate the Sample Purification Beads slowly and dispense them slowly
106. uide 1 01 Supporting Information Table 26 TruSeq Nano DNA HT Sample Prep Kit Indexed Adapter 2 Sequences Adapter Sequence Adapter Sequence D501 TATAGCCT AGGCGAAG D502 ATAGAGGC D506 TAATCTTA D503 CCTATCCT o pP CAGGACGT D504 GGCTCTGA D508 GTACTGAC 1 02 Part 15041110 Rev B Index A Acronyms 87 Add ATL 27 65 Add LIG 30 69 Add STL 32 70 ALP 19 57 Amp PCR 39 77 ATL 26 64 B Best Practices 6 C CAP 29 67 CEP 19 57 CFP 13 51 Clean Up ALP 32 70 Clean D IMP 21 59 Clean Up PCR 39 77 cluster generation 2 46 84 Covaris instrument 14 52 Covaris shearing 13 51 Covaris tubes 14 52 CPP 74 CSP 13 51 customer support 105 D DAP 28 66 DCT 43 81 DNA Adapter Indices 28 66 DNA Plate DNA 13 51 DNA sequencing 2 documentation 105 E EPM 36 74 ERP2 19 57 TruSeq Nano DNA Sample Preparation Guide xapul EtOH 14 19 29 52 57 67 experienced user card EUC 7 Fragment DNA 15 53 G gDNA 2 H help technical 105 no Sample HS 3 HSP 3 IEM 7 IMP 13 51 Incubate 1 ALP 27 65 Incubate 1 IMP 21 59 Incubate 2 ALP 32 70 indexed adapter 100 101 insert size 13 51 L lab tracking form LIF 7 LIG2 28 66 Low Sample LS 3 M Make CFP 15 53 Make DCT 44 82 Make IMP 20 58 Make PCR 38 76 Make PDP 45 83 master mixed reagents 2 micro plate shaker 3 microheating system 3 103 Index 104 MIDI 3 N normalize gDNA 15 53 P ai
107. ume up and down 10 times to mix thoroughly Incubate the CSP plate at room temperature for 2 minutes Place the CSP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear Transfer 60 ul of the clear supernatant from each well of the CSP plate to the corresponding well of the new 0 3 ml PCR plate labeled with the IMP barcode Take care not to disturb the beads NOTE Make sure that you use a 0 3 ml PCR plate because IMP plate volumes are greater than a 0 2 ml PCR plate Final volumes during size selection are up to 260 ul per well Proceed immediately to Perform End Repair and Size Selection on page 19 Part 15041110 Rev B Perform End Repair and Size Selection This process converts the overhangs resulting from fragmentation into blunt ends using End Repair Mix 2 The 3 to 5 exonuclease activity of this mix removes the 3 overhangs and the 5 to 3 polymerase activity fills in the 5 overhangs Following end repair the appropriate library size is selected using different ratios of the Sample Purification Beads Consumables Item End Repair Mix 2 ERP2 Resuspension Buffer RSB Sample Purification Beads SPB Barcode labels for ALP Adapter Ligation Plate CEP Clean Up End Repair Plate 15 ml conical tube when processing gt 6 samples ata time or 1 7 ml microcentrifuge tube when processing lt 6 samples ata time 96 well 0 3 ml PCR plates Freshly prep
108. use of this Product in any manner or for any purpose outside the scope of research use purposes ii the use of this Product in any manner not in accordance with its Specifications its Documentation the rights expressly granted to Purchaser hereunder or any breach by Purchaser of these terms and conditions iii the use of this Product in combination with any other products materials or services not supplied by Illumina iv the use of this Product to perform any assay or other process not supplied by Illumina or v Illumina s compliance with specifications or instructions for this Product furnished by or on behalf of Purchaser each of i v is referred to as an Excluded Claim Indemnification by Purchaser Purchaser shall defend indemnify and hold harmless Illumina its affiliates their non affiliate collaborators and development partners that contributed to the development of this Product and their respective officers directors representatives and employees against any claims liabilities damages fines penalties causes of action and losses of any and every kind including without limitation personal injury or death claims and infringement of a third party s intellectual property rights resulting from relating to or arising out of i Purchaser s breach of any of these terms and conditions ii Purchaser s use of this Product outside of the scope of research use purposes iii any use of this Product not in accordance with
109. using the Low Sample Protocol Chapter 3 High Sample HS Protocol explains how to perform the TruSeq Nano DNA Sample Preparation using the High Sample Protocol Equivalent results can be expected from either protocol however the HS protocol can yield more consistent results between samples Their distinguishing elements are as follows Table 1 Protocol Features Low Sample High Sample LT Kit Number of samples 24 with indexed gt 24 with indexed processed at one time adapter tubes adapter tubes HT Kit Number of samples 24 with indexed gt 24 with indexed processed at one time adapter plate adapter plate Plate Type 96 well 0 3 ml PCR 96 well HSP 96 well MIDI Incubation Equipment 96 well thermal cycler Microheating systems Mixing Method Pipetting Microplate shaker Each TruSeq Nano DNA LT Sample Prep Kit contains enough reagents to prepare up to 24 samples When used together TruSeq Nano DNA LT Sample Prep Kits A and B allow for pooling up to 24 samples using the 12 different indices in each kit Illumina does not recommend preparing more than 24 samples at a time using the LS protocol An alternative to using the HS protocol for more than 24 samples is to perform separate library preparations to ensure robust performance The TruSeq Nano DNA Sample Preparation fragmentation process is optimized to obtain final libraries with the following average insert size Table2 Insert Size Options Insert Size 350 bp 550 bp Input DNA
110. ve it store the components at 2 C to 8 C TruSeq Nano DNA Sample Preparation Guide Q 3 SJUSJUOD 1M Supporting Information 94 Figure 14 TruSeq Nano DNA HT Sample Prep Kit 96 Samples SP Beads Box 2 of 2 part 15041878 Slot Reagent Part Description 1 4 SPB 15041032 Sample Purification Beads Part 15041110 Rev B Consumables and Equipment Check to make sure that you have all of the necessary user supplied consumables and equipment before starting the TruSeq Nano DNA Sample Preparation protocol The requirement for some supplies is dependent upon the protocol performed LS or HS and these items are specified in separate tables lay NOTE The TruSeq Nano DNA Sample Preparation protocol has been optimized and validated using the items listed Comparable performance is not guaranteed when using alternate consumables and equipment Table 17 User Supplied Consumables Consumable Supplier 1 7 ml microcentrifuge tubes General lab supplier 15 ml conical tubes General lab supplier 10 ul barrier pipette tips General lab supplier 10 ul multichannel pipettes General lab supplier 10 ul single channel pipettes General lab supplier 1000 ul barrier pipette tips General lab supplier 1000 ul multichannel pipettes General lab supplier 1000 ul single channel pipettes General lab supplier 20 ul barrier pipette tips General lab supplier 20 ul multichannel pipettes General lab supplier 20 ul single channel pipettes General lab suppli
111. xture for 5 seconds to make sure that the beads are evenly dispersed Add 160 ul of the diluted bead mixture to each well of the IMP plate containing 100 ul of the end repaired sample Mix thoroughly as follows a Seal the IMP plate with a Microseal B adhesive seal b Shake the IMP plate on a microplate shaker at 1800 rpm for 2 minutes J NOTE Aspirate the diluted bead mixture slowly and dispense it slowly due to the viscosity of the solution Changes in the volume of the diluted bead mixture affect the insert size of your library Part 15041110 Rev B NOTE j Vortex the diluted bead mixture frequently Illumina recommends the following If using a single channel pipette vortex the mixture after processing four samples If using a multichannel pipette vortex the mixture after processing four columns If the mixture is in a reagent reservoir mix with a 1000 ul pipette Incubate the IMP plate at room temperature for 5 minutes Centrifuge the IMP plate at 280 x g for 1 minute Remove the adhesive seal from the IMP plate o N A OF Place the IMP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear 9 Usea 200 ul single channel or multichannel pipette set to 125 ul to transfer 125 ul of the supernatant containing the DNA of interest from each well of the IMP plate to the corresponding well of the new MIDI plate labeled with the CEP barcode Take care not to disturb the beads NOT

TruSeq Nano DNA Sample Preparation Guide 15041110 B

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