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TruSight One Sequencing Panel Library - Support

1. ILLUMINA PROPRIETARY Catalog FC 141 9001DOC Part 15046431 Rev A October 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 lumina 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 CONNE
2. Library Pool Total DNA Library Complexity Mass ng 1 plex 500 3 plex 1500 5 plex 2500 7 plex 3500 9 plex 4500 11 plex 5500 NOTE The 9 sample TruSight One kit is intended to support 3 samples per enrichment The 36 sample TruSight One kit can support up to 12 samples per enrichment TruSight One Library Preparation Guide P T UOUSZIDUOAH 18414 Protocol Procedure 1 28 Thoroughly vortex the Enrichment Hybridization Buffer tube until the solution is completely resuspended Visually make sure that no crystal structures are present NOTE If crystals and cloudiness are observed vortex the Enrichment Hybridization Buffer tube until it appears clear Add the following reagents in the order listed to each well of the new 96 well HSP plate labeled NEH1 Reagent Volume ul DNA library sample or library pool from NLS plate 40 Enrichment Hybridization Buffer 50 TruSight One Oligos 10 100 Total Volume per Sample Mix thoroughly as follows a Seal the NEH1 plate with a Microseal B adhesive seal Make sure that the plate is tightly sealed to prevent potential evaporation Use an adhesive seal roller to apply force to the seal and make sure that the seal is secured b Shake the NEHI plate on a microplate shaker at 1200 rpm for 1 minute Centrifuge the NEHI plate to 280 x g for 1 minute Place the sealed NEHI plate on the pre programmed thermal cycler Close the lid then select and run the NRC HYB program I
3. High enrichment rates low duplicates and exceptional coverage uniformity Efficient use of sequencing Reliable variant calling Reduced hands on time with the most cost effective high throughput workflow 2 Part 15046431 Rev A DNA Input Recommendations TruSight One library preparation uses an enzymatic DNA fragmentation step and thus can be more sensitive to DNA input compared to mechanical fragmentation methods The ultimate success of enrichment strongly depends on using an accurately quantified amount of input DNA Therefore accurate quantitation of the gDNA is essential Illumina recommends quantifying the starting g DNA using a fluorometric based method specific for double stranded DNA dsDNA and running samples in triplicate to obtain more confident measurements Methods that measure total nucleic acid content e g nanodrop or other UV absorbance methods should be avoided because common contaminants such as ssDNA RNA and oligos are not substrates for the TruSight One Sequencing Panel The TruSight One protocol has been optimized for 50 ng of total gDNA A higher mass input of g DNA may result in incomplete tagmentation and larger insert sizes which can impact enrichment performance Conversely a low mass input of gDNA or low quality gDNA in the tagmentation reaction may generate smaller than expected insert sizes which can be lost during subsequent clean up steps resulting in lower diversity To minimize gDNA sample input va
4. Index Primer N701 N705 and N709 15 C to 25 C Box 4 Oligos This box is shipped on dry ice As soon as you receive your kit store the components at 15 C to 25 C Quantity Reagent Name Storage Temperature 1 tube TruSight One Content Set 15 C to 25 C MiSeq Reagent Kit v3 Box 1 This box is shipped on dry ice As soon as you receive your kit store the components at 15 C to 25 C Quantity Reagent Name Storage Temperature 3 Hybridization Buffer 15 C to 25 C 3 MiSeq V3 Cartridge 15 C to 25 C MiSeq Reagent Kit v3 Box 2 This box is shipped on dry ice As soon as you receive your kit store the components at 15 C to 25 C Quantity Reagent Name Storage Temperature 3 MiSeq Flowcell 15 C to 25 C 3 PR2 Bottle 15 C to 25 C Part 15046431 Rev A TruSight One Sequencing Panel Contents 86 Samples FC 141 1007 TG 141 1007 Box 1 Rapid Capture Reagents This box is shipped at room temperature As soon as you receive your kit store the components as specified Quantity Acronym Reagent Name Storage Temperature 2 Sample Purification Beads 2 C to 8 C 2 Streptavidin Magnetic Beads 2 C to 8 C 1 Elute Target Buffer 2 2 C to 8 C 1 15 C to 30 C Box 2 Rapid Capture Reagents This box is shipped on dry ice As soon as you receive your kit store the components at 15 C to 25 C Quantity Acronym Reagent Name Storage Temperature TDE1 Tagment DNA Enzyme 15 C to 25 C 1
5. all of the supernatant from each well of the NEW1 plate 10 Remove the NEW1 plate from the magnetic stand 11 Repeat steps 2 10 one time for a total of two Enrichment Wash Solution washes First Elution de 1 Add the following reagents in the order listed in a new 1 7 ml microcentrifuge tube to create the elution pre mix Multiply each volume by the number of pooled samples being prepared The volumes include an excess amount for processing multiple samples Reagent Volume ul Enrichment Elution Buffer 1 28 5 HP3 1 5 Total Volume per Sample 30 Vortex the elution pre mix tube then add 23 5 ul of the mix to each well of the NEW1 plate Mix thoroughly as follows a Seal the NEW1 plate with a Microseal B adhesive seal b Shake the NEW1 plate on a microplate shaker at 1800 rpm for 2 minutes Let the NEW1 plate stand at room temperature for 2 minutes Centrifuge the NEW1 plate to 280 x g for 1 minute Carefully remove the adhesive seal from the NEW1 plate to avoid spilling the contents of the wells Place the NEW1 plate on the magnetic stand for 2 minutes or until the liquid appears clear Part 15046431 Rev A 10 11 12 Transfer 21 ul of clear supernatant from each well of the NEWT plate to the corresponding well of the new HSP plate labeled NEH2 Take care not to disturb the beads A NOTE Illumina recommends using a 20 ul single channel or multichannel pipette set to 10 5 ul to perform two consecutive
6. barrier pipette tips 1000 ul multichannel pipettes 1000 ul single channel pipettes Adhesive seal roller 96 well storage plates round well 0 8 ml MIDI plate Hard Shell 96 well PCR Plates HSP plate TruSight One Library Preparation Guide Supplier General lab supplier General lab supplier General lab supplier General lab supplier General lab supplier General lab supplier General lab supplier General lab supplier General lab supplier General lab supplier General lab supplier Fisher Scientific part AB 0859 Bio Rad part HSP 9601 65 jueuudinb4 pue sejqeuunsuoy Supporting Information Consumable Optional Amicon Ultra 0 5 centrifugal filter unit 0 5 ml 30 kDa Note Used to concentrate a pooled library Another option is to use a vacuum concentrator Ethanol 200 proof absolute for molecular biology 500 ml Microseal A film Microseal B adhesive seals RNase DNase free eight tube strips and caps RNase DNase free multichannel reagent reservoirs disposable Tris HCl 10 mM pH 8 5 Optional TruSeq Index Plate Fixture Kit Note Recommended for setting up indexed PCR primers This part is reusable PCR grade water Table 5 User Supplied Equipment Equipment Optional 2100 Bioanalyzer Desktop System Optional DNA 1000 Chip Optional High Sensitivity DNA Chip Supplier Millipore part UFC503008 Sigma Aldrich part E7023 Bio Rad part MS
7. case the upgraded Hardware shipped to Purchaser comes with a Base Hardware Warranty c Exclusions from Warranty Coverage The foregoing warranties do not apply to the extent a non conformance is due to i abuse misuse neglect negligence accident improper storage or use contrary to the Documentation or Specifications ii improper handling installation maintenance or repair other than if performed by Illumina s personnel iii unauthorized alterations iv Force Majeure events or v use with a third party s good not provided by Illumina unless the Product s Documentation or Specifications expressly state such third party s good is for use with the Product d Procedure for Warranty Coverage In order to be eligible for repair or replacement under this warranty Purchaser must i promptly contact Illumina s support department to report the non conformance ii cooperate with Illumina in confirming or diagnosing the non conformance and iii return this Product transportation charges prepaid to Illumina following Illumina s instructions or if agreed by Illumina and Purchaser grant Illumina s authorized repair personnel access to this Product in order to confirm the non conformance and make repairs e Sole Remedy under Warranty Illumina will at its option repair or replace non conforming Product that it confirms is covered by this warranty Repaired or replaced Consumables come with a 30 day warranty Hardware may be repaired or replac
8. final concentration is desired dilute the post enriched dilution to a final concentration of 1 0 nM Prepare a fresh 0 1N NaOH solution using HP3 by taking 10 ul HP3 190 ul water Combine 10 ul of 1 25 nM library 10 ul 0 1N NaOH and mix well Incubate the sample at room temp for 5 minutes Following incubation add 980 ul HT1 and mix well by vortexing ON 0 m W N Place the denatured DNA on ice until you are ready to load into the MiSeq cartridge Part 15046431 Rev A 7 Load the entire sample onto the MiSeq cartridge and move forward with sequencing TruSight One Library Preparation Guide 5 3 e uo Bulousnbas 104 seueJqr ino Buuedejg Protocol Sequence Library Proceed to cluster generation For more information see the cluster generation section of the user guide for your Illumina platform When quantifying a TruSight One post enriched library using a fluorometric method clustering at 12 5 pM generates cluster densities in the range of 1 200 k 1 400 k clusters mm2 using the MiSeq V3 software and reagents Results may vary based on your method of quantification Illumina recommends that you determine the library concentration to cluster density relationship based on your lab instrumentation Review the procedures in the IEM quick reference card on how to create a sample sheet for the TruSight One Sequencing Panel See Additional Resources on page 5 for information on how to download IEM software and TruSight One appropri
9. plate is stored for more than 7 days requantify your library to guarantee the accuracy of your enrichment results Part 15046431 Rev A Validate Library Ilumina recommends performing the following procedures for quality control analysis and quantification of your enriched library Quantify Libraries In order to achieve the highest data quality on Illumina sequencing platforms it is important to create optimum cluster densities across every lane of every flow cell Optimizing cluster densities requires accurate quantitation of DNA library templates Ilumina recommends a fluorometric dsDNA assay to quantitate dsDNA samples because it can quantitate small DNA volumes and measure DNA directly Other techniques can pick up contamination such as RNA and proteins Illumina recommends using a spectrofluorometer because fluorometry provides DNA specific quantification Spectrophotometry can also measure RNA and yield values that are too high If quantifying a 3 12 plex library before quantification dilute the post enriched library by adding 2 ul library to 28 ul Resuspension Buffer in a new tube or well Use this dilution for quantification and quality assessment as well as sequencing For 1 or 2 plex enrichments a dilution is not needed 4 NOTE Use the following formula to convert from ng ul to nM Assume a 650 bp library size or calculate based on the average size of the enriched library concentration in ng ul x 10 6 concentratio
10. provided in this user guide New or less experienced users are strongly advised to follow this user guide and not the EUC and LTF Click Documentation amp Literature on the TruSight One Sequencing Panel Support page 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 the TruSight One Sequencing Panel Support page To download the documentation click Documentation amp Literature on the TruSight One Sequencing Panel Support page SOSDINOSSY JCUOIPPV Part 15046431 Rev A Protocol Introduction NNNM 8 Library Prep Workflow 9 Tagmen Genomic DNA outra AE 10 Clean Up Tagmented DNA 13 First PCR Amplification aa R RR RRR RR R Raa Ra 16 PirstiPGR CleanUp EE 20 First ElybridizatlOri EEN 25 First Gapt re uses een data ale 29 Second Hybridization 2 2 2 2 2c coco e cece eee cece eeeeeeceeeceeeeeeeeeeeees 34 Second Capite ee Ee Eh 36 Capture Sample Cleanlp c eee e cece emen RR rl lille ell 41 Second PCR Amplification 2 2 2 2 cece ccc n cece cee ceeeeececeeeeeeeeeeeeeeeeee 44 Second PCR Clean Up EZ ad eiae ccc ec eee cee eee ee eee eee ence eee Tai 46 Validate Library EE 49 Preparing Your Libraries for Sequencing on a MiSeq 222222 e eee eee cece cece eeeeeeee 52 Sequence UDan 54 A zo nr off Sea df A An A TGC S cA rta vegas rc 1C4
11. solution Then gently pipette the entire volume of each well up and down 10 times to ensure complete resuspension of the sample 4 NOTE Proper resuspension of the Streptavidin Magnetic Beads is needed to ensure efficient removal of non specific DNA from the reaction which otherwise results in poor enrichment statistics Seal the NEW1 plate with a Microseal B adhesive seal Place the sealed NEW1 plate on the pre heated microheating system Close the lid and incubate at 50 C for 30 minutes NOTE i The Enrichment Wash steps are key to ensuring high enrichment specificity Illumina recommends using a microheating system with a MIDI plate insert for these steps to make sure that samples are maintained at the desired temperature Too low or too high temperatures can result in lower percent enrichments and decreased yields If a microheating system is not available a thermal cycler can be used with modifications by following the directions in Alternative Thermal Cycler Steps for Successful Enrichment on page 59 This approach requires additional sample transfers Place the magnetic stand next to the microheating system for immediate access TruSight One Library Preparation Guide 31 391n1de 18 114 Protocol Remove the NEW1 plate from the microheating system and immediately place it on the magnetic stand for 2 minutes or until the liquid appears clear Remove the adhesive seal from the NEW1 plate Immediately remove and discard
12. this Product when used for research use purposes in accordance with these terms and conditions and in accordance with this Product s Documentation and Specifications infringes the valid and enforceable intellectual property rights of a third party and ii pay all settlements entered into and all final judgments and costs including reasonable attorneys fees awarded against Purchaser in connection with such infringement claim If this Product or any part thereof becomes or in Illumina s opinion may become the subject of an infringement claim Illumina shall have the right at its option to A procure for Purchaser the right to continue using this Product B modify or replace this Product with a substantially equivalent non infringing substitute or C require the return of this Product and terminate the rights license and any other permissions provided to Purchaser with respect this Product and refund to Purchaser the depreciated value as shown in Purchaser s official records of the returned Product at the time of such return provided that no refund will be given for used up or expired Consumables This Section states the entire liability of Illumina for any infringement of third party intellectual property rights b Exclusions to Illumina Indemnification Obligations Illumina has no obligation to defend indemnify or hold harmless Purchaser for any Illumina Infringement Claim to the extent such infringement arises from i the use of Tr
13. volumes into each of the wells of an eight tube strip Pre program the thermal cycler with the following program and save as NLM AMP Choose the pre heat lid option and set to 100 C 72 C for 3 minutes 98 C for 30 seconds 10 cycles of 98 C for 10 seconds 60 C for 30 seconds 72 C for 30 seconds 72 C for 5 minutes Hold at 10 C ley NOTE Ilumina has optimized the number of recommended PCR cycles for enrichment assays based on the level of pre enrichment sample pooling and the size of the oligonucleotide set Do not add or reduce the cycles of PCR as it can compromise data quality Setup Index Primers 1 Vortex the index primer tubes for 5 seconds 2 Centrifuge the index primer tubes to 600 x g for 5 seconds Use empty 1 7 ml microcentrifuge tubes as tube adapters for the microcentrifuge TruSight One Library Preparation Guide 1 rj uoneoyiduav HOd 18414 Protocol 3 Arrange the index primers in a rack on ice i e the TruSeq Index Plate Fixture using the following arrangement a Arrange the Index 1 Primer tubes orange caps vertically aligned with columns 1 12 b Arrange the Index 2 Primer tubes white caps horizontally aligned with rows A H Figure 2 Index Plate Fixture A Index 1 Primer tubes orange caps B Index2 Primer tubes white caps C NLA plate 1 8 Part 15046431 Rev A Procedure NOTE When pooling libraries before enrichment with the 9 sample kit it is recommended to pool libraries so all Index
14. 1 i7 indices are unique Choose Index 1 and Index 2 primers for PCR accordingly For pooling 3 samples in one enrichment for sequencing on a MiSeq Illumina recommends using Index 1 Primers N701 N705 and N709 along with either Index 2 Primer for all three samples For pooling with the 36 sample kit make sure that each sample has a unique combination of Index 1 and Index 2 sequences Add 5 ul Index 1 primer to each well of the NLA plate Add 5 ul Index 2 primer to each well of the NLA plate Add 20 ul Nextera Library Amplification Mix to each well of the NLA plate BOO Mon Mix thoroughly as follows a Seal the NLA plate with a Microseal A film b Shake the NLA plate on a microplate shaker at 1200 rpm for 1 minute 5 Centrifuge the NLA plate to 280 x g for 1 minute 6 Place the sealed NLA plate on the pre programmed thermal cycler Close the lid then select and run the NLM AMP program using a heated lid SAFESTOPPING POINT If you do not plan to proceed immediately to First PCR Clean Up on page 20 the NLA plate i can remain on the thermocycler overnight If you are stopping replace the Microseal A with a Microseal D adhesive seal and store the NLA plate at 2 C to 8 C for up to two days TruSight One Library Preparation Guide 1 9 uoneoyidulv yO d 18414 Protocol 20 First PCR Clean Up This process uses Sample Purification Beads to purify the library DNA and remove unwanted products Consumables Item Resuspens
15. 280 x g for 1 minute Remove the adhesive seal from the NEA plate Transfer the entire contents from each well of the NEA plate to the corresponding well of the new 96 well MIDI plate labeled NEC2 Vortex the Sample Purification Beads until the beads are well dispersed Add 90 ul well mixed Sample Purification Beads to each well of the NEC2 plate containing 50 ul of PCR amplified library Mix thoroughly as follows a Seal the NEC2 plate with a Microseal B adhesive seal b Shake the NEC2 plate on a microplate shaker at 1800 rpm for 1 minute Incubate the NEC2 plate at room temperature for 10 minutes Centrifuge the NEC2 plate to 280 x g for 1 minute Remove the adhesive seal from the NEC2 plate Place the NEC2 plate on the magnetic stand at room temperature for 2 minutes or until the liquid appears clear Carefully remove and discard all of the supernatant from each well of the NEC2 plate NOTE Leave the NEC2 plate on the magnetic stand while performing the following 80 EtOH wash steps 12 14 With the NEC2 plate on the magnetic stand slowly add 200 ul freshly prepared 80 EtOH to each well without disturbing the beads Incubate the plate at room temperature for 30 seconds Remove and discard the 80 EtOH from each well of the NEC2 plate Repeat steps 12 13 one time for a total of two 80 EtOH washes Using a 20 ul single channel or multichannel pipette remove any remaining 80 EtOH from each well of the NEC2 plate witho
16. 3 15 C to 30 C User Preparation Procedure Review Best Practices for Handling Magnetic Beads See Additional Resources on page 5 for information on how to access TruSight One Best Practices on the Illumina website Make sure that the Resuspension Buffer and Sample Purification Beads are at room temperature Label a new 96 well HSP plate NEA Nextera Enrichment Amplification with a smudge resistant pen Remove the adhesive seal from the NECI plate Vortex the Sample Purification Beads tube until the beads are well dispersed then add 45 ul well mixed Sample Purification Beads to each well of the NECI plate Mix thoroughly as follows a Seal the NECI plate with a Microseal B adhesive seal b Shake the NECI plate on a microplate shaker at 1800 rpm for 1 minute Incubate the NECI plate at room temperature for 10 minutes TruSight One Library Preparation Guide 41 dN uea o ajdwes anides Protocol 42 10 11 12 13 14 15 16 17 18 19 20 Centrifuge the NECI plate to 280 x g for 1 minute Remove the adhesive seal from the NECI plate Place the NECI plate on the magnetic stand for 2 minutes or until the liquid appears clear Remove and discard all of the supernatant from each well of the NECI plate NOTE Leave the NEC1 plate on the magnetic stand while performing the following 80 EtOH wash steps 9 11 With the NECI plate on the magnetic stand slowly add 200 ul freshly made 8076 EtOH to each well witho
17. 5 C to 25 C TD amant DNABuffer ACOSO 15 C to 25 C 15 C to 25 C 15 C to 25 C 15 C to 25 C 15 C to 25 C 15 C to 25 C 15 C to 25 C NI RL b i F i F i i F i F i F i FA TruSight One Library Preparation Guide 6 3 S1U91U02 UN Supporting Information 64 Box 3 Indices This box is shipped on dry ice As soon as you receive your kit store the components at 15 C to 25 C Quantity Reagent Name Storage Temperature 1 tube Index Primer E502 to E505 15 C to 25 C 2 tubes Index Primer N701 to N712 15 C to 25 C Box 4 Oligos This box is shipped on dry ice As soon as you receive your kit store the components at 15 C to 25 C Quantity Reagent Name Storage Temperature 1 tube TruSight One Content Set 15 C to 25 C Part 15046431 Rev A Consumables and Equipment Check to make sure that you have all of the necessary user supplied consumables and equipment before proceeding to the library preparation and enrichment procedures E NOTE The TruSight One protocol has been optimized and validated using the items listed Comparable performance is not guaranteed when using alternate consumables and equipment Table 4 User Supplied Consumables Consumable 1 7 ml microcentrifuge tubes 20 ul barrier pipette tips 20 ul multichannel pipettes 20 ul single channel pipettes 200 ul barrier pipette tips 200 ul multichannel pipettes 200 ul single channel pipettes 1000 ul
18. A 5001 Bio Rad part MSB 1001 General lab supplier VWR part 89094 658 General lab supplier Illumina catalog FC 130 1005 General lab supplier Supplier Agilent part G2940C A Agilent part 5067 1504 Agilent part 4067 4626 Part 15046431 Rev A Equipment DNA Engine Multi Bay Thermal Cycler See Thermal Cyclers on page 68 High Speed Microplate Shaker Magnetic stand 96 Microcentrifuge Microheating System SciGene TruTemp Heating System Microplate centrifuge MIDI plate insert for microheating system Fluorometric quantitation with dsDNA binding dye reagents Optional Vacuum concentrator Note Used to concentrate a pooled library Another option is to use Amicon Ultra 0 5 centrifugal filter units Vortexer TruSight One Library Preparation Guide Supplier Bio Rad part PTC 0240G or PTC 0220G with Alpha Unit part ALS 1296GC VWR catalog 13500 890 110 V 120 V or 14216 214 230 V Life Technologies part 4 AM10027 General lab supplier Illumina catalog SC 60 503 115 V Or SC 60 504 220 V General lab supplier Illumina catalog BD 60 601 General lab supplier General lab supplier General lab supplier 67 jueuudinb4 pue se qeuunsuo Supporting Information 68 Thermal Cyclers The following table lists the recommended settings for the Illumina recommended thermal cycler as well as other comparable models If
19. A Ee 60 Consumables and Equipment 000 c eee e cece ee eeeeeeeees 65 Index Sequences eee cece cece cece cece eee I esr RR e eres esses eres 69 Technical Assistance T1 TruSight One Library Preparation Guide VI Part 15046431 Rev A Overview Introduction cnc cc cocos DNA Input Recommendations 3 Critical Steps for Successful Enrichment and Coverage cece eeeeee eee 4 Additional Resources aant ger nl TruSight One Library Preparation Guide 46e i IT fi Tc SR RS m ran rare e as LJe1deuo Introduction This protocol explains how to prepare up to 36 indexed paired end libraries followed by enrichment using the TruSight One Sequencing Panel and reagents provided in an Illumina TruSight One Sequencing Panel kit The goal of this protocol is to fragment and add adapter sequences onto template DNA to generate indexed libraries that can be carried through enrichment for targeted resequencing applications Overview The TruSight One Sequencing Panel protocol offers Fast and easy sample preparation Prepare up to 36 enriched libraries in approximately 1 5 days with approximately 5 hours of hands on time High throughput automation friendly procedures with no fragmentation bottlenecks Low DNA input and excellent data quality Excellent data quality with low input of 50 ng Access precious samples with no affect on performance Ability to archive samples for subsequent analysis
20. AACCT TAAGAT TACTTGATCCACTGAT TCAACGTACCG TAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCG TCTTCTGT TAACCTTAAGAT TACTTGATCCACTGAT TCAACG STACCGT ee ME MEA A ATA NA ATCAATTG CUTE TTAAGAGCTACCGTGCAACGACGAAAAGAATGATAACAGTAACACACTTCTGTTAACCT TA NE A LA CGTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGCTTCTGTTAACCT T RSS TE SAT NEE TCAATTGAGACTAGCAACGACG e eO al aL RE EEN Elek GTAACGAACGTATCAAT TGAGACTAAATATTAACGTACCAT TAAGAGC TACC SATAA AQTRACACAC TTCTGTTAACCTTAAGAT TACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAA ATTARRALC TACCGTCTTCTGTIAACCTIAAGATIACTTGA ee RAT STACCGTAACGAACGIA TCAT TAAGAT TACTTGATCCACT GATT CAACGTACCGTAACGAACGTAT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAGC TACCGTGCAACGACGAAAAGAATGATAACAG TAACACACT TCTGTTAACC T T IAAAAGAATGATAACAGTAACACACT TCTGTTAACC T TAAGAT TACTTGAT CCACTGAT TCAACGTACCGTAAAGAT TAC TT AT CACTGATTCAACGTAGCOTAACGAACETATOAATTGAGAC AAATAT TAA C GIA OATTAROAGU TAC CCAT TAAGAGCTACCGTGCAACAGTAACACACT TCTGTTAACCT TAAGATTACT TGATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTGCAACGACGAAAAGAAT GATAA 3ATAACAGTAACACACTTCTGTTAACCT TAAGATTACTTGATCCACTGATTCAACGTACCG TAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGC TACCG TCTTCTGTTAACCTTAAGAT TACTTGATCCACTGAT TCAACG ZLTGATCCACTGATTCAACGTTAAGAT TACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGCT TCTGT TAACCTTAAGAT TACT TGATCCACTGAT TCAACG TACCGTAACGAACGTAT CAAT T GAGAC TAGCAACGACQ S ETATE EET T E E ee N RTNCAGTA GTACCGTAACGAACGTATC
21. AATAT TAACG TACCA WA D es CGACG TTICTGTT PROS CM us GAGCTACCGTGCAACGAAAATAACCTTAAGATTACT TGATCCACTGAT TCAACGTACTTCTGTTAACCTTAAGAT TACTTGATCCACTGAT TCAACGTACCGTAACGAACGTA TRATIGAGACTAAGCIACCOTECAACGA CGAAAAGAATGAT RC GAAAAGAAT GATAACAGTAACAGACTTG TCH TAACG TIAAGATIAC LIGATCCACTGATT CARGO TACCG TANAGATIACTTGATCOACTCATICAASGIACCCTAASGAACCIATGRATT GAGACTAANTAT E AATGATAACAGTAACACACT TCTGTTAACCTTAAGA quoda aes CCGTAACGAACG TATCAAT TGAGACT Res a ee MCI E CGACGAAAAGAA UES para ag pr ge Aa GTACCAT TAAGAGC TACCGTGCAACAGTAACACACT TCTGT TAACCTTAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TGAGAC TAAATAT TAACGTACCAT TAAGAGCTACCGTGCAACGACGAAAAGAATGATAACA NAT QATAACAGTAACAGACTICNG IAAGGLAAGATTAC LIGATCCAGTGATI CANCG TACCO TAA GAACGTAT GANTT GAGACTAAATATTAAGGTACCATAAGHOCTASCOTCTTOTO MAACO TIAAGATIAGH GATGCACTGATIGANCGTA ACGTACCGTAACGAACGTATCAT TAAGATTA Eeler EERO a CGAAAAGAATGA er CTTCTGTTAACCTTAAC I TACTTGATCCACTGATTCAACGTTAAGAT TACTTGATCCACTGATTICAACGTACCGTAACGAACG AT CAAT TGAGCTTCTGTTAACCT TAAGAT TACT TGATCCACT RO AE T L A TCAATTGAGACTAGCAACGACGAA ACGAAAAGAATGATAACAGTAACACAC T TCTGTTAACCT TAAGAT TACTT GATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGAT TCAACG TACCGTAACGAACG TATCAAT TGAGAC TAAATATTAACGTACCAT TAAGAGC TACCGT Nee a ee ee EE ALT ARE ATA TTAACGTAC DEE E VAS CLP E Ee ACCGTAACGAACGTATCAATTGAGACTAAATAT TAACG TACCAT TAAGAGC TACCGTGCAACGACGAAAAGAATGATAACAG TAACACACT TCTGTTAACCTT ACGAAAAGAATGATAACAGTAACACACT TCTGT TAACCT TAAGAT TACTT GAT CCACTGATTCAACGTACC
22. ATAT TAACG TACCAT TAAGAGCTA TTCTG CCTTAAGATTACTTGATCCACTGA CCAT TAAGAGC TACCGTGCAACTTAACCTTAAGAT TACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACG TACCAT TAAGAGCTACCGTGCAACGACGAACTTCTGT TAACCT TAAGAT TACT TGAT CTAGOGIGOAAGGAAAR AAGC IARGATIACTTGATGGAGIGALIGAAGG AGITGLG TAAGGTTAAGATTACT IGATCCAGT GAL CAACGACOGTAACGAACG EE EE AAAAGAATGA Ee sea L T AACCTTAAGATTACTT SEI GMT S M AAAGATTACTTGAT SR ACTGATICAACGTACCGTAACGAACG TA TTGAGACTAAATAT TAACGTACCAT TAAGAGCTACC 3ATAACAGTAACACACTTCTGTTAACCT TAAGAT TAC TT PORT OCACTGALICAACGT ACCGTAACGAA ACTA TCAATTGAGACTAAATAT TAACGT EE AOGA CEAMAAGAA GA TAACAGTAACACACTTCTGTI CCATTAAGAGCTACEGT GU AACAGTAACAGACTTCTGT IAACCT TAAGATTACT GATCCAG EE EE 3ATAACAGTAACACACT TCTGT TAACCT TAAGAT TACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGAC TAAATAT TAACGTACCAT TAAGAGC TACCGTCTTCT AAGAT T POLI DR GA TATE ONE aTACCGT AA ANC ATCATTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGAC TAAATAT TAACG TACCATTAAGAGC TACCGTGCAACGA IIT NA TAACAGTAACACACTTCTGTTAACCT TT ITEM 21T SDH EE E EE CARTTGAGACTAGGAACGACG IAAAAGAATGATAACAGTAACACACT TCTGTTAACCT TAAGAT TACTT GATCCACTGAT T CAACGTACCGTAAAGAT TACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACC Illumina San Diego California 92122 U S A 1 800 809 ILMN 4566 1 858 202 4566 outside North America techsupport illumina com www illumina com
23. ATTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACG TATCAAT TGAGA U ACCGTGCAACGACGAAAAGAATGATAACAG TAACACACTTCTGT TAACCT A ZTTGATCCACTGATTCAACGTTAAGAT TACTTGATCCACTGATTCAACGTACCGTAACGAACG TATCAAT TGAGCTTCTGT TAACCT TAAGAT TACT TGAT CCACTGAT TCAACG TACCGTAACGAACGTATCAAT TGAGACTAGCAACGACG IAAAAGAATGATAACAGTAACACAC T TCTGT TAACCT TAAGAT TACTT GATCCACT GATT CAACGTACCGTAAAGAT TACTTGATCCACTGAT TCAACGTACCGTAACGAACG TATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACC 3ATAACAGTAACACACT TCTGTTAACCT TAAGAT TA quU A edi eie S ILU RE I Alay lA ee ATTACTT PEE Oe cU OE ZACTGATTCAACGTACCAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGAC TAAATAT TAACGTACCAT TAAGAGCTACCGTCTTCTGT TAACCT TAAGAT TACT TGATCCACTGATTCAACGTACCG TAACGA AAAAGANTGATAACAGTAAGAGAGTIGTGY IAACCLTAAGATIACT TGATCGACT GAT I CAACGTAGCG AAAGATTACT e IARGAGG AGO ee ER L GTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACG TATCAA ET AL EU n ER GATTACTTGATCCACTGATTCAACG TGAGACTAAATAT TAACGTTGTTAACCTTAAGAT TACT TGATCCACTGATTCAACGTACCGTAACGAACGTAT CAAT TGAGACTAAATA Et GCTICTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGIA ATCAATTGAGA CTAANTALT AACGTACTTAACCTIAAGAT TACT TGATCCACTGATTCAACGTACCGTAACGAACG TCTTCTGT TAACCT TAAGAT T GR ACCGTAACGAACGTATCAAT TGAGACTAACGACGA SATAACAGTAACAGAG LT CTGLIAAGG TAAGATTACTGATGOAGTGAT GAACGIAGGGTAAGGAAGGTATCAAT GAGAG AAATAT TAACGTACGA TAAGAGGTAGGGCT TCT GTTAAGG TANGATIACT IGATCUACT GAT IGAAGG 3ATAACAGTAACACACTT AACCTTAAGATTACTTGATCCACTGATTCAACG TACCG TAACGAACG TATCAAT T GAGAC TAA
24. CTION WITH CUSTOMER S ACQUISITION OF SUCH PRODUCT S FOR RESEARCH USE ONLY 2013 Illumina Inc All rights reserved Illumina IlluminaDx BaseSpace Bead Array 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 Part 15046431 Rev A 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 TruSight One Library Preparation Guide 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 Cor
25. GTAAAGAT TACT TGATCCACTGAT I CAACGTACCGTAACGAACG TATCAAT TGAGACTAAATAT TAACG Oe GTACCAT TAAGAGCTACCGTGCAACAGTAACACACTT CT GT TAACCTTAAGAT TACTT GATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGT GCAACGACGAAAAGAAT GATAACA AATGATAACAGTAACACACT TCTGTTAACCTTAAGATTACTTGATCCACTGAT TCAACGTA NW RU TIME EE el AAATAT TAACGTACCAT TAAGAGC TACCG TCTTCTGTTAACCT TAAGAT TACTTGATCCACTGAT TCAACGTA TTACTTGATCCACTGATTCAACGTTAAGAT TACTT GAT CCACT GATT CAACGTACCGTAACGAACGTATCAATT GAGCT TCTGT TAACCTTAAGAT TACTT GATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT I GAGACTAGCAACGACGAA TATCAATTGAGACTAAATATTAACGTACCAT TAAGAGTCTGT TAACCT TAAGAT TACTTGATCCACTGATTCAACG TACCGTAACGAACG TATCAAT TGAGAC TAAATAT TAACGTACCAT TAAGAGC TACCGT GCAACGAAAAGAAT GATAACAGTAAC ACGTACCGTAACGAACGTATCAT TAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACG TATCAAT TGAGAC TAAATAT TAACGTACCAT TAAGAGCTACCG TGCAACGACGAAAAGAAT GATAACAG TAACACACT TC TGT TAACCT TAAC I TACTTGATCCACTGATTCAACGTTIAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTAT CAATTGAGCTTCTGTTAACCT TAAGAT TACT TGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAGCAACGACGAA AAA ELIT ALIAS E EE AATGATAACAGTAACACACT TC TGTTAACCT TAAGAT TA EE I CTAAATAT TAACGTACCAT TAAGAGCTACCGTCTTCTGTTAACCTTAAGAT TACTTGATCCACTGAT TCAACGTACCGTAA NTCCACTGATTCAACGTACCAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TGAGAC TAAATAT TAACGTACCAT TAAGAGC TACCGTCTTCTGT TAACCT TAAGAT TACT TGATCCACTGATTCAACGTACCGTAACGAAC ACGAAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGAT TACTTGATCCAC
26. HIRD PARTY ARISING OUT OF OR 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 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 Product is 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 and ii any
27. HP3 Enrichment Elution Buffer 1 and Enrichment Wash Solution from 15 C to 25 C storage and thaw at room temperature Remove the Elute Target Buffer 2 and Streptavidin Magnetic Beads from 2 C to 8 C storage and let stand at room temperature Part 15046431 Rev A NOTE Make sure that you use the Streptavidin Magnetic Beads 2 ml tube and not the Sample Purification Beads 15 ml tube for this procedure Pre heat the microheating system to 50 C NOTE The Enrichment Wash steps are key to ensuring high enrichment specificity Ilumina recommends using a microheating system with a MIDI plate insert for these steps to make sure that samples are maintained at the desired temperature Too low or too high temperatures can result in lower percent enrichments and decreased yields If a microheating system is not available a thermal cycler can be used with modifications by following the directions in Alternative Thermal Cycler Steps for Successful Enrichment on page 59 This approach requires additional sample transfers Label a new 96 well MIDI plate NEW2 Nextera Enrichment Wash 2 with a smudge resistant pen Label a new 96 well MIDI plate NEC1 Nextera Enriched Clean Up 1 with a smudge resistant pen Second Bind 1 Remove the NEH2 plate from the thermal cycler 2 Centrifuge the room temperature NEH2 plate to 280 x g for 1 minute 3 Remove the adhesive seal from the NEH2 plate Take care when removing the seal to avoid spillin
28. NA This process tagments tags and fragments the gDNA by the Nextera transposome The Nextera transposome simultaneously fragments the gDNA and adds adapter sequences to the ends allowing amplification by PCR in subsequent processes Consumables Item Quantity Storage Supplied By Sample Purification Beads SPB 1 tube 2 C to 8 C Ilumina Stop Tagment Buffer ST 1 tube ISAC me SORT Illumina Tagment DNA Buffer TD 1 tube 15 C to 25 C Ilumina Tagment DNA Enzyme TDE1 1 tube 15 C to 25 C Ilumina 96 well MIDI plate 1 15 C to 30 C User gDNA 5 ng ul 50 ng sl re 2528 User Ice bucket 1 15 C to 30 C User Microseal B adhesive seals 2 JI me OCC User PCR grade water 10 ul per sample 15 C to 30 C User RNase DNase free eight tube 3 IBC me SOME User strips and caps for multi sample processing RNase DNase free reagent 1 15 C to 30 C User reservoir for multi sample processing Tris HCl 10 mM pH 8 5 As needed IAC me SOE User Preparation Remove the Tagment DNA Buffer Tagment DNA Enzyme 1 and gDNA from 15 C to 25 C storage and thaw on ice 1 O Part 15046431 Rev A After thawing make sure that all reagents are adequately mixed Gently invert the tubes 3 5 times followed by a brief spin in a microcentrifuge Remove the Sample Purification Beads from 2 C to 8 C storage and let stand to bring to room temperature Make sure that the Stop Tagment Buffer has no precipitate If there is precipitate vortex until all pa
29. TAAGAGCTACCGTCTTCTGTTAACCTTAAGAT TACT TGATCCACTGAT TCAACGTACCGT SNE E eg TAM BOCA TAA ST OST ESTER DAT TRA E BET CART TEMO 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 TCT TCTGTTAACCT TAAGAT TACTT GATCCACTGATTCAACGTACCGTAACGAACGTAT CAAT T GAGAC TAACGACGA SAC TAAATAT TAACGTACCAT TAAGAGCTACAACCT TAAGAT TACT TGATCCACTGATTCAACGTACCGTAACGAACG AT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAGC TACCGTGCAACGACGAAAAGAAT GATAACAGTAACACA CCA CCTTAAGATTACTT C A T TGAGACTAAATAT TAACG A AA CGAACTTCTGTTAA 3CIACCGTGCAACGAAAATAACCTTAAGATTACT TGATCCACTGAT TCAACGTACTTCTGTTAACCTTAAGAT TACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATT GAGACTAAGCTACCGTGCAACGACGAAAAGAATGAT EE 3ATAACAGTAACACACT TCTGTTAACCT T Ze TCAATTGAGACT m un CGACGAAAAGAATGATAACAGTAACACAC TTC TGT I CCAT TAAGAGC TACCGTGCAACAGTAACACACT TCTGTTAACCTTAAGAT TACT TGATCCACTGAT TCAACGTACCG TAACGAACGTAT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTGCAACGACGAAAAGAAT GATAA 3aATAACAGTAACACACT TCTGTT
30. TGATTCAACGTACCGTAAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGAC TAAATATTAACGTACCAT TAAGAGCTACCGT AATGATAACAG TAACACACT TCTGTTAACCTTAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACG TATCAAT TGAGACTAAATATTAACGTA DEN ec Lo ARE ERSTE AAGATTACTTGATCCACTGATTCAACGTA AAT TGAGACTAAATAT TAACGTTGTTAACCTTAAGATTACTTGATCCACTGAT TCAACG TACCGTAACGAACG TATCAATTGAGACTAAATATTAACGTACCAT TAAGAGCT TCTGT TAACCT TAAGAT TACTTGATCCACTGAT TCAACGTACCGTAAC CGTATGAAT GAGACTAAATAT TAACGTAGLTAACG TARGA TACT GATCCAGTGATT CAACG IACCGIAAGGAACG ICT EE E GAGACTAAATAT TAACGTACCAT TAAGAGCTACAACCTTAAGA BEACH SE EEE GE TCAAT TGAGACTAAATAT TAACG TACCAT TAAGAGCTACCGTGCAACGACGAAAAGAAT GA E AATGATAACAGTAACACACT TCTGTTAACCTTAAGAT TACTTGATCCACTGATTCAACGTACCG TAACGAACG TA TO GA RAT LIRAC QUA CAT DAC CIC G S GTC E AACG GTACCATTAAGAGCTACCGTGCAACTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAA ATTA ACCTTAA E AACCTTIAAGATTACTTGATCCACTGATTCAACGTACTTCTGTTAACCTTAAGAT TACTTGATCCAC ACGAAAAGAATGATAACAG TAACACACTTCTGT TAACCTTAAGAT TACTTGATCCACTGATTCAACGTACCGTAAAGAT TACT T GAT C AATGATAACAGTAACACACTTCTGT TAACCTTAAGAT TACTTGATCCACTGAT TCAACGTACCGTAACGAACG TATCAAT TGAGACTA uu m PRS LLL RAE Ree AATGATAACAGTAACACACT TCTGTTAACCTT TTGATCCACTGATTCAACGTACCGTAACGAA TCAATTGAGACTA AEC UAAC GANGET CAT TAAGA OTTOA GOAGTGATT ACTA Ee A Maea EE ele TATCAATTGAGCTICTGTTAA ACGAAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATC FOR RESEARCH USE ONLY
31. al A film with a Microseal D adhesive seal and store the NEA plate at 2 C to 8 C for up to two days TruSight One Library Preparation Guide A 5 uoneoyiduv yO d puooes Q Second PCR Clean Up O gt CL unwanted products Consumables Item Resuspension Buffer RSB Sample Purification Beads SPB 96 well HSP plate 96 well MIDI plate Freshly prepared 8076 ethanol EtOH Microseal B adhesive seals Preparation Quantity 1 tube 1 tube 1 1 400 ul per sample Storage 2 C to 8 C IT io BAC 15 C to 30 C DS to 302e 15 C to 30 C JEC me S0 This process uses Sample Purification Beads to purify the enriched library and remove Supplied By Ilumina Ilumina User User User Review Best Practices for Handling Magnetic Beads See Additional Resources on page 5 for information on how to access TruSight One Best Practices on the Illumina website Make sure that the Resuspension Buffer and Sample Purification Beads are at room temperature Remove the NEA plate from 2 C to 8 C storage if it was stored at the conclusion of Second PCR Amplification and let stand to bring to room temperature Label a new 96 well MIDI plate NEC2 Nextera Enriched Clean Up 2 with a smudge resistant pen Label a new 96 well HSP plate NES Nextera Enrichment Sample with a smudge resistant pen 46 Part 15046431 Rev A Procedure 10 11 12 13 14 15 16 Centrifuge the NEA plate to
32. ate IEM documentation from the Illumina website A short sequencing run on a MiSeq can also be performed to optimize cluster density prior to performing a high density sequencing run TruSight One prepared libraries contain dual 8 bp indexes Depending on the combination of indexes used in your library pool configure the sequencing run for either single or dual 8 bp index reads When clustering TruSight One libraries on the cBot and sequencing on the HiSeq 1000 2000 GAILx or in High Output mode on the HiSeq 1500 2500 new primers are required whether performing a non indexed single indexed or dual indexed run Use the TruSeq Dual Index Sequencing Primer Kit for Paired End runs catalog PE 121 1003 which is good for a single run and contains the required primers for sequencing HP10 HP11 HP12 These primers are included with MiSeq and HiSeq 1500 2500 rapid run reagent kits For sequencing TruSight One libraries lumina recommends a paired end 151 cycle sequencing run Due to the library sizes generated in TruSight One sequencing at longer read lengths can lead to an increase in the likelihood of sequencing into the flanking adapter sequence 5 A Part 15046431 Rev A Supporting Information Introduction EE 56 ACOMYS ofits oleh EA AE EE EE Ee 57 Alternative Thermal Cycler Steps for Successful Enrichment 59 IRIE COMPAS er en en EE ee 60 Consumables and Equipment o 22 22 c cece eceeeeccccccecccc
33. ction PCR Primer Cocktail Resuspension Buffer Streptavidin Magnetic Beads Sample Purification Beads Stop Tagment Buffer Tagment DNA Buffer Tagment DNA Enzyme 1 TruSight One Oligos Part 15046431 Rev A Alternative Thermal Cycler Steps for Successful Enrichment The Enrichment Wash steps remove non specific DNA bound to the Streptavidin Magnetic Beads and their success require that samples are maintained at a specific temperature Too low or too high temperatures can result in lower percent enrichments due to non specific binding and decreased yields from loss of targeted regions Illumina recommends using a microheating system for these steps If a microheating system is not available a thermal cycler can be used Follow these steps when using a thermal cycler 1 Wu VD CON QQ Transfer the sample and beads resuspended in Enrichment Wash Solution to a PCR plate approximate 200 ul in volume Seal the PCR plate with a Microseal B adhesive seal Use an adhesive seal roller to apply force to the seal and make sure that the seal is secured Incubate the PCR plate on the thermal cycler at 42 C for 30 minutes with a heated lid set to 100 C NOTE For optimal results it is important that the thermal cycler lid is heated to 100 C Place the magnetic stand next to the thermal cycler for immediate access Remove the PCR plate from the thermal cycler and immediately place it on the magnetic stand for 2 minutes until the li
34. e IP are separate non overlapping subsets of all Illumina owned or 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 Sp
35. e Purification Beads until the beads are well dispersed Add 90 ul well resuspended Sample Purification Beads to each well of the NLC plate Mix thoroughly as follows a Seal the NLC plate with a Microseal B adhesive seal b Shake the NLC plate on a microplate shaker at 1800 rpm for 1 minute Incubate the NLC plate at room temperature for 10 minutes Centrifuge the NLC plate to 280 x g for 1 minute Remove the adhesive seal from the NLC plate Place the NLC plate on the magnetic stand for 2 minutes or until the liquid appears clear Using a 200 ul single channel or multichannel pipette set to 140 ul remove and discard all of the supernatant from each well of the NLC plate NOTE Leave the NLC plate on the magnetic stand while performing the following 80 EtOH wash steps 12 14 With the NLC plate on the magnetic stand slowly add 200 ul freshly prepared 80 EtOH to each well without disturbing the beads Incubate at room temperature for 30 seconds Remove and discard the 80 EtOH from each well of the NLC plate Repeat steps 12 and 13 one time for a total of two 80 EtOH washes Using a 20 ul single channel or multichannel pipette remove any remaining 80 EtOH from each well of the NLC plate without disturbing the beads TruSight One Library Preparation Guide 2 1 dN uee o HOd 18414 Protocol 22 16 17 18 19 20 21 22 23 24 25 26 Let the NLC plate stand at room temperature for 10 minutes t
36. e in specific applications may be protected by patents Customers are advised to determine whether they are required to obtain licenses from the party that owns or controls such patents in order to use the product in customer s specific application 6 O Part 15046431 Rev A TruSight One Sequencing Panel Contents 9 Samples FC 141 1006 TG 141 1006 Box 1 Rapid Capture Reagents This box is shipped at room temperature As soon as you receive your kit store the components as specified Quantity Acronym Reagent Name Storage Temperature 1 Sample Purification Beads 2 C to 8 C 2 Streptavidin Magnetic Beads 2 C to 8 C 1 Elute Target Buffer 2 2 C to 8 C 1 15 C to 30 C Box 2 Rapid Capture Reagents This box is shipped on dry ice As soon as you receive your kit store the components at 15 C to 25 C Quantity Acronym Reagent Name Storage Temperature TDE1 Tagment DNA Enzyme 15 C to 25 C 15 C to 25 C TD amant DNABuffer ACOSO 15 C to 25 C 15 C to 25 C 15 C to 25 C 15 C to 25 C 15 C to 25 C 15 C to 25 C 15 C to 25 C NIE RL b i b i b i b i b i F i F i Fi TruSight One Library Preparation Guide 6 1 S1U91U02 UN Supporting Information 62 Box 3 Indices This box is shipped on dry ice As soon as you receive your kit store the components at 15 C to 25 C Quantity Reagent Name Storage Temperature 1 tube Index Primer E503 to E504 15 C to 25 C 1 tubes
37. ecceececceceeceeceeceeteceeeees 65 Index Sequencas Ee EE 69 P nn iff STAT el arrasa ra e We TruSight One Library Preparation Guide 5 5 v xipuaddawy 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 5 6 Part 15046431 Rev A Acronyms Table 2 TruSight One Sequencing Panel Acronyms Acronym dsDNA EEN EHB EH EWS gDNA HP3 HSP NEA NEC1 NEC2 NEH1 NEH2 NEM NEW NEW NLA Definition Double stranded DNA Enrichment Elution Buffer 1 Enrichment Hybridization Buffer Elute Target Buffer 2 Enrichment Wash Solution Genomic DNA 2N NaOH Hard Shell Plate Nextera Enrichment Amplification Plate Nextera Enriched Clean Up Plate 1 Nextera Enriched Clean Up Plate 2 Nextera Enrichment Hyb Plate 1 Nextera Enrichment Hyb Plate 2 Nextera Enrichment Amplification Mix Nextera Enrichment Sample Plate Nextera Enrichment Wash Plate 1 Nextera Enrichment Wash Plate 2 Nextera Library Amplification Plate Nextera Library Clean Up Plate TruSight One Library Preparation Guide 5 T sw uoIoy Supporting Information 58 Acronym NLM NLS NLT PCR PRE RSB SMB SPB Sii HUP TI TOO Definition Nextera Library Amplification Mix Nextera Library Sample Plate Nextera Library Tagment Plate Polymerase Chain Rea
38. ecifications means Illumina s written specifications for this Product in effect on the date that the Product ships from Ilumina 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 Products 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 licens
39. eck the size of the library for a distribution of DNA fragments with a size range from approximately 300 bp 1 kb It is not necessary to have a sharp peak but rather more important that most of the fragments fall within the 300 bp 1 kb range The traces can be variable from prep to prep The traces show some examples of possible distributions but is not inclusive of successful preps Part 15046431 Rev A Figure 3 Example TruSight One Post PCR Pre Enriched Library Distributions TruSight One Library Preparation Guide 23 dN uee o HOd 18414 Protocol 24 300 400 500 600 1000 2000 SAFE STOPPING POINT If you do not plan to proceed immediately to First Hybridization on page 25 you can safely stop the protocol here If you are stopping seal the NLS plate with a Microseal B adhesive seal and store it at 15 C to 25 C for up to 14 days Part 15046431 Rev A First Hybridization This process mixes the DNA library with capture probes to targeted regions of interest The recommended hybridization time makes sure that targeted regions bind to the capture probes thoroughly This process also describes how to combine multiple libraries with different indices into a single pool before enrichment Consumables Item Quantity Storage Supplied By TruSight One Oligos TOO 1 tube 15 C to 25 C Illumina Enrichment Hybridization 1 tube Se Illumina Buffer EHB 96 well HSP plate 1 15 C to 30 C User Optional Amicon U
40. ed to Purchaser and not sold Except as expressly stated in this Section no right or license under any of Illumina s intellectual property rights is or are granted expressly by implication or by estoppel Purchaser is solely responsible for determining whether Purchaser has all intellectual property rights that are necessary for Purchaser s intended uses of this Product including without limitation any rights from third parties or rights to Application Specific IP Illumina makes no guarantee or warranty that purchaser s specific intended uses will not infringe the intellectual property rights of a third party or Application Specific IP Regulatory This Product has not been approved cleared or licensed by the United States Food and Drug Administration or any other regulatory entity whether foreign or domestic for any specific intended use whether research commercial diagnostic or otherwise This Product is labeled For Research Use Only Purchaser must ensure it has any regulatory approvals that are necessary for Purchaser s intended uses of this Product Unauthorized Uses Purchaser agrees a to use each Consumable only one time and b to use only Illumina consumables reagents with Illumina Hardware The limitations in a b do not apply if the Documentation or Specifications for this Product state otherwise Purchaser agrees not to nor authorize any third party to engage in any of the following activities i disassemble reverse engin
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43. erage library size In the first example a second minor peak at 2000 bp is visible Do not include this in the determination of average library size The presence of these larger fragments does not affect downstream clustering and sequencing of your enriched library TruSight One Library Preparation Guide 5 1 AjeJqr e1epijeA Protocol Preparing Your Libraries for Sequencing ona MiSeq e Illumina recommends using the following protocol to achieve optimal cluster density when sequencing a TruSight One library on a MiSeq Optimization of loading concentrations may be necessary to achieve desired cluster densities Consumables Item Quantity Storage Supplied By Hybridization Buffer HT1 1 tube 15 C to 30 C Ilumina 2N NaOH HP3 1 tube 15 C to 30 C User PCR grade water 15 C to 30 C User PhiX Optional 15 C to 30 C User Prepare a Fresh Dilution of NaOH 1 Dilute the post enriched dilution that was used for fluorometric quantification to a final concentration of 1 25 nM using RSB buffer 10 ul of diluted material is needed per MiSeq cartridge Try to use a minimum of 4 ul of library to ensure aspirating the appropriate volumes L NOTE Following this protocol achieves a final concentration of 12 5 pM of library loaded onto the MiSeq by creating a post enriched dilution of 1 25 nM If an 8 pM final concentration is desired dilute the post enriched dilution to a final concentration of 0 8 nM Alternatively if a 10 0 pM
44. esuspension Buffer to each well of the NLT plate Do not touch the beads with the pipette tips Mix thoroughly as follows a Seal the NLT plate with a Microseal B adhesive seal b Shake the NLT plate on a microplate shaker at 1800 rpm for 1 minute Incubate the NLT plate at room temperature for 2 minutes Centrifuge the NLT plate to 280 x g for 1 minute Place the NLT plate on the magnetic stand for 2 minutes or until the liquid appears clear Remove the adhesive seal from the NLT plate Transfer 20 ul of clear supernatant from each well of the NLT plate to the corresponding well of the new HSP plate labeled NLA Take care not to disturb the beads NOTE Ilumina recommends using a 20 ul single channel or multichannel pipette set to 10 ul to perform two consecutive transfers of 10 ul This technique reduces sample loss by making sure that all of the liquid is transferred without disturbing the beads TruSight One Library Preparation Guide 1 5 VNG pajuaube dN uea Protocol First PCR Amplification 16 This process amplifies the purified tagmented DNA via a 10 cycle PCR program It also adds index 1 i7 and index 2 i5 sequences needed for sequencing as well as common adapters P5 and P7 required for cluster generation and sequencing It is critical to use the full amount of recommended input DNA It is imperative that no extra cycles are added to the PCR process to ensure the generation of libraries that produce hig
45. 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 IV Part 15046431 Rev A 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
46. g the contents of the wells 4 Transfer the entire contents 100 ul from each well of the NEH2 plate to the corresponding well of the new 96 well MIDI plate labeled NEW2 A NOTE It is normal to see a small degree of sample loss after overnight hybridization However if the sample loss is greater than 15 Illumina does not recommend proceeding with the sample preparation Poor sealing or not heating the lid can cause this amount of loss D Vortex the Streptavidin Magnetic Beads tube until the beads are well dispersed then add 250 ul well mixed Streptavidin Magnetic Beads to the wells of the NEW2 plate 6 Mix thoroughly as follows a Seal the NEW2 plate with a Microseal B adhesive seal b Shake the NEW2 plate on a microplate shaker at 1200 rpm for 5 minutes 7 Let the NEW2 plate stand at room temperature for 25 minutes TruSight One Library Preparation Guide 37 eJnjde puooes Protocol 8 Centrifuge the NEW2 plate to 280 x g for 1 minute 9 Remove the adhesive seal from the NEW2 plate 10 Place the NEW2 plate on the magnetic stand for 2 minutes at room temperature or until the liquid appears clear 11 Carefully remove and discard all of the supernatant from each well of the NEW2 plate without disturbing the beads 12 Remove the NEW2 plate from the magnetic stand Second Wash 1 Make sure that the Enrichment Wash Solution tube is at room temperature then thoroughly vortex the tube L NOTE It is normal that the Enrichment Wa
47. h quality sequencing results Consumables Item Index 1 primers i7 N701 N712 Index 1 Tube Caps Orange Index 2 primers i5 E502 E505 Index 2 Tube Caps White Nextera Library Amplification Mix NLM 1 7 ml microcentrifuge tubes Microseal A film Microseal B adhesive seal RNase DNase free eight tube strips and caps for multi sample processing Optional TruSeq Index Plate Fixture Kit Quantity 1 tube each index 1 per Index 1 primer tube 1 tube each index 1 per Index 2 primer tube 1 tube 1 per index primer tube 1 1 Storage 15 C to 25 C IEC me SAC 15 C to 25 C JUSC tio SORT 15 C to 25 C IEC me SOC 15 C to 30 C ISAC tt SAC 15 C to 30 C ISAC me SAC Supplied By Ilumina Illumina Illumina Ilumina Ilumina User User User User User Part 15046431 Rev A Preparation Remove the Nextera Library Amplification Mix from 15 C to 25 C storage and thaw on ice Remove the following from 15 C to 25 C storage and thaw at room temperature Index 1 primers i7 N7xx only remove primers being used Index 2 primers i5 E5xx only remove primers being used NOTE TruSight One Sequencing Panel kits are designed to work only with Index 2 primers with the E prefix Do not use Index 2 primers from other sample prep kits For multi sample processing Use a multichannel pipette Dispense the Nextera Library Amplification Mix in equal
48. his Product not in accordance with 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 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 15046431 Rev A Revision History Part Revisi
49. hod See DNA Input Recommendations on page 3 If you are pooling libraries combine 500 ng of each DNA library Ensure that each library in the pool has a unique index If the total volume is greater than 40 ul concentrate the pooled sample Use either a vacuum concentrator or Amicon Ultra 0 5 centrifugal filter unit 0 5 ml 30 kDa according to the manufacturer s instructions If you are using a vacuum concentrator Illumina recommends concentrating samples with a no heat and medium drying rate setting If you are using an Amicon Ultra 0 5 centrifugal filter unit 0 5 ml 30 kDa it is not required to pre rinse the device before use Most of the volume filters through in 5 minutes but up to 30 minutes can be required depending on the starting volume If the pooled sample volume after concentrating is less than 40 ul bring the volume up to 40 ul with Resuspension Buffer The recommended pre enrichment pooling strategy is to pool libraries so that each contains a unique Index 1 i7 index With this pooling approach samples can be sequenced using a single index read workflow as described in the HiSeq and GAIIx user guides If Index1 i7 indices are not unique make sure that libraries with different Index 2 i5 indices are included e g N703 E503 and N703 E504 With this approach Part 15046431 Rev A sequence samples using a dual index read workflow as described in the HiSeq and GAIIx user guides Table 1 DNA Libraries for Enrichment
50. illumina TruSight One Sequencing Panel Library Preparation Guide ACGAAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGT AATGATAACAGTAACACACT TC TGTTAACCT TAAGATTACTTGT TGATCCACTGAT TCAACGTACCG TATCAAT TGAGAC TAAATAT TAACGTACCAT TAAGAGCTACCGTCTTCTGTTAACCTTAAGAT TACT TGATCCACTGAT TCAACGTACCGTAA ete SM eM AETS TOTEE CTOATT CANGA ARET AACGAACGT ee ACCATTAAGAGCTACCGTCTTCTGT T AIMER RT UAE RAT C e EE ACCGTAACGAAC ACGAAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGAT T ATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAA TTGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGT AAT EATAACAGTRACAGAS TIGI GTIN AGAT ACIOA CACT GATIGAAGGTACCGTAACGAACG TAT GANTT GAGAC TAAATAT TAACGTACCATTAAGAGCIACUGTO IC TOT TAAGO TAAGATTAC TIGATCCAC TAT CAACTA Ee AACCTTAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAATTGAGACTAAATATTAACGTACCAT TAAGAGCT TCTGTTAACCT TAAGAT TACTTGATCCACTGATTCAACGTACCGTAAC CGTATCAATTGAGAC TAAATAT TAACGTACT TAACCT TAAGAT TACT TGATCCACTGATTCAACGTACCGTAACGAACG TCT TC TGT TAACC T TAAGAT TAC T TGATCCACTGATTCAACGTACCGTAACGAACG TATCAAT TGAGAC TAACGACGAAA GAGAC TAAATAT TAACGTACCAT TAAGAGCTACAACCT TAAGAT TACT TGATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TGAGACTAAATAT TAACG TACCAT TAAGAGC TACCGTGCAACGACGAAAAGAAT GATAACAGTAACACACT AA SAEC TARDA E LIAC TE Ee GE e ee TAG TI ATE I GTACCATTAAGAGC TACCGTGCAACTTAACCTTAAGAT TACTTGATCCACT GAT TCAACGTACCGTAACGAACGTATCAATT GAGACTA
51. ion Buffer RSB Sample Purification Beads SPB 96 well HSP plate 96 well MIDI plate Freshly prepared 80 ethanol EtOH Microseal B adhesive seals RNase DNase free reagent reservoirs for multi sample processing Preparation Quantity 1 tube 1 tube 1 il 400 ul per sample Storage 2 C to 8 C IT io BAC 15 C to 30 C DS to 302e 15 C to 30 C AE me AE 15 C to 30 C Supplied By Ilumina Ilumina User User User User User Review Best Practices for Handling Magnetic Beads See Additional Resources on page 5 for information on how to access TruSight One Best Practices on the Illumina website Make sure that the Resuspension Buffer and Sample Purification Beads are at room temperature For multi sample processing Use a multichannel pipette Pour the Resuspension Buffer Sample Purification Beads and 80 EtOH into separate multichannel reagent reservoirs Label a new 96 well MIDI plate NLC Nextera Library Clean Up with a smudge resistant pen Part 15046431 Rev A Procedure 10 11 12 13 14 15 Label a new 96 well HSP plate NLS Nextera Library Sample with a smudge resistant pen Remove the NLA plate from the thermocycler and centrifuge to 280 x g for 1 minute Remove the adhesive seal from the NLA plate Transfer 50 ul of clear supernatant from each well of the NLA plate to the corresponding well of the new 96 well MIDI plate labeled NLC Vortex the Sampl
52. it is normal to see a small degree of sample loss However if the sample loss is greater than 15 Illumina does not recommend proceeding with the sample preparation Poor sealing or not heating the lid can cause this amount of loss Vortex the Streptavidin Magnetic Beads tube until the beads are well dispersed then add 250 ul well mixed Streptavidin Magnetic Beads to the wells of the NEW1 plate Mix thoroughly as follows a Seal the NEW1 plate with a Microseal B adhesive seal b Shake the NEW1 plate on a microplate shaker at 1200 rpm for 5 minutes Let the NEW1 plate stand at room temperature for 25 minutes Centrifuge the NEWI plate to 280 x g for 1 minute Part 15046431 Rev A 10 11 12 First Wash 1 6 Remove the adhesive seal from the NEW1 plate Place the NEW1 plate on the magnetic stand for 2 minutes at room temperature or until the liquid appears clear Carefully remove and discard all of the supernatant from each well of the NEW1 plate without disturbing the beads Remove the NEW1 plate from the magnetic stand Make sure that the Enrichment Wash Solution tube is at room temperature then thoroughly vortex the tube NOTE A It is normal that the Enrichment Wash Solution can be cloudy after vortexing Add 200 ul Enrichment Wash Solution to each well of the NEW1 plate Mix thoroughly and resuspend the bead pellet by repeatedly dispensing the wash solution over the bead pellet until it is immersed in the
53. ke sure that you use the Streptavidin Magnetic Beads 2 ml tube and not the Sample Purification Beads 15 ml tube for this procedure TruSight One Library Preparation Guide p 9 391n1de 18 114 Protocol First Bind 30 Pre heat the microheating system to 50 C NOTE The Enrichment Wash steps are key to ensuring high enrichment specificity Ilumina recommends using a microheating system with a MIDI plate insert for these steps to make sure that samples are maintained at the desired temperature Too low or too high temperatures can result in lower percent enrichments and decreased yields If a microheating system is not available a thermal cycler can be used with modifications by following the directions in Alternative Thermal Cycler Steps for Successful Enrichment on page 59 This approach requires additional sample transfers Label a new 96 well MIDI plate NEW1 Nextera Enrichment Wash 1 with a smudge resistant pen Label a new 96 well HSP plate NEH2 Nextera Enrichment Hyb 2 with a smudge resistant pen Remove the NEH1 plate from the thermal cycler Centrifuge the NEHI plate to 280 x g for 1 minute Remove the adhesive seal from the NEH1 plate Take care when removing the seal to avoid spilling the contents of the wells Transfer the entire contents 100 ul from each well of the NEH1 plate to the corresponding well of the new 96 well MIDI plate labeled NEW1 E NOTE If an overnight First Hybridization was performed
54. ltra 0 5 1 per pooled sample IC tore User centrifugal filter unit 0 5 ml 30 kDa Microseal B adhesive seal 1 15 C to 30 C User RNase DNase free eight tube 2 IC 10 SORT User strips and caps for multi sample processing Preparation Remove the following from 15 C to 25 C storage and thaw them at room temperature Enrichment Hybridization Buffer TruSight One Oligos For multi sample processing Use a multichannel pipette Distribute the TruSight One Oligos and Enrichment Hybridization Buffer into separate eight tube strips dispensing equal volumes into each of the wells Remove the NLS plate from 15 to 25 C storage if it was stored at the conclusion of First PCR Clean Up and thaw on ice TruSight One Library Preparation Guide 2 5 uonezipuq AH 18 14 Protocol Centrifuge the thawed NLS plate to 280 x g for 1 minute Remove the adhesive seal from the thawed NLS plate Pre program the thermal cycler with the following program and save as NRC HYB a Choose the pre heat lid option and set to 100 C b 95 C for 10 minutes c 18 incubations of 1 minute each starting at 94 C then decreasing 2 C per incubation d 58 C for forever Label a new 96 well HSP plate NEH1 Nextera Enrichment Hyb 1 with a smudge resistant pen Pool Libraries 26 Reference Table 1 for the amount of DNA library to use for enrichment Illumina recommends using 500 ng of each DNA library quantified by a fluorometric dsDNA quantification met
55. m each well of the NEW2 plate to the corresponding well of the new MIDI plate labeled NEC1 Take care not to disturb the beads NOTE Ilumina recommends using a 20 ul single channel or multichannel pipette set to 10 5 ul to perform two consecutive transfers of 10 5 ul This technique reduces sample loss by making sure that all of the liquid is transferred without disturbing the beads Add 4 ul Elute Target Buffer 2 to each well of the NEC1 plate containing samples to neutralize the elution Mix thoroughly as follows a Seal the NECI plate with a Microseal B adhesive seal b Shake the NECI plate on a microplate shaker at 1800 rpm for 1 minute Centrifuge the NECI plate to 280 x g for 1 minute Store the remaining reagents as follows a Place the Elute Target Buffer 2 and Streptavidin Magnetic Beads tubes in 2 C to 8 C storage b Place the HP3 Enrichment Elution Buffer 1 and Enrichment Wash Solution tubes in 15 C to 25 C storage c Discard any remaining elution pre mix Part 15046431 Rev A Capture Sample Clean Up This process uses Sample Purification Beads to purify the captured library before PCR amplification Consumables Item Quantity Storage Supplied By Resuspension Buffer RSB 1 tube 2 C to 8 C Ilumina Sample Purification Beads SPB 1 tube PAC WO IC Illumina 96 well HSP plate 1 15 C to 30 C User Freshly prepared 80 ethanol 400 ul per sample 15 to Sun User EtOH Microseal B adhesive seals
56. m the microheating system and immediately place it on the magnetic stand for 2 minutes or until the liquid appears clear Remove the adhesive seal from the NEW2 plate Immediately remove and discard all of the supernatant from each well of the NEW2 plate 10 Remove the NEW2 plate from the magnetic stand 11 Repeat steps 2 10 one time for a total of two Enrichment Wash Solution washes Second Elution 1 Add the following reagents in the order listed in a new 1 7 ml microcentrifuge tube to create the elution pre mix Multiply each volume by the number of pooled samples being prepared The volumes include an excess amount for processing multiple samples Reagent Volume pl Enrichment Elution Buffer 1 28 5 HP3 1 5 Total Volume per Sample 30 Vortex the elution pre mix tube then add 23 5 ul of the mix to each well of the NEW2 plate Mix thoroughly as follows a Seal the NEW2 plate with a Microseal B adhesive seal b Shake the NEW2 plate on a microplate shaker at 1800 rpm for 2 minutes Let the NEW2 plate stand at room temperature for 2 minutes Centrifuge the NEW2 plate to 280 x g for 1 minute Carefully remove the adhesive seal from the NEW2 plate to avoid spilling the contents of the wells Place the NEW2 plate on the magnetic stand for 2 minutes or until the liquid appears clear TruSight One Library Preparation Guide 3 9 eJnjde puogas Protocol 40 10 11 12 Transfer 21 ul of clear supernatant fro
57. mends using the same dilution of final library for both quantification and clustering Inaccurate quantification can result in lower than targeted cluster densities less reads passing filter and or inefficient demultiplexing if overclustered Illumina recommends targeting 1200k 1400k clusters mm2 raw density on MiSeq v3 runs though the optimal cluster density can vary between instruments A Part 15046431 Rev A Additional Resources The following resources are available for TruSight One Sequencing Panel protocol guidance and sample tracking Access these and other resources on the Illumina website at support illumina com sequencing kits ilmn Then select TruSight One Sequencing Panel Support Resource Best Practices TruSight One Sequencing Panel Experienced User Card and Lab Tracking Form part 15046433 Illumina Experiement Manager IEM IEM TruSight One or TruSight Rapid Capture Quick Reference Card part 15048138 TruSight One Library Preparation Guide Description Provides best practices specific to this protocol Review this before starting sample preparation Topics include Consistency Handling Magnetic Beads Avoiding Cross Contamination Washing During SPB Clean Up Freeze thawing for Small Number of Samples Preventing PCR Product Contamination Click Best Practices on the TruSight One Sequencing Panel Support page Provides protocol instructions but with less detail than what is
58. ment Hybridization 1 tube 15 C to 25 C Ilumina Buffer EHB Resuspension Buffer RSB 1 tube 2 C to 8 C Illumina Microseal B adhesive seal 1 TIC ee XO CC User Preparation Remove the following from 15 C to 25 C storage and thaw them at room temperature Enrichment Hybridization Buffer TruSight One Oligos Make sure that the Resuspension Buffer is at room temperature Remove the NEH2 plate from 15 C to 25 C storage if it was stored at the conclusion of First Capture and thaw on ice Centrifuge the thawed NEH2 plate to 280 x g for 1 minute Procedure 34 1 Thoroughly vortex the Enrichment Hybridization Buffer tube until the solution is completely resuspended Visually make sure that no crystal structures are present NOTE i If crystals and cloudiness are observed vortex the Enrichment Hybridization Buffer tube until it appears clear 2 Remove the adhesive seal from the NEH2 plate Part 15046431 Rev A Add the following reagents in the order listed to each well of the NEH2 plate Reagent Volume ul Resuspension Buffer 15 Enrichment Hybridization Buffer 50 TruSight One Oligos 10 Mix thoroughly as follows a Seal the NEH2 plate with a Microseal B adhesive seal Make sure that the plate is tightly sealed to prevent potential evaporation Use an adhesive seal roller to apply force to the seal and make sure that the seal is secured b Shake the NEH2 plate on a microplate shaker at 1200 rpm for 1 minute Cen
59. mm TruSight One Library Preparation Guide T c Je1deuo Protocol Introduction This chapter describes the TruSight One protocol Review Best Practices before proceeding See Additional Resources on page 5 for information on how to access TruSight One Sequencing Panel Best Practices on the Illumina website Follow the protocols in the order shown using the specified volumes and incubation parameters 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 well formed sample sheets for Illumina sequencers and analysis software Detailed procedures on how to create a sample sheet for the TruSight One Sequencing Panel are available in an IEM quick reference card See Additional Resources on page 5 for information on how to download IEM software and TruSight One appropriate IEM documentation from the Illumina website Each column should contain a common index This will facilitate pipetting operations when dispensing indexed adapters and pooling indexed libraries later in the protocol 8 Part 15046431 Rev A Library Prep Workflow The following diagram illustrates the workflow using a TruSight One Sequencing Panel kit Safe stopping points are marked between steps Figure 1 TruSight One Workflow 50 ng Genomic DNA 5 ng ul MOJ P4JJOM dead MLT TruSight One Library Preparation Guide 9 Protocol Tagment Genomic D
60. mple Purification Beads SPB 1 tube 96 well hard shell plate HSP 1 Freshly prepared 80 ethanol 400 ul per sample EtOH Microseal B adhesive seals 2 RNase DNase free reagent 3 reservoirs for multi sample processing Preparation Storage 15 C to 25 C AC BAC 15 C to 30 C ISAC me HOKE 15 C to 30 C IC ie S0 Supplied By Ilumina Illumina User User User User Remove the Resuspension Buffer from 15 C to 25 C storage and thaw at room temperature be NOTE The Resuspension Buffer can be stored at 2 C to 8 C after the initial thaw Review Best Practices for Handling Magnetic Beads See Additional Resources on page 5 for information on how to access TruSight One Best Practices on the Illumina website Make sure that the Sample Purification Beads are at room temperature For multi sample processing Use a multichannel pipette Pour the Resuspension Buffer Sample Purification Beads and 8076 EtOH into separate multichannel reagent reservoirs TruSight One Library Preparation Guide 19 WNC pajuaube dN uea Protocol Procedure 14 VD 0 N O OF 10 11 12 13 Label a new 96 well HSP plate NLA Nextera Library Amplification with a smudge resistant pen Remove the adhesive seal from the NLT plate Vortex the room temperature Sample Purification Beads until they are well dispersed NOTE Keep the Sample Purification Beads tube at room temperature for later use in
61. n in nM 660 g mol average library size For example 15 ng ul x10 6 34 9nM 660 g mol 650 Alternatively you can quantitate libraries using qPCR according to the Sequencing Library qPCR Quantification Guide part 11322363 NOTE You can download the Sequencing Library qPCR Quantification Guide from the Illumina website at support illumina com sequencing kits ilmn Click TruSight One Sequencing Panel Support Then click Documentation amp Literature on the TruSight One Sequencing Panel Support page TruSight One Library Preparation Guide A 9 AjeJqr eyepije Protocol Assess Quality Optional o0 To assess library quality load 1 ul of diluted post enriched library on an Agilent Technologies 2100 Bioanalyzer using an Agilent High Sensitivity DNA Chip Check the size of the library for a distribution of DNA fragments with a size range from approximately 200 bp 1 kb Depending on the level of indexing insert size distribution can vary slightly however the library peak must not be significantly shifted compared to the examples in Figure 4 Figure 4 Example TruSight One Post Enrichment Library Distributions SC i a IA Dm T T aes T T T T T E Y 35 100 150 200 300 400 500 600 1000 2000 Part 15046431 Rev A Region 1 je T T Tr 200 300 400 500 700 NOTE The blue lines indicate the boundaries that were manually created to determine av
62. ncubate the plate at the 58 C holding temperature for at least 90 minutes and up to a maximum of 24 hours Do not remove the plate from 58 C incubation until you are ready to proceed to First Capture on page 29 Part 15046431 Rev A First Capture This process uses streptavidin beads to capture probes hybridized to the targeted regions of interest Two heated wash procedures remove non specific binding from the beads The enriched library is then eluted from the beads and prepared for a second round of hybridization The use of the correct equipment and temperatures is needed to ensure removal of non specific DNA as well as retention of the target regions Consumables Item Quantity Storage Supplied By 2N NaOH HP3 1 tube 15 C to 25 C Illumina Elute Target Buffer 2 ET2 1 tube to Ilumina Enrichment Elution Buffer 1 EE1 1 tube 15 C to 25 C Ilumina Enrichment Wash Solution EWS 1 tube 15 C to 25 C Illumina Streptavidin Magnetic Beads SMB 1 tube 2 C to 8 C Ilumina 1 7 ml microcentrifuge tube il IC to 30 C User 96 well HSP plate 1 15 C to 30 C User 96 well MIDI plate 1 ISS me SUE User Microseal B adhesive seals 6 15 C to 30 C User Preparation Remove the HP3 Enrichment Elution Buffer 1 and Enrichment Wash Solution from 15 C to 25 C storage and thaw at room temperature Remove the Elute Target Buffer 2 and Streptavidin Magnetic Beads from 2 C to 8 C storage and let stand at room temperature Ir NOTE Ma
63. o dry on the magnetic stand Remove the NLC plate from the magnetic stand Add 27 5 ul Resuspension Buffer to each well of the NLC plate Do not touch the beads with the pipette tips Mix thoroughly as follows a Seal the NLC plate with a Microseal B adhesive seal b Shake the NLC plate on a microplate shaker at 1800 rpm for 1 minute Incubate the NLC plate at room temperature for 2 minutes Centrifuge the NLC plate to 280 x g for 1 minute Remove the adhesive seal from the NLC plate Place the NLC plate on the magnetic stand for 2 minutes or until the liquid appears clear Transfer 25 ul of clear supernatant from each well of the NLC plate to the corresponding well of the new HSP plate labeled NLS Take care not to disturb the beads NOTE Illumina recommends using a 20 ul single channel or multichannel pipette set to 12 5 ul to perform two consecutive transfers of 12 5 ul This technique reduces sample loss by making sure that all of the liquid is transferred without disturbing the beads Quantify the library in the NLS plate using a fluorometric quantification method that uses dsDNA binding dyes 1 NOTE Accurate library quantification is needed to achieve even pooling for enrichment Inaccurate quantitation and pooling can result in higher representation of some samples compared to others in the same pool Optional Load 1 ul of the library on an Agilent Technologies 2100 Bioanalyzer using an Agilent DNA 1000 Chip Ch
64. on Date 15046433 A October 2013 TruSight One Library Preparation Guide Description of Change Initial Release 40 SIH UOISIA8H Table of Contents Revision History vii Table of Contents viii Chapter 1 Overview 1 treie ere MERERI 2 DNA Input Recommendations 3 Critical Steps for Successful Enrichment and Coverage 4 Additional Resources 5 Chapter 2 Protocol s oo ar do LEAD ILU 7 treie AA A A EE 8 Library Prep Workflow ole ee ccc cece eee IIR IRR 9 Tagment Genomic DNA 10 Clean Up Tagmented DNA ss RR RR RR cceceeeees 13 First PCR Amplification 2 22 00 0 000000 ccc c cece ccc messis 16 First PCR OeanUlp ce eee ce ononon ronnan 20 First Hybridization 00000 o cece ccc cece cee cece ee cece eeeeeeeeeeeeeeees 25 First Capture 29 Second Hybridization 2 2 0 2 2 cece cece cece cccccceceecccccceceeeecucceeeeeeeeeee 34 Second Capture 36 Capture Sample CleanUp c cece nennen nennen 41 Second PCR Amplification Rmi 44 Second PCR Cleanlp IIl lIIIIIIllllllll 46 Validate f gr 49 Preparing Your Libraries for Sequencing on a MiSeq 52 Sequence Libary dd A RE See e ee 54 Appendix A Supporting Information 55 Introduction 56 ACTON MS H erario N ee 57 Alternative Thermal Cycler Steps for Successful Enrichment 59 Kit CONTENTS ic tt S
65. ond Capture and thaw on ice Centrifuge the thawed NEA plate to 280 x g for 1 minute Remove the adhesive seal from the thawed NEA plate A A Part 15046431 Rev A Pre program the thermal cycler with the following program and save as NEM AMP10 Choose the pre heat lid option and set to 100 C 98 C for 30 seconds 10 cycles of 98 C for 10 seconds 60 C for 30 seconds 72 C for 30 seconds 72 C for 5 minutes Hold at 10 C E NOTE Illumina has optimized the number of recommended PCR cycles for enrichment assays based on the level of pre enrichment sample pooling and the size of the oligonucleotide set Do not add or reduce the cycles of PCR as it may compromise data quality Procedure 1 Add 5 ul PCR Primer Cocktail to each well of the NEA plate 2 Add20 ul Nextera Enrichment Amplification Mix to each well of the NEA plate 3 Mix thoroughly as follows a Seal the NEA plate with a Microseal A film Use an adhesive seal roller to apply force to the film and make sure that the film is secured b Shake the NEA plate on a microplate shaker at 1200 rpm for 1 minute 4 Centrifuge the NEA plate to 280 x g for 1 minute 5 Place the sealed NEA plate on the pre programmed thermal cycler Close the lid then select and run the NEM AMP10 program SAFESTOPPING POINT If you do not plan to proceed immediately to Second PCR Clean Up on page 46 the NEA plate can remain on the thermocycler overnight If you are stopping replace the Microse
66. quid appears clear Remove the adhesive seal from the PCR plate Immediately remove and discard all of the supernatant from each well Remove the PCR plate from the magnetic stand Add 200 ul Enrichment Wash Solution to each sample well of the PCR plate Gently pipette the entire volume up and down 10 20 times Mix thoroughly and avoid excessive bubbling or foaming Make sure that the beads are fully resuspended 10 Repeat steps 2 8 one time 11 Continue to the elution step TruSight One Library Preparation Guide 5 9 JO SASIS JAJ9 D jewu y eAjeuadej v Supporting Information Kit Contents Check to make sure that you have all of the reagents identified in this section before proceeding to the library preparation procedures TruSight One Sequencing Panel kits are available in the following configurations Table 3 TruSight One Sequencing Panel Kits Kit Name Catalog TG Catalog TruSight One Sequencing Panel 9 Samples FC 141 1006 TG 141 1006 TruSight One Sequencing Panel 36 Samples FC 141 1007 TG 141 1007 NOTE TG labeled consumables include features intended to help reduce the frequency of revalidation They are available only under supply agreement and require you to provide a binding forecast Please contact your account manager for more information Note regarding biomarker patents and other patents unique to specific uses of products Some genomic variants including some nucleic acid sequences and their us
67. riability into the tagmentation step Illumina strongly recommends a two step method of gDNA normalization After the initial quantification gDNA samples are first normalized to 10 ng ul Samples are then re quantified using a similar fluorometric based method and normalized to a final 5 ng ul TruSight One Library Preparation Guide 3 suonepueuuulooeH 1ndu VNA Overview Critical Steps for Successful Enrichment and Coverage To ensure robust performance from the TruSight One Sequencing Panel Illumina recommends using a microheating system with a MIDI plate insert for the Enrichment Wash steps The Enrichment Wash steps reduce non specific DNA binding and require that samples are maintained at the indicated temperature Too low or too high temperatures can result in lower percent enrichments and decreased yields If a microheating system is not available a thermal cycler can be used with some modifications See Alternative Thermal Cycler Steps for Successful Enrichment on page 59 for instructions using a thermal cycler Obtaining Desired Reads Per Sample The number of resulting reads for each sample of a pool are dependent on the following factors Accurate quantification of tagmented samples before pooling for enrichment Inaccurate quantification can lead to uneven pooling between samples in the enrichment and can result in less than expected reads for a given sample Accurate quantification of final enriched library pools Illumina recom
68. rticulates are resuspended For multi sample processing Use a multichannel pipette Distribute the Stop Tagment Buffer Tagment DNA Buffer and Tagment DNA Enzyme 1 into separate eight tube strips dispensing equal volumes into each of the wells Pour the Sample Purification Beads into a multichannel reagent reservoir Place a MIDI plate insert on the microheating system Pre heat the microheating system to 58 C Label a new 96 well MIDI plate NLT Nextera Library Tagment with a smudge resistant pen Use the Illumina Experiment Manager to determine the index primers to be used For more information on IEM see Additional Resources on page 5 ly WARNING Allowing the Tagment DNA Enzyme 1 to warm up to room temperature might result in decreased activity Procedure NOTE Make sure that the reaction is assembled in the order described for optimal kit performance It is not necessary to assemble the reaction on ice 1 Perform the following steps to normalize your gDNA samples a Quantify your gDNA samples using a fluorometric method such as QuantiFluor or Qubit b Normalize your gDNA samples in Tris HCl 10 mM pH 8 5 to 10 ng ul c Requantify the 10 ng ul normalized sample using the same fluorometric quantification method d Based on the quantification further dilute your gDNA samples in Tris HCl 10 mM pH 8 5 to a final volume of 10 ul at 5 ng ul 50 ng total TruSight One Library Preparation Guide 1 1 WNC olluouesy jUaube Pro
69. sh Solution can be cloudy after vortexing 2 Add 200 ul Enrichment Wash Solution to each well of the NEW2 plate 3 Mix thoroughly and resuspend the bead pellet by repeatedly dispensing the wash solution over the bead pellet until it is immersed in the solution Then gently pipette the entire volume of each well up and down 10 times to ensure complete resuspension of the sample E NOTE Proper resuspension of the Streptavidin Magnetic Beads is needed to ensure efficient removal of non specific DNA from the reaction which otherwise results in poor enrichment statistics 4 Seal the NEW2 plate with a Microseal B adhesive seal 5 Incubate the NEW2 plate on the pre heated microheating system with the lid closed at 38 50 C for 30 minutes NOTE The Enrichment Wash steps are key to ensuring high enrichment specificity Ilumina recommends using a microheating system with a MIDI plate insert for these steps to make sure that samples are maintained at the desired temperature Too low or too high temperatures can result in lower percent enrichments and decreased yields If a microheating system is not available a thermal cycler can be used with modifications by following the directions in Alternative Thermal Cycler Steps for Successful Enrichment on page 59 This approach requires additional sample transfers Part 15046431 Rev A Place the magnetic stand next to the microheating system for immediate access Remove the NEW2 plate fro
70. the protocol Add 65 ul well resuspended Sample Purification Beads to each well of the NLT plate Mix thoroughly as follows a Seal the NLT plate with a Microseal B adhesive seal b Shake the NLT plate on a microplate shaker at 1800 rpm for 1 minute Incubate the NLT plate at room temperature for 8 minutes Centrifuge the NLT plate to 280 x g for 1 minute Remove the adhesive seal from the NLT plate Place the plate on the magnetic stand for 2 minutes or until the liquid appears clear Using a 200 ul single channel or multichannel pipette set to 130 ul remove and discard all of the supernatant from each well of the NLT plate NOTE Leave the NLT plate on the magnetic stand while performing the following 80 EtOH wash steps 10 12 With the NLT plate on the magnetic stand slowly add 200 ul freshly prepared 80 EtOH to each well without disturbing the beads Incubate the plate at room temperature for 30 seconds Remove and discard the 80 EtOH from each well of the NLT plate Repeat steps 10 and 11 one time for a total of two 80 EtOH washes Using a 20 ul single channel or multichannel pipette remove any remaining 80 EtOH from each well of the NLT plate without disturbing the beads Part 15046431 Rev A 14 15 16 17 18 19 20 21 22 With the NLT plate on the magnetic stand incubate the plate at room temperature for 10 minutes to dry Remove the NLT plate from the magnetic stand Add 22 5 ul R
71. tocol ON 0 m Q 10 11 12 13 14 15 12 Add 10 ul gDNA at 5 ng ul 50 ng total to each well of the new 96 well MIDI plate labeled NLT Add 25 ul Tagment DNA Buffer to each well of the NLT plate Add 5 ul Tagment DNA Enzyme 1 to each well of the NLT plate Add 10 ul PCR grade water to each well of the NLT plate Mix thoroughly as follows a Seal the NLT plate with a Microseal B adhesive seal b Shake the NLT plate on a microplate shaker at 1800 rpm for 1 minute Centrifuge the NLT plate to 280 x g for 1 minute Place the sealed NLT plate on the pre heated microheating system Close the lid and incubate at 58 C for 10 minutes Remove the NLT plate from the microheating system Remove the adhesive seal from the NLT plate Add 15 ul Stop Tagment Buffer to each well of the NLT plate Mix thoroughly as follows a Seal the NLT plate with a Microseal B adhesive seal b Shake the NLT plate on a microplate shaker at 1800 rpm for 1 minute Centrifuge the NLT plate to 280 x g for 1 minute Incubate the NLT plate at room temperature for 4 minutes Proceed to Clean Up Tagmented DNA on page 13 Part 15046431 Rev A Clean Up Tagmented DNA This process purifies the tagmented DNA from the Nextera transposome It is critical because the Nextera transposome can bind tightly to DNA ends and interfere with downstream processes when it is not removed Consumables Item Quantity Resuspension Buffer RSB 1 tube Sa
72. transfers of 10 5 ul This technique reduces sample loss by making sure that all of the liquid is transferred without disturbing the beads Add 4 ul Elute Target Buffer 2 to each well of the NEH2 plate containing samples to neutralize the elution Mix thoroughly as follows a Seal the NEH2 plate with a Microseal B adhesive seal b Shake the NEH2 plate on a microplate shaker at 1200 rpm for 1 minute Centrifuge the NEH2 plate to 280 x g for 1 minute Store the remaining reagents as follows a Place the Elute Target Buffer 2 and Streptavidin Magnetic Beads tubes in 2 C to 8 C storage b Place the HP3 Enrichment Elution Buffer 1 and Enrichment Wash Solution tubes in 15 C to 25 C storage c Discard any remaining elution pre mix SAFESTOPPING POINT If you do not plan to proceed immediately to Second Hybridization on page 34 you can safely 1 stop the protocol here If you are stopping seal the NEH2 plate with a Microseal B adhesive seal and store it at 15 C to 25 C for up to 7 days TruSight One Library Preparation Guide 3 3 391n1de 18 114 Protocol Second Hybridization This process combines the eluted DNA library from the first enrichment round with additional capture probes to targeted regions of interest This second hybridization is required to ensure high specificity of the captured regions Consumables Item Quantity Storage Supplied By TruSight One Oligos TOO 1 tube 15 C to 25 C Illumina Enrich
73. trifuge the NEH2 plate to 280 x g for 1 minute Place the sealed NEH2 plate on the pre programmed thermal cycler Close the lid then select and run the NRC HYB program Incubate the plate at the 58 C holding temperature overnight for at least 14 5 hours and up to a maximum of 24 hours Do not remove the plate from 58 C incubation until you are ready to proceed to Second Capture on page 36 TruSight One Library Preparation Guide 3 5 UONEZIPUG H puooes Protocol Second Capture 36 This process uses streptavidin beads to capture probes hybridized to the targeted regions of interest Two heated wash procedures remove non specific binding from the beads The enriched library is then eluted from the beads and prepared for sequencing The use of the correct equipment and temperatures is needed to ensure removal of non specific DNA as well as retention of the target regions ly NOTE These procedures are similar to the First Capture on page 29 Consumables Item Quantity Storage Supplied By 2N NaOH HP3 1 tube 15 C to 25 C Illumina Elute Target Buffer 2 ET2 1 tube PACOS Ilumina Enrichment Elution Buffer 1 EE1 1 tube 15 C to 25 C Ilumina Enrichment Wash Solution EWS 1 tube 15 C to 25 C Illumina Streptavidin Magnetic Beads SMB 1 tube 2 C to 8 C Illumina 1 7 ml microcentrifuge tube 1 15 C to 30 C User 96 well MIDI plates 2 15 C to 30 C User Microseal B adhesive seals 6 AC Ge SOC User Preparation Remove the
74. turbing the beads SAFE STOPPING POINT If you do not plan to proceed immediately to Second PCR Amplification on page 44 you can ji safely stop the protocol here If you are stopping seal the NEA plate with a Microseal B adhesive seal and store it at 15 C to 25 C for up to 7 days TruSight One Library Preparation Guide A 3 dN uee o ajdwes ande Q Second PCR Amplification 2 O This process amplifies the captured library via a 10 cycle PCR program It is critical to use eg the full amount of recommended input DNA and not add extra PCR cycles to ensure libraries produce high quality sequencing results Consumables Item Quantity Storage Supplied By Nextera Enrichment 1 tube 15 C to 25 C Illumina Amplification Mix NEM PCR Primer Cocktail PPC 1 tube 15 C to 25 C Ilumina Microseal A film 1 15 C to 30 C User Microseal B adhesive seal il IC me SIG User Preparation Remove the Nextera Enrichment Amplification Mix and PCR Primer Cocktail from 15 C to 25 C storage and thaw on ice Briefly centrifuge the thawed Nextera Enrichment Amplification Mix and PCR Primer Cocktail tubes for 5 seconds NOTE If you do not intend to consume the Nextera Enrichment Amplification Mix and PCR Primer Cocktail in one use dispense the reagents into single use aliquots Freeze the aliquots in order to avoid repeated freeze thaw cycles Remove the NEA plate from 15 C to 25 C storage if it was stored at the conclusion of Sec
75. uSight One Library Preparation Guide V 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 t
76. uSight One kits are identical to S500 series Index 2 i5 sequences in other kits However the Index 2 i5 adapters are not interchangeable across kits Part 15046431 Rev A Technical Assistance For technical assistance contact Illumina Technical Support Table 7 Illumina General Contact Information Illumina Website www illumina com Email techsupport illumina com Table8 Ilumina 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 MSDSs Material safety data sheets MSDSs are available on the Illumina website at www illumina com msds Product Documentation 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 TruSight One Library Preparation Guide E S0ue sISsy JEIIUUOS AAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATATTAACGTACCATTAAGAGCTACC 3aATAACAGTAACACACT TCTGTTAACCT TAAGAT TACT TGT TGATCCACTGATTCAACGTACCGTATCAAT TGAGAC TAAATAT TAACGTACCAT
77. ut disturbing the beads Let the NEC2 plate stand at room temperature for 10 minutes to dry on the magnetic stand TruSight One Library Preparation Guide AT dn ueso HOd PUO98S Protocol 17 18 19 20 21 22 23 24 25 48 Remove the NEC2 plate from the magnetic stand Add 32 5 ul Resuspension Buffer to each well of the NEC2 plate Do not touch the beads with the pipette tips Mix thoroughly as follows a Seal the NEC2 plate with a Microseal B adhesive seal b Shake the NEC2 plate on a microplate shaker at 1800 rpm for 1 minute Incubate the NEC2 plate at room temperature for 2 minutes Centrifuge the NEC2 plate to 280 x g for 1 minute Remove the adhesive seal from the NEC2 plate Place the NEC2 plate on the magnetic stand for 2 minutes or until the liquid appears clear Transfer 30 ul of clear supernatant from each well of the NEC2 plate to the corresponding well of the new HSP plate labeled NES Take care not to disturb the beads i NOTE Ilumina recommends using a 20 ul single channel or multichannel pipette set to 15 ul to perform two consecutive transfers of 15 ul This technique reduces sample loss by making sure that all of the liquid is transferred without disturbing the beads Seal the NES plate with a Microseal B adhesive seal SAFESTOPPING POINT If you do not plan to proceed immediately to Validate Library on page 49 store the sealed NES plate at 15 C to 25 C for up to 7 days If the
78. ut disturbing the beads Incubate the plate at room temperature for 30 seconds Remove and discard the 80 EtOH from each well of the NECI plate Repeat steps 9 and 10 one time for a total of two 8076 EtOH washes Using a 20 ul single channel or multichannel pipette remove any remaining 80 EtOH from each well of the NECI plate without disturbing the beads Let the NECI plate stand at room temperature for 10 minutes to dry on the magnetic stand Remove the NECI plate from the magnetic stand Add 27 5 ul Resuspension Buffer to each well of the NECI plate Do not touch the beads with the pipette tips Mix thoroughly as follows a Seal the NECI plate with a Microseal B adhesive seal b Shake the NECI plate on a microplate shaker at 1800 rpm for 1 minute Incubate the NECI plate at room temperature for 2 minutes Centrifuge the NECI plate to 280 x g for 1 minute Remove the adhesive seal from the NECI plate Place the NECI plate on the magnetic stand for 2 minutes or until the liquid appears clear Part 15046431 Rev A 21 Transfer 25 ul of clear supernatant from each well of the NECI plate to the corresponding well of the new HSP plate labeled NEA Take care not to disturb the beads NOTE CG Ilumina recommends using a 20 ul single channel or multichannel pipette set to 12 5 ul to perform two consecutive transfers of 12 5 ul This technique reduces sample loss by making sure that all of the liquid is transferred without dis
79. your lab has a thermal cycler that is not listed validate the thermal cycler before performing the TruSight One protocol Thermal Cycler Temp Mode Lid Temp Vessel Type Bio Rad DNA Engine Calculated Heated Constant Polypropylene plates Tetrad 2 at 100 C and tubes MJ Research DNA Calculated Heated Plate Engine Tetrad Eppendorf Mastercycler Gradient S Heated Plate ProS Simulated Tube Part 15046431 Rev A Index Sequences The following list of index sequences is provided for generating sample sheets to demultiplex the samples A dual indexing strategy uses two 8 base indices Index 1 i7 next to the P7 sequence and Index 2 i5 next to the P5 sequence Dual indexing is enabled by adding a unique Index 1 i7 and Index 2 i5 to each sample N refers to Nextera E refers to Enrichment 7 refers to Index 1 i7 5 refers to Index 2 i5 01 12 refers to the Index number soouenbes xepu Table 6 TruSight One Index Adapter Sequences Index 1 i7 Sequence Index 2 i5 Sequence N701 TAAGGCGA E502 CTCTCTAT N702 CGTACTAG E508 TATCCICT N703 AGGCAGAA E504 AGAGTAGA N704 TCCTGAGC E505 GTAAGGAG N705 GGACTCCT N706 TAGGCATG N707 CTCTCTAC N708 CAGAGAGG N709 GCTACGCT N710 CGAGGCTG N711 AAGAGGCA N712 GTAGAGGA Only available in the TruSight One Sequencing Panel Kit 36 Samples TruSight One Library Preparation Guide 6 9 Supporting Information 70 NOTE The E500 series Index 2 i5 sequences in the Tr

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