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TruSeq Synthetic Long-Read DNA Library Prep - Support

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1. 2 58 Size Selection o ee a abe 63 Validate Final Product 1 66 if v ES 5 SSA em S _ SS TE e Oa if 28 n TS itt WA panataan eS o s n v Se TruSeq Synthetic Long Read DNA Library Prep Guide T ca deuo Protocol Introduction This chapter describes the TruSeq Synthetic Long Read DNA Library Prep protocol Use IEM to create a sample sheet for Illumina sequencing systems and analysis software See Additional Resources on page 5 for information about IEM documentation on the Illumina website Use BaseSpace to organize samples libraries pools and runs for Illumina sequencing systems and analysis software See Additional Resources on page 5 for information about BaseSpace documentation on the Illumina website Follow the protocol in the order shown using the specified volumes and incubation parameters y NOTE The library prep procedures are described using a 96 well PCR plate However due to the small number of samples they can be performed with an eight tube strip or a previously unused well of a plate Review best practices before proceeding See Additional Resources on page 5 for information about TruSeq Synthetic Long Read DNA Library Prep best practices on the Illumina website Review
2. A ARA aa tr A AAA AL MARA MARA T ST GTACCATTAAGAGCTACCGTGCAACTTAACCTTAAGAT TACTTGATCCACT GAT TCAACGTACCGTAACGAACGTATCAATT GAGACTAAATAT TAACGTACCA 2277 CGACG TICTGTT 252177 GAGCTACCGTGCAACGAAAATAACCTIAAGAT TACTTGATCCACTGATTCAACGTACTTCTGTTAACCTIAAGAT TACTTGATCCACTGAT TCAACGTACCGTAACGAACGTA TRATIGAGACTAARCIACCGTECAACGA CGAAAAGAAT GATA ACEAAAAGAATGATAACAGIAAGACAGTIGTGT IAACCTTAAGATTACT TGATCCACTGATTCAACKTACCS AAAGATTAC TGATCCAGTGAt CAAGG ACCS IAACGAACGTAT GATT GAGACTAAATATTAACGIACCAT TAA GAGO TACCA AATGATAACAGTAACACACTICTGTTAACCTTAAGA 2155 CCGTAACGAACGTATCAAT TGAGACT 77UU707 7 U7 CGACGAAAAGAA 277 7 O 5 GTACCAT TAAGAGCTACCGTGCAACAGTAACACACTTCTGT TAACCTTAAGATTACTIGATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGT GCAACGACGAAAAGAATGATAACA AAT QATAACAGTAACAGACTICNG LIGATCCAGT GAT CANCG TACCO TAACGAACGTAT GANTT GAGA TAAATATTAAGGTACCATAAGHOCTASCOT CTT OTC MAACO TIAAGATIAGH GATCCACTGATTCARCOTA ACGTACCGTAACGAACGTATCAT TAAGATTA ig ALG ar CGAAAAGAATGA P CTTCTGTTAACC TTAAC 25 CGTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGIATCAATTGAGCTICTGTTAACCTTAAGATTACTTGATCCACT ao y A TCAAT TGAGACTAGCAACGACGAA ACGAAAAGAAT GATAACAGTAACACACTICTGTIAACCTTIAAGATIACTTGATCCACTGATT CAACGTACCGTAAAGATIACTTGATCCACTGAT TCAACG TACCGTAACGAACGTATCAAT TGAGACTAAATATTAACGTACCAT TAAGAGC TACCGT AATGATAACAG TAACACACTICTGTTAACCT TAAGAT TACT TG
3. Create a scatter plot of the average Cq of the qPCR standards on the X axis and the log base 2 value of the DNA concentration pg ul of the qPCR standards on the Y axis For example Concentration Log Example Avg Cq pg uD Concentration Std1 10 3 321928 9 98 Std2 1 0 13 03 Std3 0 1 3 32193 16 69 Std4 0 01 6 64386 20 56 Determine the equation of the best fit line for the qPCR standard curve values which is in the format of y mx b This is equivalent to log base 2 DNA concentration slope x Cq y_int Part 15047264 Rev B Confirm that the qPCR reaction efficiency the slope in the equation in step c is 50 100 which is a typical reaction efficiency of a long qPCR amplicon Successive 10 fold dilutions of Standard should have Cq values evenly spaced approximately 3 2 cycles apart Pay particular attention to the spacing between Std1 and Std2 and Sample dilutions in this concentration range Examine the amplification plots to make sure that early amplification has not interfered with the automatic baseline determination subtraction on your instrument For more information see your qPCR instrument specific instructions Confirm that the R of the best fit line is gt 0 97 Poor R values can indicate a dilution error in the standard curve or poor amplification of one or more of the standards If so repeat the protocol Determine the value of y in y mx b by using the average Cq of each 1 100 dilution of s
4. Pre program the thermal cycler with the following program and save as ATAIL Choose the pre heat lid option and set to 100 C 37 C for 30 minutes Hold at 4 C 16 Part 15047264 Rev B Add ATL Centrifuge the thawed A Tailing Mix tube at 600 x g for 5 seconds Add 12 5 ul thawed A Tailing Mix to each well of the LFP2 plate Set a 20 ul pipette to 20 ul then gently pipette the entire volume up and down 10 times to mix thoroughly Seal the plate with a Microseal B adhesive seal Return the A Tailing Mix tube to 25 C to 15 C storage Incubate 1 LFP2 1 2 Centrifuge the plate at 280 x g for 1 minute Place the sealed plate containing 30 ul of each sample on the pre programmed thermal cycler Close the lid then select and run the ATAIL program a Choose the pre heat lid option and set to 100 C 37 C for 30 minutes c Hold at 4 C When the thermal cycler temperature is 4 C remove the LFP2 plate from the thermal cycler then proceed immediately to Ligate Adapters on page 18 TruSeq Synthetic Long Read DNA Library Prep Guide 4 rs spuq aje huspy Protocol Ligate Adapters This process ligates adapters to the ends of the long DNA fragments These adapters are used as markers in downstream data analysis processes to denote the end of a contig Consumables Item Quantity Storage Supplied By TruSeq Synthetic Long Read DNA Library Prep Kit contents e Ligation Mix LIG 1 tube per 4
5. SNB 58 software downloads 5 SPB 12 18 63 SYBR Green 31 T TDE 51 technical assistance 103 training 5 Part 15047264 Rev B Technical Assistance For technical assistance contact Illumina Technical Support Table 6 Illumina General Contact Information Illumina Website www illumina com Email techsupport illumina com Table 7 Illumina Customer Support Telephone Numbers Region Contact Number Region Contact Number North America 1 800 809 4566 Italy 800 874909 Austria 0800 296575 Netherlands 0800 0223859 Belgium 0800 81102 Norway 800 16836 Denmark 80882346 Spain 900 812168 Finland 0800 918363 Sweden 020790181 France 0800 911850 Switzerland 0800 563118 Germany 0800 180 8994 United Kingdom 0800 917 0041 Ireland 1 800 812949 Other countries 44 1799 534000 Safety Data Sheets Safety data sheets SDSs are available on the Illumina website at support illumina com sds html Product Documentation Product documentation in PDF is available for download from the Illumina website Go to support illumina com select a product then click Documentation amp Literature TruSeq Synthetic Long Read DNA Library Prep Guide 1 O 3 OUE1SISSV EOIUYD AAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATATTAACGTACCATTAAGAGCTACC SATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGT TGATCCACTGATTCAACGTACCGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTCTTCTGT
6. Dye Mix 44 ul 52 ul 58 ul 68 ul 1 5x SYBR Green with optional 10x ROX MasterAmp Extra long DNA Polymerase 9 ul 10 5 ul 11 5 ul 13 5 ul Mix Total volume 352 ul 416 5 ul 463 5 ul 543 5 ul NOTE Reference Figure 7 while performing steps 3 12 Add 16 ul master mix to each required well of the plate labeled with the QLP barcode TruSeq Synthetic Long Read DNA Library Prep Guide 3 5 UO Jenjueno YOdb Protocol 36 VD ON BD A 11 Figure 7 Example QLP Plate Setup for 1 Sample I O0 TJn mM U DP Std 1 Std 2 Std 3 Std 4 NTC 1 100 Dilution sample mTmuou gt Remove the cap from the standard eight strip tube from step 9 of Dilute qPCR Standard on page 33 Add 4 ul Std 1 to each well in rows C E column 4 Add 4 ul Std 2 to each well in rows C E column 5 Change the tip Add 4 ul Std 3 to each well in rows C E column 6 Change the tip Add 4 ul Std 4 to each well in rows C E column 7 Change the tip Add 4 ul NTC to each well in rows C E column 8 Change the tip Remove the cap from the microcentrifuge tube that contains each one 1 100 Dilution sample Add 4 ul of one 1 100 Dilution sample to each well in rows C E column 9 Part 15047264 Rev B 12 15 14 15 16 17 Analysis Add 4 ul of each additional 1 100 Dilution sample to each well in rows C E adding one column per sample Cap and store the sample name 1 100 Dilution tubes at 2 C to 8 C for subsequent use in this
7. 1 tube per 4 reactions 1 label per plate 2 400 ul per sample As needed 2 Storage 25 C to 15 C 2 C to 8 C AE O 15 C to 30 C T do Oe 15 C to 30 C 25 C to 15 C 15 C to 30 C Supplied By Illumina Ilumina Illumina Illumina User User User User This procedure is described using 96 well PCR plates however RNase DNase free eight tube strips with caps can be used in this procedure in place of the plates Preparation Prepare an ice bucket Part 15047264 Rev B Make LFP Remove the End Repair Mix from 25 C to 15 C storage Thaw it at room temperature and then place it on ice Review best practices for handling magnetic beads See Additional Resources on page 5 for information about TruSeq Synthetic Long Read DNA Library Prep best practices on the Illumina website Remove the Sample Purification Beads and Resuspension Buffer from 2 C to 8 C storage and let stand for at least 30 minutes to bring them to room temperature Remove the gDNA 4200 x g tube from 2 C to 8 C storage if it was stored at the conclusion of Fragment DNA on page 10 Let the tube stand to bring it to room temperature Centrifuge the tube at 280 x g for 1 minute Remove the cap from the tube Pre program the thermal cycler with the following program and save as ERP Choose the thermal cycler pre heat lid option and set to 100 C 30 C for 30 minutes Hold at 4 C Apply an LFP ba
8. 12 Load all of the Diluted 2 Log DNA Ladder into the well of lane 1 of a E Gel EX 1 Figure 10 E Gel EX Agarose Gel 1 Loading Layout Lane M Resuspension Buffer Lane 1 Diluted 2 Log DNA Ladder Lane 2 GST1 Lane 3 GST2 Lane 4 GST3 Lane 5 GST4 Lane 6 Pooled sample Lane 7 QC sample Lane 8 Resuspension Buffer Lane 9 Resuspension Buffer Lane 10 Resuspension Buffer Part 15047264 Rev B 13 14 15 16 17 18 19 20 21 Load 20 ul from the GST1 tube into the well of lane 2 of the gel Load 20 ul from the GST2 tube into the well of lane 3 of the gel Load 20 ul from the GST3 tube into the well of lane 4 of the gel Load 20 ul from the GST4 tube into the well of lane 5 of the gel Load 20 ul pooled sample into the well of lane 6 of the gel Load 20 ul quality control sample from the conclusion of Long Amp Quality Control on page 45 into the well of lane 7 of the gel Load each of the empty wells with 20 ul Resuspension Buffer lanes M 8 10 Select and run the E Gel EX 1 2 program The run time is 10 minutes View the gel on a Dark Reader transilluminator The pooled sample and QC sample bands should migrate the same distance and the pooled sample intensity should be between the intensity of GST1 0 1 ng ul and GST4 0 0125 ng ul The Gel Standard migrates in the gel as a single band at 10 kb NOTE If the band is dimmer than GST4 or brighter than GST1 the quantifi
9. 15 C 1 25 C to 15 C 1 25 C to 15 C 1 25 C to 15 C 1 EPM 15046175 Long amp Primer Mix 25 C to 15 C DNA Polymerase Mix i RSB 15047457 Resuspension Buffer 25 C to 15 C 1 Sample Neutralization 2 C to 8 C Buffer 1 25 C to 15 C Barcode Kit 4 Samples Box C Store at room temperature This box is shipped at room temperature As soon as you receive it store the components at room temperature Figure 25 TruSeq Synthetic Long Read DNA Barcode Kit 4 Samples Box C part 15048205 Slot Part Description 1 4 15052780 Collection Plate 5 8 15055281 Fragmentation Plate Part 15047264 Rev B TruSeq Synthetic Long Read DNA Accessory Kit Store at room temperature The TruSeq Synthetic Long Read DNA Accessory Kit contains one box shipped at room temperature As soon as you receive it store the components at room temperature TruSeq Synthetic Long Read DNA Accessory Box part 15048204 Quantity Part Description 4 15044644 Alignment Ring Fixture TruSeq Synthetic Long Read DNA Library Prep Guide 81 614 1 11 Consumables and Equipment Check to make sure that you have all of the necessary user supplied consumables and equipment before proceeding to the TruSeq Synthetic Long Read DNA Library Prep protocol 2 i The TruSeq Synthetic Long Read DNA Library Prep protocol has been optimized and validated using the items listed Comparable performance is not guaranteed when using
10. BC135 BC136 BC137 BC138 BC139 BC140 BC141 Barcode CTGTCTGA GGCAATCT TGGTTAGG GAGTTCGT CGCAATAC TCTCGCTA TCATCTGG CGAAGATC TTACCTCC CAAGTCGA CAAGTGAC GTGTTACG GCACTCAA TGAAGAGG TGAGACGA CATCTGAG GTCTGGTA GGATGTTC GAAGCTGA CCGTTAAC TGGCAATC Name BC142 BC143 BC144 BC145 BC146 BC147 BC148 BC149 BC150 BC151 BC152 BC153 BC154 BC155 BC156 BC157 BC158 BC159 BC160 BC161 BC162 Barcode TCGGTATG CHNEETET GGTATGGA CACATTGG TACTGTGC CTTGATCG TCCTAAGG TCGCAGAA CTCTGTAG CAAGCCAT GCGAATAG TCGTTGAG TGACACTG GATGGACT CCTTGACT TCTGTTGG CCAACTAG CATCACTC GGATCTAG TCAGGCAA CGCTTATG Name BC163 BC164 BC165 BC166 BC167 BC168 BC169 BC170 BC171 BC172 BC173 BC174 BC175 BC176 BC177 BC178 BC179 BC180 BC181 BC182 BC183 Barcode CGAGTGTT CHANGES GTTACCAC CGATACAG TGGTCTCA TTAGGCTG GCCTCATA CCATGGTT TAGACGCT TTGGCAAC TCGAGACT TTGACCGT CATCTACC GCATTGTC GTGGATGT CTTGAGAC TCGTCAGT TTCGTGCA GACTAGGA GGTCGTAA GATAGGCA Part 15047264 Rev B Name BC184 BC185 BC186 BC187 BC188 BC189 BC190 BC191 BC192 BC193 BC194 BC195 BC196 BC197 BC198 BC199 BC200 BC201 BC202 BC203 BC204 Barcode CACAGCAT TGTGGCTT TTCGTTGC GTTGTCAG ECCT GATCGATC CCTAGGAA CCTCTTCA GGAAGGTA TCACGTGA CACCAGAT TCTGAGAG TATAGCGG TTGCGTAC GGTAGAAG CAAGCATC CACGACTT GAAGAAGG GACCATAG GACGGATA CAGGAGAA Name BC205 BC206 BC207 BC208 BC209 BC210
11. BC211 BC212 BC213 BC214 BC215 BC216 BC217 BC218 BC219 BC220 BC221 BC222 BC223 BC224 BC225 TruSeq Synthetic Long Read DNA Library Prep Guide Barcode TAACGGAC TAGGCGTA GGTCCATA TGCACACT GATTCGAG CGGAACAA TGGAGAAC CCACATCT CATTCCGT GTGGTGTT CTCTAACG TGCTATCC GTCGGTAA GCTTCTTG CCAAGAGT CTGATCCT GCAACATC TCCAGGTA TGCCAGTA GTCAACAG GCGTCTAT Name BC226 BC227 BC228 BC229 BC230 BC231 BC232 BC233 BC234 BC235 BC236 BC237 BC238 BC239 BC240 BC241 BC242 BC243 BC244 BC245 BC246 Barcode TGCTGATC TICCGTGT GATTACGG CAGTAGGT GGTATTCG CGGTTCTA TTAGAGCC CGCGAATA TCTACCAG TIGCGATG CCATCCAA CTTCCATC TGTAAGCC CTCAAGTC CCTGTGTA CATGAAGC GGATTCTG GAATGCCA GTGATGCA TCTTGGCA CGTCTAGA 91 s u nb pooleq Supporting Information 92 Name BC247 BC248 BC249 BC250 BC251 BE252 BC253 BC254 BC255 BC256 BC257 BC258 BC259 BC260 BC261 BC262 BC263 BC264 BC265 BC266 BC267 Barcode TCGTGTTG TGTCCTGA CCTATCGT GTCTTCGA GTACCAAG CCTTATCC TATTCGCC CACTGGAA CTCAACCA GCGTGATT TGAGTGAG CTCACAAC CGCTGTTA CTAAGGCA CAACTAGG GTGCTTGA Name BC268 BC269 BC270 BC271 BC272 BC273 BC274 BC275 BC276 BC277 BC278 BC279 BC280 BC281 BC282 BC283 BC284 BC285 BC286 BC287 BC288 Barcode TCCGATTC CTAACCTC GCATCACT TACGATGG CTACAACC CTAGCTAC TAGCGCTT
12. CAGGATCT CACTTAGC TIGGTCGA CCAATGGA TAGGCTAG TGTTGGAG GGTACTTC GCTTACCT CGAGGTAA CTCGTCAA CAATGTGG CAACCACA TACGTCAC CAGACTTC Name BC289 BC290 BC291 BC292 BC293 BC294 BC295 BC296 BC297 BC298 BC299 BC300 BC301 BC302 BC303 BC304 BC305 BC306 BC307 BC308 BC309 Barcode CATTGCAG TGGAATCG GTGAACGA TGCAGTAG GCGTATTC CGGTAAGA GGTTCAAC TAGAGGTG TGGAAGGA TGGTTGCT CTGGAATC CGTGTAAG TCGCTTGT GGAATGCT TCCACATG TGAGCTTC CTCTACGT GTTAAGCG GAAGGTAC GAGCGTTA Part 15047264 Rev B Name BC310 BC311 BC312 BC313 BC314 BC315 BC316 BC317 BC318 BC319 BC320 BC321 BC322 BC323 BC324 BC325 BC326 BC327 BC328 BC329 BC330 Barcode GTGAAGAC GATCACCA GAACCTIC GCTCGAAT CCTAACAC GACCTAGA CATGGTGA GGCAAGAA CGAATCAC GTTCCTCA TATCGACG TCCTTACC GAAGCGTT GTAGGATC GTGCTAAC CTAACAGG GGCTTGTT TTGCCAGA GAATCCAC CTGATTGC CCAGTTGT Name BC331 BC332 BC333 BC334 BC335 BC336 BC337 BC338 BC339 BC340 BC341 BC342 BC343 BC344 BC345 BC346 BC347 BC348 BC349 BC350 BC351 TruSeq Synthetic Long Read DNA Library Prep Guide Barcode CGAGAAGT CCTTCAGA CTGACGTA TCCGTTCT css ms GCAAGTCT TACCTCCA TGAAGCCA CATCCTAC CCTATACG CTCACTCT GAGCTGAA CCATGAAG TCACCAAC GGAACCAA CCGTGTAA GGTGAACA GTGAGTTG CAGTGATG TGGTACGT GGACTICA Name BC352 BC353 BC354 BC355 BC356 BC357 BC358 BC359 BC360 BC361 BC362 BC363 BC364 BC365 BC366
13. Part 15047264 Rev B 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 Consumablets means Illumina branded reagents and consumable items that are intended by Illumina for use with and are to be consumed through the use of Hardware Documentation means Illumina s user manual for this Product including without limitation package inserts and any other documentation that accompany this Product or that are referenced by the Product or in the packaging for the Product in effect on the date of shipment from Illumina Documentation includes this document Hardware means Illumina branded instruments accessories or peripherals Illumina means Illumina Inc or an Illumina affiliate as applicable Product means the product that this document accompanies e g Hardware Consumables or Software Purchaser is the person or entity that rightfully and legally acquires this Product from Illumina or an Illumina authorized dealer Software means Illumina branded software e g Hardware operating software data analysis software All Software is licensed and not sold and may be subject to additional terms found in the Software s end user license agreement Specifications means lllumina s written specifications for this Pro
14. alternate consumables and equipment Supporting Information Table 4 User Supplied Consumables Consumable 10 ul barrier pipette tips 10 ul multichannel pipettes 10 ul single channel pipettes 1000 ul barrier pipette tips 1000 ul multichannel pipettes 1000 ul single channel pipettes 20 ul barrier pipette tips 20 ul multichannel pipettes 20 ul single channel pipettes 200 ul barrier pipette tips 200 ul electronic eight channel pipette 200 ul multichannel pipettes 200 ul single channel pipettes 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 General lab supplier General lab supplier 1 Kb DNA Extension Ladder Invitrogen part 10511 012 8 Part 15047264 Rev B Consumable 2 Log DNA Ladder 0 1 10 0 kb 2 propanol Isopropanol 15 ml conical tube 15 ml RNase DNase free reagent reservoirs 50 ml conical tube 96 well 0 3 ml skirtless PCR plates or Twin Tec 96 well PCR plates 96 well storage plates round well 0 8 ml MIDI plate Axygen 384 well PCR microplate Compression Mats for PCR Plates Dimethy sulfoxide DMSO DNA Binding Buffer DNA Wash Buffer E Gel EX Agarose Gels 1 E Gel NGS 0 8 Agarose Gels Ethanol 200 proof absolute for molecular biology 500 ml g TUBE Ice bucket KAP
15. b Draw two horizontal lines on the plastic gel cassette to mark the position of the 10 kb and 8 kb bands of the ladder c Repeat step a and b on the other side of plastic cassette because the gel can stick to either side of cassette when it is opened Figure 4 1 kb Extension Ladder Line dravvn to the left of the sample Line dravvn to the right of the sample Line dravvn at 10 kb band Line dravvn at 8 kb band Part 15047264 Rev B Purify Gel The following procedures are from the QlAquick Gel Extraction handbook Make sure to use Isopropanol as specified Contact the kit manufacturer for technical support Carefully open the gel with the Novex Gel Knife E NOTE i For information on how to open the gel see the Novex Gel Knife manufacturer instructions View the gel on a Dark Reader transilluminator to confirm the correct placement of the lines drawn on the plastic gel cassette at the 8 10 kb region of interest Carefully adjust the position of the gel if it has shifted relative to the marked region Place an x tracta tool on the gel between the marked region of interest on the plastic gel cassette and press the tool into the gel Rock the x tracta tool side to side to extract the desired gel slice 3 NOTE View the gel on a Dark Reader transilluminator to confirm that the entire region of interest is extracted Place the x tracta tool over the appropriately labeled microcentrifuge tube a
16. 1 per LAP plate lb o 056 Illumina Microseal B adhesive seals 3 15 C to 30 C User Preparation Remove the IDP plate from 25 C to 15 C storage and thaw at room temperature for at least 10 minutes Pre program the thermal cycler with the following program and save as PostTagAmp Choose the thermal cycler pre heat lid option and set to 100 C 94 C for 1 minute 10 cycles of 94 C for 15 seconds 65 C for 4 minutes Hold at 4 C Procedure 1 Centrifuge the LAP plate at 500 x g for 1 minute 2 Centrifuge the IDP plate at 500 x g for 1 minute 5 A Partit 15047264 Rev B Make sure that the droplets are at the bottom of each well of the IDP plate Remove the adhesive seal from the LAP plate Remove the foil seal from the IDP plate Hn 0 A Q Figure 13 Alignment Ring on LAP A Alignment ring B LAP C Aligned corner notches 7 Invert the IDP plate TruSeq Synthetic Long Read DNA Library Prep Guide 5 5 Optional Place the alignment ring on the LAP plate so that the notched corners align Protocol 56 8 10 11 12 13 Carefully place the inverted IDP plate on top of the LAP plate so that the corner notches and wells of both plates align Make sure that both plates snap together tightly Figure 14 IDP on LAP A IDP B LAP 2 i Surface tension holds the liquid in the IDP plate so liquid does not come out of the plate when it is turned upside down Cent
17. 15 C e Long amp Master Mix 1 tube 25 C to 15 C LMM e Long amp Primer Mix LPM 1 tube 25 C to 15 C Supplied By Illumina Ilumina Ilumina Part 15047264 Rev B Hem Quantity Storage Supplied By z e MasterAmp Extra Long DNA 1 tube 25 C to 15 C Ilumina Q Polymerase Mix JJ e Resuspension Buffer RSB 1 tube 236 O AE Illumina 5 LAP Long Fragment 1 label per plate 15 C to 30 C Ilumina O Amplification Plate barcode U label E Gel EX Agarose Gel 196 il IEE to BOC User J 2 Log DNA Ladder 1 15 C to 30 C User 15 ml conical tube 1 ISAC do SOME User 96 well PCR plate or 1 15 C to 30 C User RNase DNase free eight tube strip with caps 384 well PCR plate 1 to s re User Ice bucket As needed 25 C to 15 C User Microcentrifuge tubes 6 AS O SAS User Microseal B adhesive seals 4 15 C to 30 C User Needle 22 1 2 gauge 1 per sample 15 C to 30 C User RNase DNase free eight tube 2 15 C to 30 C User strips with caps RNase DNase free reagent 1 IDECE User reservoir Preparation Prepare an ice bucket Remove the following from 25 C to 15 C storage and thaw at room temperature Place the tubes on ice Gel Standard TruSeq Synthetic Long Read DNA Library Prep Guide A Protocol MasterAmp Extra Long DNA Polymerase Mix Long amp Master Mix Long amp Primer Mix Remove the Resuspension Buffer from 2 C to 8 C storage and bring it to room temperature For the Phasing wo
18. 50x As specified by 29 to 15 User manufacturer SYBR Green 10 000x 5 ul 25 C to 15 C User Preparation Prepare an ice bucket Remove the following from 25 C to 15 C storage and thaw them at room temperature Place the tubes on ice MasterAmp Extra Long DNA Polymerase Mix qPCR Long amp Primer Mix qPCR Master Mix qPCR Standard Remove the SYBR Green 10 000x from 25 C to 15 C storage and thaw it room temperature Do not place it on ice 4 DMSO thaws slowly so make sure that the SYBR Green 10 000x is completely thawed before using Remove the Resuspension Buffer from 2 C to 8 C storage and bring it to room temperature Set up your qPCR instrument See gPCR Systems on page 87 for a list of validated qPCR systems for this protocol Select the SYBR DNA Binding Dye Assay workflow Determine if ROX is needed for your instrument See your instrument guide for the standard quantification process Pre program the qPCR instrument with the following program 94 C for 1 minute 40 cycles of 94 C for 30 seconds 65 C for 30 seconds 68 C for 10 minutes Optional Melting Curve setting suggested by qPCR instrument TruSeq Synthetic Long Read DNA Library Prep Guide 31 uolyeyJUeND YOdb Protocol Apply a QLP barcode label to a new plate for the qPCR instrument Label new microcentrifuge tubes with a smudge resistant pen as follows QST 1 100 Dilution One tube for each sample with the sample name 1 100 Dilution Lab
19. Add ATL 17 Add LIG 19 ATL 16 B barcode sequences 88 BaseSpace 5 8 best practices 5 BluePippin System 22 95 C Clean Up LFP 14 Clean Ub LFP2 20 CLP 18 cluster generation 69 customer support 103 D DMSO 30 98 documentation 5 103 E E Gel EX Agarose Gel 1 41 E Gel iBase Power System 23 E Gel NGS 0 8 Agarose 22 ERP 12 EtOH 12 18 63 experienced user card EUC 5 TruSeq Synthetic Long Read DNA Library Prep Guide xapul FAQs 5 FPM 51 fragment 10 Fragmented gDNA 22 24 FSP 63 G TUBE 10 ST 40 H help technical 103 High Sensitivity DNA Kit 66 IDP 54 IEM 5 8 Incubate 1 LFP2 17 Incubate 2 LFP2 20 Incubate LFP 14 Isopropanol 22 K KAPA Library Quantification Kit 66 L Lab pen 22 LAD 18 LAP 41 LFP 12 LFP2 12 LIG 18 LMM 40 Long Read workflow 43 45 46 LPM 40 101 Index M W Make LFP 13 workflow diagram 9 MasterAmp 41 MasterAmp Extra Long DNA X Polymerase Mix 30 X tracta 22 N Z Novex Gel Knife 27 Zymo Spin V Column with P Reservoir 58 Zymo DNA Binding Buffer 58 PAP 58 Zymo DNA Wash Buffer 58 PCR grade water 30 98 Phasing workflow 42 43 45 46 Q QlAquick Gel Extraction Kit 22 QLP 30 QMM 30 7 66 PM 87 QST 30 quality control 29 quantify libraries 29 66 uantitation 3 bit dsDNA BR Assay Kit 10 Qubit dsDNA HS Assay Kit 10 66 R requirements and compatibility 5 ROX 31 RSB 10 12 18 22 30 41 58 63 66 Ruler 22 S
20. BC056 TIGCCGAT BC019 TICGGAAG BC038 TTCACGAG BC057 8 8 Part 15047264 Rev B Name BC058 BC059 BC060 BC061 BC062 BC063 BC064 BC065 BC066 BC067 BC068 BC069 BC070 BC071 BC072 BC073 074 BC075 BC076 BC077 BC078 Barcode GTTCAAGG GGCCTAAT TGATGCAC TGGTGGTA GCCGAATT se Le re TTGAGCTC GTTGGAGA GAAGTGCA TCAACGCA GCTAGTGA CAGGTAGT TGACTTGG GGACGATT GTATCAGC GGTGATTG CTGTAGCA CATAGGTC GAGTCCTA TCCATCTC Name BC079 BC080 BC081 BC082 BC083 BC084 BC085 BC086 BC087 BC088 BC089 BC090 BCO91 BC092 BC093 BC094 BC095 BC096 BC097 BC098 BC099 TruSeq Synthetic Long Read DNA Library Prep Guide Barcode GAAGACTC CTGGTTAG GCAGGTTA CTAGACCT CACTTCCT GCCATAAC CAATCCTG GGTTGTCT CGTACCAT GTTAGCGT ee usm CACTAGTG CCACTATC GCTAACTG TAACTCGC CGGTCAAT GCGTACAA CCTGGTAT CCTGAATG CGTTACCA EIMENCEN Name BC100 BC101 BC102 BC103 BC104 BC105 BC106 BC107 BC108 BC109 BC110 BC111 BC112 BC113 BC114 BC115 BC116 BC117 BC118 BC119 BC120 Barcode TGTCGAGT TCTCAGCT GGAATTGC CTGTTGTG GTACGACA CTTGCACT TAGGTTCC TCTGACCA GGTCAGAT CTCATGGT TTAGCGGA TCACAACG CAGCCATT TGTGCGAA CTECATGA CGACTACT TGATAGCG GGACAAGA ilin ms GATGCATG CTTCCGAA 89 saouenbes pooleq Supporting Information 90 Name BC121 BC122 BC123 BC124 BC125 BC126 BC127 BC128 BC129 BC130 BC131 BC132 BC133 BC134
21. BC367 BC368 BC369 BC370 BC371 BC372 Barcode GTTGCGAT GCATAGGT CGTTGTAC TICCGCAA TCAAGGAG TICCTGTC GAGGCAAT TCGGACAT TATGTCCG TAGACCAC GTAGTTGG TGGACCTA GATCTCAC GCTAGGTT GACGACAA CAATCGCT TGTTGCGA TTGTGACC TGTCTGCA CCAATTCC TGTGTACC 22 saouenbes pooleq Supporting Information 94 Name Barcode Name Barcode ame Barcode scars TACocG BCYB TOTCCAAG seme Ticacrec cu Teccaaca nes acrrancc Part 15047264 Rev B BluePippin Size Selection Gel based size selection of ligated products is required to ensure stringent purification of long fragments and the elimination of smaller fragments that can bias the results The BluePippin System can be used as an alternative to the gel based method of size selection of ligated products described in Purify Ligation Products and Size Selection on page 22 The BluePippin system performs pulsed field electrophoresis for resolving and automatically collecting high molecular weight DNA It has the advantage of automated size selection and purification and little risk to cross contamination from sample to sample in the same cassette For detailed instructions on operating the instrument see BluePippin documentation or contact Sage Science y NOTE This procedure requires using 1 ug of genomic DNA normalized to 20 ng ul as input This is to compensate for lower DNA recovery with this alternate protocol than the E Gel based size selection
22. E Gel NGS 0 8 Agarose Loading Layout 232856768 8 1562 E E A A E E Lane M Resuspension Buffer Lane 1 Resuspension Buffer Lane 2 Resuspension Buffer Lane 3 Resuspension Buffer Lane 4 Sample from CLP plate Lane 5 Resuspension Buffer Lane 6 Diluted 1 Kb DNA Extension Ladder Lane 7 Resuspension Buffer Lane 8 Diluted fragmented gDNA Lane 9 Resuspension Buffer Lane 10 Resuspension Buffer 6 Repeat step 5 for each sample loading a single sample into lane 4 of each gel TruSeq Synthetic Long Read DNA Library Prep Guide 2 5 UOH y s 3ZIS pue si9npoJg uorjebr7 Hund Protocol 26 10 11 12 Load 20 ul diluted 1 Kb DNA Extension Ladder into lane 6 of each gel x NOTE Do not overload the DNA ladder Without clear and distinct bands it is difficult to excise the correct fragment size Also an overloaded ladder might run faster than the DNA sample library Load 20 ul diluted fragmented gDNA sample into lane 8 of each gel Load each empty well of each gel with 20 ul Resuspension Buffer lanes M 1 2 3 5 7 9 and 10 On the E Gel iBase Power System select and run the E Gel 0 8 2 program Set the run time to 26 minutes View the gel on a Dark Reader transilluminator Use a lab pen and ruler to mark the 8 10 kb region of interest for precise gel excision as follows a Draw two vertical lines on the plastic gel cassette to mark the left and right side of the sample well
23. FOR RESEARCH USE ONLY ILLUMINA PROPRIETARY Catalog 8 126 9001 Part 8 15047264 Rev B September 2014 This document and its contents are proprietary to Illumina Inc and its affiliates Illumina and are intended solely for the contractual use of its customer in connection with the use of the product s described herein and for no other purpose This document and its contents shall not be used or distributed for any other purpose and or otherwise communicated disclosed or reproduced in any way whatsoever without the prior written consent of Illumina Illumina does not convey any license under its patent trademark copyright or common law rights nor similar rights of any third parties by this document The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order to ensure the proper and safe use of the product s described herein All of the contents of this document must be fully read and understood prior to using such product s FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN MAY RESULT IN DAMAGE TO THE PRODUCT S INJURY TO PERSONS INCLUDING TO USERS OR OTHERS AND DAMAGE TO OTHER PROPERTY ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT S DESCRIBED HEREIN INCLUDING PARTS THEREOF OR SOFTWARE OR ANY USE OF SUCH PRODUCT S OUTSIDE THE SCOPE OF THE EXPRESS WRITTEN LICENSES OR PERMISS
24. Library Prep Guide 3 3 UO e ueno YOdb Protocol 2 Add Resuspension Buffer to the labeled tubes as follows Tube Label Tube Type Resuspension Buffer Volume ul QST 1 100 Dilution Microcentrifuge 495 Std2 Eight tube strip 45 Std3 Eight tube strip 45 Std4 Fight tube strip 45 NTC no template control Fight tube strip 50 Sample name 1 100 Dilution Microcentrifuge 495 Add 5 ul qPCR Standard to the OST 1 100 Dilution tube for a total of 10 pg ul 10 000 fg ul Using a 1000 ul single channel or multichannel pipette gently pipette the entire volume up and down 6 8 times to mix thoroughly Centrifuge the QST 1 100 Dilution tube at 600 x g for 5 seconds Transfer 50 ul from the QST 1 100 Dilution tube to the Std 1 tube in the eight tube strip Change the tip Transfer 5 ul from the 5641 tube to the Std2 tube for a total of 1 pg ul 1000 fg ul Using a 200 ul single channel pipette set to 45 ul gently pipette the entire volume up and down 6 8 times to mix thoroughly Change the tip Transfer 5 ul from the Std2 tube to the Std3 tube for a total for a total of 0 1 pg ul 100 fg ul Using a 200 ul single channel pipette set to 45 ul gently pipette the entire volume up and down 6 8 times to mix thoroughly Change the tip Transfer 5 ul from the Std3 tube to the Std4 tube for a total of 0 01 pg ul 10 fg ul Using a 200 ul single channel pipette set to 45 ul gently pipette the entire volume up and down 6 8 times to mix thorough
25. ONG GTACCGT AACOMACOT ATCATTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATATTAACGTACCATTAAGAGCTACCGTGCAACGA CORABACAA GENY GAIT GAACG TTAAGATTACTTGATCCACTGATT GAACGTACCGTAACGAACGTATCAATT GAGCTTCTG HACC AAGAT TACT IGATCCAG TGA CAAGOTACCGTAACGAACG TA CARTTGAGACTAGGAACGACG AAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATATTAACGTACCATTAAGAGCTACC lllumina 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
26. 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 TruSeq Synthetic Long Read DNA Library Prep Guide V causes of action and losses of any and every kind including without limitation personal injury or death claims and infringement of a third party s intellectual property rights resulting from relating to or arising out of i Purchaser s breach of any of these terms and conditions ii Purchaser s use of this Product outside of the scope of research use purposes iii any use of this
27. of a long qPCR amplicon Successive 10 fold dilutions of the Standard should have Cq values evenly spaced approximately 3 2 cycles apart Pay particular attention to the spacing between Std1 and Std2 and Sample dilutions in this concentration range Examine the amplification plots to make sure that early amplification has not interfered with the automatic baseline determination subtraction on your instrument For more information see your qPCR instrument specific instructions Confirm that the R of the best fit line is gt 0 97 Poor R values can indicate a dilution error in the standard curve or poor amplification of one or more of the standards If so repeat the protocol Use the average of the triplicate data points corresponding 1 100 sample dilution to calculate the concentration of the sample N CAUTION Unexpected results such as delayed amplification no amplification or poor R can be due to the inhibition of qPCR by SYBR Green If you get unexpected results Ilumina recommends diluting the 100x SYBR Green to 50x and repeating qPCR Quantitation 2 If you are using a graphing program to manually calculate sample concentration a Calculate the average Cq value of the qPCR standards and 1 100 dilution of sample from triplicate wells in the QLP plate If one of the three replicates appears to be an outlier it can be omitted from the calculation If more than one of the three replicates appear to be outliers repeat the assay
28. protocol The samples can be stored for up to 30 days Mix the plate thoroughly as follows a Seal the plate with an appropriate adhesive seal for the plate b Shake the plate on a microplate shaker at 1600 rpm for 30 seconds Centrifuge the plate at 280 x g for 1 minute Place the sealed plate on the qPCR instrument Close the lid then and run the instrument as follows a 94 C for 1 minute b 40 cycles of 94 C for 30 seconds 65 C for 30 seconds 68 C for 10 minutes Optional Melting Curve setting suggested by qPCR instrument Remove the plate from the qPCR instrument Assess the quality of the qPCR run and calculate the DNA concentration of your unknown samples using the Cq values from the qPCR run Do one of the following 1 If you are using qPCR instrument software to annotate standards and sample concentration a Calculate the average Cq value of the gPCR standards and 1 100 dilution of sample from triplicate wells in the QLP plate If one of the three replicates appears to be an outlier it can be omitted from the calculation If more than one of the three replicates appear to be outliers repeat the protocol b Use the qPCR instrument software to annotate standards as follows Concentration pg ul Std1 10 Std2 1 Std3 0 1 Std4 0 01 TruSeq Synthetic Long Read DNA Library Prep Guide 37 uonenueno YOdb Protocol Confirm that the gPCR reaction efficiency is 50 100 which is a typical reaction efficiency
29. thermal cycler pre heat lid option and set to 100 C 30 C for 10 minutes Hold at 4 C Apply a CLP barcode label to a new 96 well PCR plate Centrifuge the Long Fragment Adapters tube at 600 x g for 5 seconds Immediately before use remove the Ligation Mix tube from 25 C to 15 C storage Centrifuge the LFP2 plate at 280 x g for 1 minute Remove the adhesive seal from the plate Add 5 ul Long Fragment Adapters to each sample well of the plate Add 2 5 ul Ligation Mix to each sample well of the plate Set a 200 ul pipette to 30 ul then gently pipette the entire volume up and down 10 times to mix thoroughly Return the Ligation Mix tube back to 25 C to 15 C storage immediately after use Seal the plate with a Microseal B adhesive seal then centrifuge the plate at 280 x g for 1 minute TruSeq Synthetic Long Read DNA Library Prep Guide 4 9 s1 depy o1e6rq Protocol Incubate 2 LFP2 1 Place the sealed plate containing 37 5 ul of each sample on the pre programmed thermal cycler Close the lid then select and run the LIG program a Choose the thermal cycler pre heat lid option and set to 100 C b 30 C for 10 minutes Hold at 4 C 2 Remove the plate from the thermal cycler when the program reaches 4 C 3 Centrifuge the plate at 280 x g for 1 minute Clean Up LFP2 1 Remove the adhesive seal from the plate 2 Vortex the Sample Purification Beads for at least 1 minute or until they are well dispersed 3 Add 37
30. 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 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 this Product in any
31. 12 ul of DNA of interest Remove the plate from the magnetic stand Add 30 ul well mixed Sample Purification Beads to each well of the plate Mix thoroughly as follows a Seal the plate with a Microseal B adhesive seal b Shake the plate on a microplate shaker at 1600 rpm for 2 minutes Incubate the plate at room temperature for 5 minutes Part 15047264 Rev B 11 12 13 14 15 16 17 18 19 20 21 22 23 Centrifuge the plate at 280 x g for 1 minute Remove the adhesive seal from the plate Place the plate on the magnetic stand for 5 minutes or until the liquid is clear Remove and discard all of the supernatant from each well of the plate Take care not to disturb the beads F NOTE Leave the plate on the magnetic stand while performing the following 80 EtOH wash steps 15 17 With the plate on the magnetic stand add 200 ul freshly prepared 80 EtOH to each well with a sample without disturbing the beads Incubate the plate at room temperature for 30 seconds and then remove and discard all of the supernatant from each well Take care not to disturb the beads Repeat steps 15 and 16 one time for a total of two 80 EtOH washes Remove and discard any remaining EtOH from each well of the plate with a 10 ul pipette Let the plate stand at room temperature for 5 minutes to dry and then remove the plate from the magnetic stand Resuspend the dried pellet in each well with 32 5 ul Resuspension Buff
32. 5 and 5 487 972 no right to perform any patented method and no right to perform commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration is conveyed expressly by implication or by estoppel This product is for research use only Diagnostic uses under Roche patents require a separate license from Roche Further information on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA Read Before Using this Product This Product and its use and disposition is subject to the following terms and conditions If Purchaser does not agree to these terms and conditions then Purchaser is not authorized by Illumina to use this Product and Purchaser must not use this Product 1 Definitions Application Specific IP means Illumina owned or controlled intellectual property rights that pertain to this Product and use thereof only with regard to specific field s or specific application s Application Specific IP excludes all Illumina owned or controlled intellectual property that cover aspects or features of this Product or use thereof that are common to this Product in all possible applications and all possible fields of use the Core IP Application Specific IP and Core IP are separate non overlapping subsets of all Illumina owned or controlled intellectual
33. 5 ul well mixed Sample Purification Beads to each sample well of the plate Set a 200 ul pipette to 65 ul and then gently pipette the entire volume up and down 10 times to mix thoroughly 4 Incubate the plate at room temperature for 5 minutes 5 Place the plate on the magnetic stand for 5 minutes or until the liquid is clear 6 Remove and discard 70 ul of the supernatant from each well of the plate Take care not to disturb the beads i NOTE Leave the plate on the magnetic stand while performing the following steps 7 12 7 With the plate on the magnetic stand add 200 ul freshly prepared 80 EtOH to each sample well without disturbing the beads 8 Incubate the plate at room temperature for 30 seconds and then remove and discard all of the supernatant from each well Take care not to disturb the beads 9 Repeat steps 7 and 8 one time for a total of two 80 EtOH washes 10 Remove and discard any remaining EtOH from each well of the plate with a 10 ul 20 pipette Part 15047264 Rev B 11 12 13 14 15 16 17 With the plate on the magnetic stand let the samples air dry at room temperature for 5 minutes With the plate on the magnetic stand add 22 5 ul Resuspension Buffer to each sample well of the plate Make sure the Resuspension Buffer runs over the beads Remove the plate from the magnetic stand Resuspend the beads in each well of the plate by repeatedly dispensing the Resuspension Buffer over the bead pelle
34. 6 8 times to mix thoroughly Change the tip Transfer 20 ul from the GST2 tube to the GST3 tube for a total of 0 025 ng ul Gently pipette the entire volume up and down 6 8 times to mix thoroughly Change the tip Transfer 20 ul from the GST3 tube to the GST4 tube for a total of 0 0125 ng ul Gently pipette the entire volume up and down 6 8 times to mix thoroughly Centrifuge the LAP plate at 500 x g for 1 minute Using 22 1 2 gauge needle carefully pierce a hole in the plate seal above four randomly selected wells of the plate Transfer 5 ul from each of four randomly selected plate wells to pool in one well of an eight tube strip Note which wells were selected from the plate y NOTE Select samples from across the entire plate and not confined to the perimeter or to a single region of the plate e Avoid selecting corner wells Sample LAP Plate Well 1 2 3 4 Place the LAP plate on ice or store the plate at 2 C to 8 C for up to 24 hours or until this Long Range PCR procedure is complete Cap the eight tube strip that contains the pooled samples briefly centrifuge the strip at 500 x g Add 0 5 ul 2 Log DNA Ladder and 19 5 ul Resuspension Buffer to the tube labeled 2 Log Ladder to dilute Gently pipette the entire volume up and down 6 8 times to mix thoroughly TruSeq Synthetic Long Read DNA Library Prep Guide ebuey 6u07 Protocol 48 NOTE Reference Figure 12 vvhile performing steps 12 19
35. A Library Quantification Kit Illumina Universal TruSeq Synthetic Long Read DNA Library Prep Guide Supplier NEB part N3200L General lab supplier General lab supplier General lab supplier General lab supplier E amp K Scientific part 480096 Eppendorf part 951020303 Fisher Scientific part AB 0859 VWR part 10011 194 or 47744 810 VWR part 10011 006 General lab supplier Zymo Research part D4003 1 25 Zymo Research part D4003 2 24 Invitrogen catalog G4020 01 Invitrogen catalog A25798 Sigma Aldrich part E7023 Covaris part 520079 General lab supplier KAPA Biosystems part KK4824 83 juaudinby pue sejqeuunsuoy Supporting Information 84 Consumable Lab pen Lab tissue low lint Microcentrifuge Tubes with Attached Flat Caps Neptune 1 6 ml Microseal B adhesive seals Needles 22 1 2 gauge PCR grade water QIAquick Gel Extraction Kit qPCR plate and seal Qubit dsDNA BR Assay Kit Qubit dsDNA HS Assay Kit RNase DNase free eight tube strips and caps RNase DNase free multichannel reagent reservoirs disposable RNaseZap to decontaminate surfaces ROX Reference Dye Ruler SYBR Green Nucleic Acid Gel Stain x tracta gel extractor Supplier General lab supplier VWR part 21905 026 or equivalent VWR part 89126 722 Bio Rad part MSB 1001 General lab supplier General lab supplier QIAGEN part 28704 General lab supplier Life Te
36. AANTALT AACGTACTTAACCTIAAGAT TACT TGATCCACTGAT T CAACGTACCGTAACGAACGTCTTCTGTTAACCTTAAGAT Ti KETIGMCCACIGATTCAACGT ACCGTAACGAACGTATCAAT TGAGACTAACGACGA SATAACAGTAACAGAG LT CTGLIAACG T TAAGA TACT GAT GOAGTGAT GAACGIAGGG TARO GAO O TALCA TGAGAG AAATAT TAACG ACCA TAAGAGCTACGGCT TCT GTTAAGG TANGATIACT IGATCUACT GAT IGAACG GATAACAGTAACACACTT AACCTIAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTA TTCTG CCTTAAGATTACT TGATCCACTGA ACCATTAAGAGCTACCGTGCAACTTAACCTTAAGATTACTTGATCCACTGATTICAACGTACCGTAACGAACGTATCAATTGAGACTAAATAT TAACGTACCATTAAGAGCTACCGTGCAACGACGAACTICTGTTAACCTTAAGATTACTIGAT TACOS TGCAACGAAAATAACC TTAAGATTACT IGATCCACTGAT ICAACGTAGTICTG TAAGGTTAAGATTACT IGATCCACT GAL CAACGACOGTAACGAACG TAI GAAT TGAGAG TAAGGAGCGIGCAACGACGAAAAGAATAT AAAAGAATGA SCA AAAGATTACTTGATI ACTGATTCAACGTACCGTAACGAACGTA TT GAGAC TAAATAT TAACGTACCAT TAAGAGCTACC PORT OCACTGALICAACGT ACTA TCAAT TGAGACTAAATAT TAACGT AC CATAAGAGCTACCGTOCAA AOGA AMAA AA GA TAACAGTAACACACTICTGTI CCATTAAGAGCTACEGT SC AACAGTAACACAC E TGTOT IAACCTTAAGATTACT GATCCAG GAT GRACG TACOS IAACGAACG IAT CAAT GAGAGTAAATAT TAACGTACGAI TAAGAGCTACCG I GCAACGACGARAAGAAT SATAN GSATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT T GAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTCTTCT AAGAT T BOT GAT RG an
37. ACACACTTCTGTIAACCTIAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATATTAACGTACCATTAAGAGCTACCGTCTTCTGTTAACCT TAAGATTACTTGATCCACTGATTCAACGTA VAATTGAGACTAAATAT TAACGTTGTTAACCTTAAGAT TACT TGATCCACTGATTCAACGTACCGTAACGAACGTAT CAAT T GAGACTAAATAT TAACGTACCAT TAAGAGCT TCTGT TAACCT TAAGAT TACTT GATCCACT GAT TCAACGTACCGTAAC TREAT ANTATT OT a renee LET CAAS Tce Greve NN ATE TCAE A ae GAGACTAAATAT TAACGTACCAT TAAGAGCTACAACCTTAAGA 125755 577 TCAATTGAGACTAAATATTAACGTACCAT TAAGAGCTACCGTGCAACGACGAAAAGAAT GA 1277 AATGATAACAGTAACACACT TCTGTTAACCTTAAGATTACI TGATCCACTGATTCAACGTACCGTAACGAACGTA TAAG G IACRATTRAGAGG TACCOTOTICTO TAAGO OA AGAR AACG GTACCAT TAAGAGCTACCGTGCAACTTIAACCTIAAGAT TACTT GATCCACT GATT CAACGTACCGTAACGAACGTATCAATT GAGACTAAATATI ALTA CA ACI TAAGATACTIGATGC 2 175755 AACCTI 1607700 CTI 25 G ACGAAAAGAATGATAACAGTAACACACTTCTGT TAACCTT AATGATAACAGTAACACACI TCTGTTAACCTTAA TACTO GALT CAACGTACCOTAACGAACG TAT CANT GAGAG TA a 2650057 AE E ES A AATGATAACAGTAACACACT TCTGTTAACCTT TTGATCCACTGATTCAACGTACCGTAACGAA TCAATTGAGACTA MA A ARCSAACOTATCATIAAGATIACTGATOCAGTOATICAACGTA COTA AAC TAT QATI GA AN CTAAATAT T ITACT TGATCCACTGATTCAACGTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTAT CAATTGAGCTTCTGTTAAC ACGAAAAGAATGATAACAGTAACACACTTCTGTTAACCT TAAGAT TACTT GATCCACTGATT CAACGTACCGTAAAGAT TACTTGATC
38. ATCCACT GATT CAACGTACCG TAACGAACGTAT CAAT T GAGA CTAAATALTAACGTACCAT AAGAGCTACCGTCITCT GI TACK IAAGATIACT Gal CCACIGATTCAACG TA a eee ACCGTAACGAACGTATCAAT T GAGACTAAATAT TAACGTACCAT TAAGAGC TACCGT GCAACGACGAAAAGAAT GATAACAGTAACACACT TCTGTTAACCTT ACGAAAAGAAT GATAACAGTAACACACTTCTGTTAACCT TAAGAT TACTT GAT CCACTGAT TCAACGTACCGTAAAGAT TACT TGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACG TKCGATTAAGAG SACO AG Ae TUT TAG TC AC er TA her AAG AGG TAC AATGATAACAGTAACACACT TCTGTTAACCTTAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAGC TACCGTCTTCTGTTAACCTTAAGAT TACTTGATCCACTGATTCAACGTA ITACTTGATCCACTGAT TCAACGTTAAGAT TACTT GAT CCACT GATT CAACGTACCGTAACGAACGTATCAATT GAGCT TCTGT TAACCTTAAGAT TACTT GATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TGAGACTAGCAACGACGAA ACC C G AACGACGAAAAGAATGAT MIACTTOMCCACTEATTCAACGTTAAGALTACTTGATCCACT GATTCAAGGTACCGTAACGAACGTATCAATIGAGCHICTOLL AACCTTAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAGCAACGACGAA Me CAG AEE eee eee A eae ELA MS ced TAAA sO Ue Ua meal AAT GATAACAGTAACACACTTCTGTIAACCT TAAGAT TA 22777 CTAAATAT TAACGTACCAT TAAGAGCTACCGTCTTCTGTTAACCTIAAGAT TACT TGATCCACTGAT TCAACGTACCGTAA TCCACTGAT T CAACGTACCAAGAT TACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAATT GAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTCTTCTGTTAACCTTAAGAT TACTTGAT CCACTGAT TCAACGTACCGTAACGAAC CORO A RA DO TENRA AE ATA TS ALCS ECA RITA AATGATAACAGTA
39. Appendix A Supporting Information before proceeding to confirm your kit contents and make sure that you have obtained all of the requisite consumables and equipment 8 Part 15047264 Rev B Library Prep Workflow The following figure illustrates the processes of the TruSeq Synthetic Long Read DNA Library Prep protocol Figure 2 TruSeq Synthetic Long Read DNA Library Prep Workflow Day 1 Purified Genomic DNA Purify Ligation Products and Size Selection Day 3 Tagmentation Consumables Fragment DNA tele Fragmentation plate Consumable Eo FPM Qubit dsDNA BR or HS Assay Kit Eeprepahel TDE QlAquick Gel Extraction Kit Plate Tube SB LAP gDNA 4200 x g Pit late CLP di Indexing PCR k 4 y Consumables Perform End Repair 4 IDP i il Plate Consumables Validate Library 7 EtOH ERP Consumable RSB Optional High Sensitivity DNA Kit v SPB Qubit dsDNA HS Assay Kit u Plates E Pool and Concentrate LFP2 na qPCR Quantitation Consumables Consumables RSB DMSO SNP 4 MasterAmp Zymo DNA Binding Buffer OMM Zyma DNA Wash Buffer a QPM Adenylate 3 Ends QST Plate ROX PAP Consumable RSB ATL SYBR Green i Plate Plate LFP2 QLP 4 Size Selection Day 2 Ligate Adapters y Consumables EtOH Consumabli RSB EtOH Long Range PCR SPP LAD UG Consumables Plate RSB 2 log ladder FSP SPB E Gel GST Plate LMM CLP LPM 4 MasterAmp v RSB 4 SYBR Green y Validate Final Product Plate WAP Consumable Library Prep Y O
40. C DNA Polymerase Mix i RSB 15047457 Resuspension Buffer 25 C to 15 C 1 Sample Neutralization 2 C to 8 C Buffer 1 25 C to 15 C Barcode Kit 1 Sample Box C Store at room temperature This box is shipped at room temperature As soon as you receive it store the components at room temperature Figure 23 TruSeq Synthetic Long Read DNA Barcode Kit 1 Sample Box C part 15048202 Description 15049001 Collection Plate 15044207 Fragmentation Plate TruSeq Synthetic Long Read DNA Barcode Kit 4 Samples The TruSeq Synthetic Long Read DNA Barcode Kit 4 Samples contains one Box A four Box Bs and one Box C T 8 Part 15047264 Rev B Barcode Kit 4 Samples Box A Store at 2 C to 8 C This box is shipped at room temperature As soon as you receive it store the components at 2 C to 8 C This box also contains plate barcode labels TruSeq Synthetic Long Read DNA Barcode Kit 4 Samples Box A part 15051788 Quantity Reagent Part Description 4 SPB 15044759 Sample Purification Beads Barcode Kit Box B Store as specified This box is shipped on dry ice As soon as you receive it store the components as specified Figure 24 TruSeq Synthetic Long Read DNA Barcode Kit Box B part 15048203 IDP Packaged Assembly TruSeq Synthetic Long Read DNA Library Prep Guide F Q s u U09 11 Supporting Information Quantity Reagent Part Description Storage Temperature 1 25 C to
41. E STOPPING POINT 1 If you do not plan to proceed immediately to Adenylate 3 Ends on page 16 you can safely stop the protocol here If you are stopping seal the LFP2 plate with a Microseal B adhesive seal and store at 25 C to 15 C for up to 7 days TruSeq Synthetic Long Read DNA Library Prep Guide 1 5 puy wooed Protocol Adenylate 3 Ends A single A nucleotide is added to the 3 ends of the blunt fragments to prevent them from ligating to one another during the adapter ligation reaction A corresponding single nucleotide on the 3 end of the adapter provides a complementary overhang for ligating the adapter to the fragment This strategy ensures a low rate of chimera concatenated template formation Consumables Item Quantity Storage TruSeq Synthetic Long Read DNA Library Prep Kit contents A Tailing Mix ATL 1 tube per 4 reactions 25 C to 15 C Ice bucket As needed 25 C to 15 C Microseal B adhesive seal 1 15 fo BSOXC Preparation Prepare an ice bucket Supplied By Illumina User User Remove the A Tailing Mix from 25 C to 15 C storage and thaw it at room temperature Place the tube on ice Remove the LFP2 plate from 2 C to 8 C storage if it was stored at the conclusion of Perform End Repair on page 12 Let the LFP2 plate stand at room temperature Centrifuge the LFP2 plate at 280 x g for 1 minute Remove the adhesive seal from the LFP2 plate
42. GAT TACTTGATCCACT GATTCAACGTACCGTAACGAACGTATCAAT TGAGCTTCTGTTAACCTTAAGAT TACTTGATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT T GAGAC TAGCAACGACGi O OPT VST CAT TAANE EET AA ON Galt ore aL GTACCGTAACGAACGTATCAT TAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAATTGAGA 1200755 ACCGTGCAACGACGAAAAGAATGATAACA G TAACACACTTCTOTTAACCTTA STTGATCCACTGATTCAACGT TAAGAT TACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGCTTCTGTTAACCTTAAGAT TACT TGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGACTAGCAACGACG iAAAAGAAT GATAACAGTAACACACTTCTGT TAACCTTAAGAT TACTT GATCCACT GATT CAACGTACCGTAAAGAT TACTTGATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACC SATAACAGTAACACACT TCTGTTAACCT TAAGAT TA A ee 217075 SACTGATTCAACGTACCAAGATTACTT GATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT T GAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTCTTCTGT TAACCTTAAGAT TACTTGATCCACTGATTCAACGTACCGTAACGA AAAAGANTGATAACAGTAAGAGAGTIGTGY IAACCLTAAGATIACT TGATCGACT GAACGTAGCG AAAGAT TACT EGATOCAC GAT ICAAGGTACCGTAACGAACGIATCAAI TGAGAC TAAATATTAACGTAGCATTAAGAGC TAGC 17 7 75 2 152555 00 0 Sp GATTACTTGATCCACTGATTCAACG TGAGACTAAATAT TAACGTTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATA LU ae aa ATCAATTGAGA CT
43. IONS GRANTED BY ILLUMINA IN CONNECTION WITH CUSTOMER S ACQUISITION OF SUCH PRODUCT S FOR RESEARCH USE ONLY 2014 Illumina Inc All rights reserved Illumina 24sure BaseSpace BeadArray BlueFish BlueFuse BlueGnome cBot CSPro CytoChip DesignStudio Epicentre GAIIx Genetic Energy Genome Analyzer GenomeStudio GoldenGate HiScan HiSeq HiSeq X Infinium iScan iSelect ForenSeq MiSeq MiSeqDx MiSeq FGx NeoPrep Nextera NextBio NextSeq Powered by Illumina SeqMonitor SureMDA TruGenome TruSeq TruSight Understand Your Genome UYG VeraCode verifi VeriSeq the pumpkin orange color and the streaming bases design are trademarks of Illumina Inc and or its affiliate s in the U S and or other countries All other names logos and other trademarks are the property of their respective owners MasterAmp is a trademark of Epicentre Madison Wisconsin Notice to Purchasers Limited License Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US 5 079 352 5 789 224 5 618 711 6 127 155 and claims outside the US corresponding to US Patent No 4 889 818 The purchase of this product includes a limited non transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own internal research No right under any other patent claim such as the patented 5 Nuclease Process claims in US Patents Nos 5 210 01
44. Product not in accordance with this Product s 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 15047264 Rev B Revision History Part Revisio
45. 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 3 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 4 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 engineer reverse compile or reverse assemble the Product ii s
46. T HOd Protocol 58 Pooland Concentrate Consumables Item TruSeq Synthetic Long Read DNA Barcode Kit contents Resuspension Buffer RSB e Sample Neutralization Buffer SNB Collection plate e PAP Pooled Amplicon Plate barcode label 50 ml conical tube 96 well MIDI plate Microcentrifuge tube Microseal B adhesive seal RNase DNase free eight tube strip and caps Zymo DNA Binding Buffer Zymo DNA Wash Buffer with ethanol added Zymo Spin V Column with Reservoir Quantity 1 tube 1 tube per 1 reaction 1 per sample 1 label per plate 1 per sample 1 2 per sample 1 1 per sample Storage 216 2 C to 8 C E o SAS 15 C to 30 C AC O SAS 15 C to 30 C ISS tro SUE 15 C to 30 C IDEELE 15 C to 30 C IPE wE 15 C to 30 C This process collects and concentrates the PCR amplified and indexed DNA from the 384 well plate into a single sample Supplied By Illumina Ilumina Ilumina Ilumina ser ser ser ser an Chen C ES ser G ser User User Part 15047264 Rev B NOTE This procedure is described using a 96 well MIDI plate However a microcentrifuge tube can be used instead of the plate Preparation Remove the following from 2 C to 8 C storage and bring it to room temperature Resuspension Buffer ja Sample Neutralization Buffer Apply a PAP barcode label to a new 96 well MIDI plate La
47. TAACCTTAAGAT TACT TGATCCACTGATT CAACGTACCGTA ee A ee A A AA AHA Ue a Aa iAAAAGAAT GATAACAGTAACACACTTCTGTTAACCTTIAAGAT TACTT GATCCACTGAT TCAACGTACCGTAAAGAT TACT TGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACC SATAACAGTAACACACT TCTGTTAACCTIAAGAT TACT TGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTCTTCTGTTAACCT TAAGATTACTTGATCCACTGAT TCAACG TGAGACTAAATAT TAACGTT GTTAACCTTIAAGAT TACT TGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT T GAGACTAAATAT TAACGTACCAT TAAGAGCT TCTGT TAACCT TAAGAT TACTT GATCCACTGAT TCAACGTACCGTA ATCAAT TGAGACTAAATAT TAACGTACTIAACCTIAAGAT TACT TGATCCACTGATT CAACGTACCGTAACGAACGTCTTCTGTTAACCTIAAGAT TACTT GATCCACTGATTCAACGTACCGTAACGAACGTAT CAAT TGAGACTAACGACGA SAC TAAATAT TAACGTACCAT TAAGAGCTACAACCT TAAGAT TACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGC TACCGTGCAACGACGAAAAGAAT GATAACAGTAACACA CCA C A TTGAGACTAAATAT TAACG A AAI CGAACTTCTGT TAA aCTACCGTGCAACGAAAATAACCTIAAGATTIACTTGATCCACTGATTCAACGTACTTCTGTTAACCTIAAGAT TACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATT GAGACTAAGCTACCGTGCAACGACGAAAAGAAT GAT eee ee eee eee ACA Ts A A HA CN Ae Ana TA WN Feet at iets 3ATAACAGTAACACACT TCTGTTAACCTT 25725 2 55 72 TCAATTGAGACT 27751 175757771 1 CCAT TAAGAGC TACCGTGCAACAGTAACACACT TCTGT TAACCTIAAGAT TACT TGATCCAC
48. TGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGT GCAACGACGAAAAGAAT GATAAC SATAACAGTAACACACT TCTGTTAACCT TAAGATTACTTGATCCACT GAT TCAACGTACCGTAACGAACGTAT CAAT T GAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTCT TCTGTTAACCTIAAGATTACTT GATCCACTGATTCAACG GTACCGT 7577 77575 5 EL a aces 22577 TTAAGAGC TACCGTGCAACGACGAAAAGAATGATAACAGTAACACACTICTGTTAACCTTA NE a Lae CGTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGCTICTGTIAACCTTY A A ORTA TADA TCAATTGAGACTAGCAACGACG ORAARARI MAACAGTAACACACT IGGL IAACOTY MARA ee ee eee ew UN roe NAG gla AN Moyle Near GTAACGAACGTATCAAT TGAGACTAAATATTAACGTACCAT TAAGAGCTACC SATAA AQTRACACAC TTCTGTTAACCTTAA AT TACTTGATCCACTGATTCAACGTACCG TAACGAACGTATCAAT T GAGACTAAA AN KAGAGC TACCGTCTTCTGTIAACCTIAAGATIACTTGA ee AW a STACCGTAACGAACGIA MV CATTAAGAT TACTTGATCCACT GATT CAACGTACCGTAACGAACG TAT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTGCAACGACGAAAAGAAT GATAACAGTAACACACTTCTGTTAACCTT 1AAAAGAATGATAACAGTAACACACT TCTGTTAACCTTAAGAT TACT TGATCCACTGATT CAACGTACCGTAAAGAT TACT Ti GATCCACTGAT I GAACCTACCGTAACGAACGTAT CAME GAGACTAAATALTAASGIRCCATIAAGAGK ACG CCAT TAAGAGCTACCGTGCAACAGTAACACACTTCTGT TAACCT TAAGAT TACTTGATCCACTGAT T CAACGTACCGTAACGAACGTAT CAATTGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGT GCAACGACGAAAAGAAT GATAA GATAACAGTAACACACTTCTGTTAACCTIAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATATTAACGTACCAT TAAGAGCTACCGTCTICTGTTAACCTTAAGAT TACTTGATCCACTGATTCAACG 2TTGATCCACTGAT TCAACGT TAA
49. TY 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 expirati
50. User supplied Consumables Item Quantity Storage 0 75 Agarose gel cassette low range S1 1 per 4 samples 15 C to 30 C TE buffer 10 ul per sample 15 C to 30 C Lab pen 1 15 C to 30 C Microcentrifuge tubes 1 per sample LHC to NAC Preparation Remove the CLP plate from 2 C to 8 C storage if it was stored at the conclusion of Clean Up LFP2 on page 20 Let the plate stand to bring it to room temperature Centrifuge the plate at 280 x g for 1 minute Remove the adhesive seal from the plate Bring the loading solution that comes with the cassette to room temperature Prepare the BluePippin instrument and cassette according to manufacturer instructions TruSeq Synthetic Long Read DNA Library Prep Guide 9 5 4011091 zis ulddid niq Supporting Information Procedure 96 DD 0 A Q Use BluePippin software v 6 0 with cassette definition 12 or higher Pre program the BluePippin with the following program and save as HF 7 11kb Lane 1 S1 Marker Option Setting Run Time 8 00 hr Ref Lane il BP Target 9000 bp BP Start 7000 bp BP End 11000 bp BP Pause 0 BP Range Flag broad Apply references to all lanes End the run when elution is complete Select the 0 75 DF 3 10kb Marker S1 Improved Recovery cassette Label one new microcentrifuge tube for each sample with size selected sample name using a smudge resistant pen Add 10 ul TE buffer to each well of the CLP plate Add 10 ul loading solution to eac
51. ample for x in the equation Calculate the concentration of each 1 100 dilution of sample in pg ul using the following equation where Concentration pg ul 2 y Figure 8 Example qPCR Standard Curve qPCR Standard Curve y 0 9354x 12 431 8 5 8 2 8 N 4 2 8 6 B s Sample average Cq 14 6 y 0 935 x 14 6 12 431 1 226 Concentration of 1 100 dilution of sample 2 1 226 0 428 pg ul N CAUTION Unexpected results such as delayed amplification no amplification or poor R can be due to the inhibition of qPCR by SYBR Green If you get unexpected results Ilumina recommends diluting the 100x SYBR Green to 50x and repeating qPCR Quantitation TruSeq Synthetic Long Read DNA Library Prep Guide 3 9 YOdb Protocol Long Range PCR 40 This process enriches long DNA fragments with the appropriate adapters The PCR starting material is diluted in a 384 well plate to limit the number of molecules in each well which enables downstream data analysis applications The PCR amplified material is subject to gel quality control to make sure that the material is not over or under amplified Figure 9 Long Range PCR Workflow Dilute Template Prepare Master Mix Long Amp Bun Quality Control Gel Quality Control Consumables Hem Quantity Storage TruSeq Synthetic Long Read DNA Barcode Kit contents Gel Standard GST 1 tube 25 C to
52. are stopping seal the PAP plate with a Microseal B adhesive seal Store the plate at 25 C to 15 C for up to 7 days or at 2 C to 8 C for up to 24 hours 6 2 Part 15047264 Rev B Size Selection This process removes adapter dimers and DNA fragments that are either too small or too large selecting for tagmented DNA in the optimal range for cluster formation Consumables Item Quantity Storage Supplied By TruSeq Synthetic Long Read DNA Barcode Kit contents e Resuspension Buffer RSB 1 tube 2 C to 8 C Illumina e Sample Purification Beads 1 tube per 24 2 C to 8 C Ilumina SPB reactions FSP Final Sample Plate 1 label per plate AC O SAS Illumina barcode label 96 well MIDI plate 1 15 C to 30 C User Freshly prepared 80 ethanol 400 ul per sample MAE to SOE User EtOH Microseal B adhesive seals 3 15 C to 30 C User 4 This procedure is described using a 96 well MIDI plate However a microcentrifuge tube or 96 well PCR plate can be used instead of the 96 well MIDI plate with a corresponding magnet Preparation Remove the PAP plate from 25 C to 15 C storage and thaw at room temperature or from 2 C to 8 C storage and let stand at room temperature if it was stored at the conclusion of Pool and Concentrate on page 58 Centrifuge the PAP plate at 280 x g for 1 minute Remove the adhesive seal from the PAP plate TruSeq Synthetic Long Read DNA Library Prep Guide 6 3 UOIJODBJ9S 9ZIS Proto
53. ations Additional Resources Library Prep VVorkflovv Ligate Adapters Purify Ligation Products and Size Selection Pool and Concentrate Consumables and Equipment Barcode Sequences BluePippin Size Selection Calibrate Diluted SYBR Green Technical Assistance Part 15047264 Rev B Overview L1a1deyo IAUOGUCTION AA A A IA 2 DNA Input Recommendations 3 AddlitionallReSQUICES evn gee bac asa 5 t Ga 4 Sheet 4 YO V zoe gt A a m o R amil x o 2 AECA TEA rOGASIEGICA 00 4 E oy x ponen 7 er TruSeq Synthetic Long Read DNA Library Prep Guide 1 Overview Introduction This protocol explains how to prepare up to four libraries of gnomic DNA gDNA using the reagents provided in the Illumina TruSeq Synthetic Long Read DNA Library Prep Kit and TruSeq Synthetic Long Read DNA Barcode Kit The libraries ar
54. before running the qPCR select only the wells with samples If the entire plate is selected the resulting file is too large to open by the SDS software For more information contact Life Technologies TruSeq Synthetic Long Read DNA Library Prep Guide 87 juaudinby pue sejqeuunsuoy Supporting Information Barcode Sequences TruSeq Synthetic Long Read DNA Barcode Kits contain the following the barcodes Name Barcode Name Barcode Name Barcode BC001 TGAACGTG BC020 TCCTTGGT BC039 TGCGCTAT BC002 BC021 BC040 BC003 TGTGAGGT 022 CIGAGGAT BC041 BC004 BC023 BC042 BC005 TGGATCAG BC024 TIGICCAG BC043 GITGTTCC BC006 BC025 BC044 CCAGAACA BC007 TAAGGAGC BC026 GCCATTCA BC045 TIGCATGG BC008 BC027 CTATGGAC BC046 CTACCGTT BC009 CCATCTIC BC028 CCTAGTTG BC047 GACGTAAG BC010 BC029 TCCAACGT BC048 BC011 GICTIGAC BC030 TTACGGCT BC049 BC012 BCO31 CGATICGT BC050 TCAATCCG BC013 GCAGTAGA BC032 BC051 CCGGATTA BC014 TAAGGTCG BC033 1 CTTGGTTC BC052 CGGTATTG BC015 TTCCAAGC BC034 CGAACTGA BC053 GGATTACC BC016 BC035 CGGATACA BC054 TGAGGACT BC017 GATCTGGT BC036 GACTAACC BC055 GIGTCGAA BCO18 GAGAACCT BC037
55. bel a new eight tube strip QC1 Pre Size Selection with a smudge resistant pen Procedure 1 Centrifuge the LAP plate at 500 x g for 1 minute 2 Remove the adhesive seal from the LAP plate then attach the collection plate to the LAP plate so that the LAP plate is covered with the collection plate Figure 15 Collection Plate Attached to LAP Plate TruSeq Synthetic Long Read DNA Library Prep Guide 5 9 2 pue OO 4 3 Invert the attached collection and LAP plates so that the sample plate wells face down into the collection plate Figure 16 Invert Collection Plate and LAP Plate Protocol 4 Centrifuge the collection and LAP plates with a balance to 500 x g for 30 seconds 5 Set up a master mix in a new sterile nuclease free 50 ml conical tube using the following Reagent Volume All of the pooled library from the collection plate 4 5 ml Sample Neutralization Buffer 200 ul Zymo DNA Binding Buffer 20 ml Total Volume 24 5 ml 6 Cap the master mix tube and invert the tube several times to mix 6 O Part 15047264 Rev B 7 8 9 14 15 Set up a Zymo Spin V column with reservoir on a vacuum manifold Figure 17 Zymo Spin V Column with Reservoir on Vacuum Manifold Turn on the vacuum and leave it on Add 12 ml master mix to the Zymo Spin V column NOTE See manufacturer instructions for recommendations for syringe or centrifuge base purification Run the master mix through the vacuum
56. cap the eight tube strip TruSeq Synthetic Long Read DNA Library Prep Guide A 5 H d ebuey 6u07 Protocol 3 Place the sealed plate or capped tube on the 96 well thermal cycler Close the lid then select and run the Phasing20QC or LongRead26QC program depending on the workflow Workflow Phasing Long Read Program Name Phasing20QC LongRead26QC Program e Choose the thermal Choose the thermal cycler pre heat lid cycler pre heat lid option and set to option and set to 95 C 100 C 95 C 100 C 94 C for 1 minute 94 C for 1 minute 20 cycles of e 26 cycles of 94 C for 30 94 C for 30 seconds seconds 65 C for 30 65 C for 30 seconds seconds 68 C for 10 e 68 C for 10 minutes minutes e 68 C for 10 minutes e 68 C for 10 minutes Hold at 4 C Hold at 4 C Hours to 4 5 5 Complete 4 Remove the LAP plate and 96 well PCR plate or eight tube strip from both thermal cyclers and place them on ice Gel Quality Control 1 Add Resuspension Buffer to the labeled microcentrifuge tubes as follows Tube Resuspension Buffer Volume ul GST1 36 GST2 20 GST3 20 GST4 20 4 6 Part 15047264 Rev B 10 11 Add 4 ul undiluted Gel Standard to the GST1 tube for a total of 0 1 ng ul Gently pipette the entire volume up and down 6 8 times to mix thoroughly Change the tip Transfer 20 ul from the GST1 tube to the GST2 tube for a total of 0 05 ng ul Gently pipette the entire volume up and down
57. cation is probably inaccurate for the long fragment To ensure optimal tagmentation performance reevaluate the qPCR Quantitation and repeat Long Range PCR using the correct dilution TruSeq Synthetic Long Read DNA Library Prep Guide A 9 H d ebuey 6u07 Protocol DO Figure 11 Gel Quality Control A Pooled sample B QC sample SAFE STOPPING POINT If you do not plan to proceed immediately to Tagmentation on page 51 you can safely stop the protocol here If you are stopping seal the LAP plate with a Microseal B adhesive seal and store at 2 C to 8 C for up to 24 hours Part 15047264 Rev B Tagmentation This process tagments tags and fragments PCR amplified long DNA fragments by adding the Nextera transposome to the 384 well plate The Nextera transposome simultaneously fragments the genomic DNA and adds adapter sequences to the ends allowing for amplification by PCR in subsequent procedures Consumables Item Quantity Storage Supplied By TruSeq Synthetic Long Read DNA Barcode Kit contents e Fragmentation plate 1 15 olsu Illumina e Fragmentation Pre Mix 1 tube per LAP plate 25 C to 15 C Ilumina FPM Tagment DNA Enzyme 1 tube per LAP plate 25 to 155G Illumina TDE Ice bucket As needed 25 C to 15 C User Microcentrifuge tube l IBC o HOKE User Microseal B adhesive seals 2 15 C to 30 C User Preparation Prepare an ice bucket Remove the Fragmentation Pre Mix from 25 C t
58. chnologies catalog Q32850 Life Technologies catalog Q32851 General lab supplier VWR part 89094 658 General lab supplier Invitrogen part 12223012 General lab supplier Invitrogen part S7585 USA Scientific catalog 5454 0100 Part 15047264 Rev B Consumable Zymo Spin V with Reservoir Optional for BluePippin size selection 0 75 Agarose gel cassettes Dye Free Low Range 10 pk S1 Optional for BluePippin size selection TE buffer Table 5 User Supplied Equipment Equipment 96 well thermal cycler with heated lid 384 well thermal cycler 2100 Bioanalyzer Desktop System Agilent High Sensitivity DNA Kit Dark reader transilluminator E Gel iBase Power System High Speed Microplate Shaker Magnetic stand 96 Microplate centrifuge Novex Gel Knife TruSeq Synthetic Long Read DNA Library Prep Guide Supplier Zymo Research part C1016 25 or C1016 50 Sage Science catalog BLF7510 General lab supplier Supplier Bio Rad part ALS 1296G or equivalent Bio Rad part 185 1138 or equivalent Agilent part G2940CA Agilent part 5067 4626 Clare Chemical Research part DR195M Life Technologies catalog G6400 VWR catalog 13500 890 110 V 120 V VWR catalog 14216 214 230 V Life Technologies catalog AM10027 General lab supplier Life Technologies catalog EI9010 85 juaudinby pue sejqeuunsuoy Supporting Information 86 Equip
59. cluster generation section of the user guide for your Illumina platform Store the sealed FSP plate at 25 C to 15 C TruSeq Synthetic Long Read DNA Library Prep Guide 6 9 J9NPOlAd 201 TEP EA Part 15047264 Rev B Supporting Information Introduction Acronyms Kit Contents Consumables and Equipment Barcode Sequences BluePippin Size Selection Calibrate Diluted SYBR Green rosa 0 f Axa 3 WAL a b E SAY AN E Nro 20 7 o A a 42 7 oe TruSeq Synthetic Long Read DNA Library Prep Guide y 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 T 2 Part 15047264 Rev B Acronyms Table 2 TruSeq Synthetic Long Read DNA Library Prep Acronyms Acronym Definition ATL A Tailing Mix Dimethyl sulfoxide Experienced User Card Final Sample Plate GST Gel Standard LAD Long Fragment Adapter Long Fragment Plate Ligation Mix Long amp Primer Mix TruSeq Synthetic Long Read DNA Library Prep Guide 7 3 sw uoIoy Supporting Information 74 Acronym PAP EGE QLP QMM QPM QST RSB SNB SPB Definition Pooled Amplicon Plate Polymerase Chain Reaction Quanti
60. col Procedure 10 64 Review best practices for handling magnetic beads See Additional Resources on page 5 for information about TruSeq Synthetic Long Read DNA Library Prep best practices on the Illumina website Remove the Sample Purification Beads and Resuspension Buffer from 2 C to 8 C storage and bring them to room temperature Apply an FSP barcode label to a new 96 well MIDI plate Vortex the Sample Purification Beads until they are well dispersed Add 67 5 ul well mixed Sample Purification Beads to each sample well of the PAP plate Mix thoroughly as follows a Seal the plate with a Microseal B adhesive seal b Shake the plate on a microplate shaker at 1600 rpm for 2 minutes or until the beads are well dispersed Incubate the plate at room temperature for 5 minutes Centrifuge the plate at 280 x g for 1 minute Remove the adhesive seal from the plate then place the plate on the magnetic stand for 5 minutes or until the liquid is clear Using a 200 ul single channel or multichannel pipette set to 106 ul transfer 106 ul of the supernatant containing the DNA of interest from each sample well of the plate to an empty well in the same plate Take care not to disturb the beads NOTE Transfer do not discard the supernatant It contains the DNA of interest Repeat step 6 one time transferring each sample to the same well that the sample was transferred to in step 6 Each plate sample well now contains a total of 2
61. d to follow this user guide and not the EUC and LTF Provide information about creating and editing appropriate sample sheets for Illumina sequencing systems and analysis software and record parameters for your sample plate Provides information about the BaseSpace sequencing data analysis tool that also enables you to organize samples libraries pools and sequencing runs in a single environment Visit the TruSeq Synthetic Long Read DNA Library Prep support page on the Ilumina website for access to requirements and compatibility additional documentation software downloads online training frequently asked questions and best practices TruSeq Synthetic Long Read DNA Library Prep Guide SOSDINOSSY IEUOH DPV Part 15047264 Rev B Protocol ntroduction 8 Library Prep Workflow 1 9 Fragment DNA 10 Perform End Repallt rinitis 12 Adenylate 3 EndS ii tibia 16 Ligate Adapters 18 Purify Ligation Products and Size Selection 22 VELERO 29 QPCR Quantitation 30 Long Range so b a b ba 40 Tagmenta ON race o o Ms 51 Indexing PCR l ll llll 54 Pool and Concentrate
62. djustment calculation Quality Control 1 Dilute the Final DNA library from the FSP plate to an optimal concentration for the Agilent Technologies 2100 Bioanalyzer using a High Sensitivity DNA chip as follows a Quant the Final DNA library using a Qubit dsDNA HS Assay Kit b Dilute 2 ul of the Final DNA library to 1 ng ul with Resuspension Buffer TruSeq Synthetic Long Read DNA Library Prep Guide 67 jonpodd 201 Protocol 68 2 Load 1 ul of the diluted Final DNA library on an Agilent Technologies 2100 Bioanalyzer using a High Sensitivity DNA chip Figure 19 Example TruSeq Synthetic Long Read DNA Library Prep Final Library Distribution for Human DNA 3 Prepare a 1 5 dilution of the QC1 Pre size selection DNA library from step 20 of Pool and Concentrate on page 58 with Resuspension Buffer Part 15047264 Rev B 4 Load 1 ul of the diluted QC1 Pre size selection DNA library on an Agilent Technologies 2100 Bioanalyzer using a High Sensitivity DNA chip Check the size of the sample for a broad distribution of DNA fragments with a size range from approximately 200 3000 bp Figure 20 Example TruSeq Synthetic Long Read DNA Library Prep QC1 Pre Size Selection Library Distribution for Human DNA 1504 Excess Primer Ll L ry T T T T T T T T T T T T T 35 100 150 200 300 400 500 600 1000 2000 10380 bp 5 Do one of the following Proceed to cluster generation For more information see the
63. duct in effect on the date that the Product ships from Ilumina 2 Research Use Only Rights Subject to these terms and conditions and unless otherwise agreed upon in writing by an officer of Illumina Purchaser is granted only a non exclusive non transferable personal non sublicensable right under Illumina s Core IP in existence on the date that this Product ships from Illumina solely to use this Product in Purchaser s facility for Purchaser s internal research purposes which includes research services provided to third parties and solely in accordance with this 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 Ilumina s Hardware does not apply if the Specifications or Documentation state otherwise or g is the transfer to a third party of or sub licensing of Software or any third party software All Software whether provided separately installed on or embedded in a Product is licensed to Purchaser and not sold Except as expressly stated in this
64. e prepared for subsequent cluster generation and DNA sequencing The kits are designed for two applications preparing DNA libraries for long read assembly and phasing analysis from whole human genome sequencing data TruSeq Synthetic Long Read DNA Library Prep leverages TruSeq and Nextera chemistries with the high accuracy of short sequencing reads to construct long synthetic fragments with high assembly accuracy or efficient phasing of whole human genome sequencing data It enables phasing of de novo mutations and the identification of co inherited alleles in a population providing greater insight into the human genome The long read application generates synthetic long read fragments that can improve the accuracy of genome construction by providing data on traditionally challenging regions such as repetitive content This application enables more accurate long contigs for de novo assembly genome finishing or metagenomics applications The phasing application is designed for preparing human DNA libraries for phasing analysis Combined with whole human genome sequencing variant data this method assigns highly accurate shorter reads into long haplotype fragments for allele specific analysis Part 15047264 Rev B DNA Input Recommendations It is important to quantitate the input DNA and assess the DNA quality before performing TruSeq Synthetic Long Read DNA Library Prep Input DNA Quantitation Follow these DNA input recommendati
65. el five tubes of an eight tube strip with a smudge resistant pen as follows Std1 Std2 Std3 Std4 NTC Prepare SYBR Green 1 Vortex the thawed SYBR Green 10 000x to mix thoroughly 2 Add 5 ul SYBR Green 10 000x and 495 ul of DMSO to a microcentrifuge tube to dilute the SYBR Green to 100x Vortex the solution to mix thoroughly 3 Measure the absorbance of 100x diluted SYBR Green on a NanoDrop instrument The ideal AbS 44 3 nm Of 100x SYBR Green stock is 0 5 0 6 which indicates that the concentration is 100x Adjust the concentration if necessary For more information see Calibrate Diluted SYBR Green on page 98 NOTE Protect the 100x diluted SYBR Green from light e You can store the 100x diluted SYBR Green at 25 C to 15 C for up to six months When removing the dilution from storage thaw it completely then mix thoroughly while protecting it from light 3 2 Part 15047264 Rev B Dilute qPCR Standard NOTE Reference Figure 6 vvhile performing the dilution procedures Figure 6 Dilute qPCR Standard and Sample QST RSB Size selected DNA 495 ul 45 pul 45 ul 45 ul 50 H 495 pl Sul 504i Sul su su QST STD1 STD2 STD3 STD4 NTC Sample 1 100 10 pil 4pg l Otpg l 0 01 pg pl 1 100 10 000 fg ul 1 000 fg p 100 fg p 10 fa 1 Using a 200 ul single channel pipette set to 20 ul gently pipette the qPCR Standard up and down 10 times to mix thoroughly then centrifuge briefly TruSeq Synthetic Long Read DNA
66. entrifuge tube labeled with the sample name and record the weight Sample Sample Name Empty Tube Weight 1 2 3 4 Label one new microcentrifuge tube for each sample with size selected sample name using a smudge resistant pen Size Separate 1 Place one E Gel NGS 0 8 Agarose per sample into an E Gel iBase Power System according to manufacturer instructions Add 0 5 ul 1 Kb DNA Extension Ladder to 19 5 ul Resuspension Buffer in a microcentrifuge tube to dilute the DNA ladder Multiply each reagent volume by the number of gels being prepared Gently pipette the entire volume up and down 6 8 times to mix thoroughly TruSeq Synthetic Long Read DNA Library Prep Guide 2 3 UOH l S ZIS pue 1 uoneSn Al nda Protocol 24 Add 5 ul of 10 ng ul fragmented DNA sample from the tube labeled gDNA 4200 x g to 15 pl Resuspension Buffer in a microcentrifuge tube to dilute the fragmented gDNA Multiply each reagent volume by the number of gels being prepared Gently pipette the entire volume up and down 6 8 times to mix thoroughly The fragmented DNA sample in the tube labeled gDNA 4200 x g is from the conclusion of Fragment DNA on page 10 and is used as a control sample Centrifuge the diluted fragmented gDNA tube at 280 x g for 1 minute Reference Figure 3 while performing steps 5 9 Load 20 ul sample from one well of the CLP plate into lane 4 of one gel Part 15047264 Rev B Figure 3
67. eparate extract or isolate components of this Product or subject this Product or components thereof to any analysis not expressly authorized in this Product s Documentation iii gain access to or attempt to determine the methods of operation of this Product or iv transfer to a third party or grant a sublicense to any Software or any third party software Purchaser further agrees that the contents of and methods of operation of this Product are proprietary to Illumina and this Product contains or embodies trade secrets of Illumina The conditions and restrictions found in these terms and conditions are bargained for conditions of sale and therefore control the sale of and use of this Product by Purchaser TruSeq Synthetic Long Read DNA Library Prep Guide Limited Liability TO THE EXTENT PERMITTED BY LAW IN NO EVENT SHALL ILLUMINA OR ITS SUPPLIERS BE LIABLE TO PURCHASER OR ANY THIRD PARTY FOR COSTS OF PROCUREMENT OF SUBSTITUTE PRODUCTS OR SERVICES LOST PROFITS DATA OR BUSINESS OR FOR ANY INDIRECT SPECIAL INCIDENTAL EXEMPLARY CONSEQUENTIAL OR PUNITIVE DAMAGES OF ANY KIND ARISING OUT OF OR IN CONNECTION WITH WITHOUT LIMITATION THE SALE OF THIS PRODUCT ITS USE ILLUMINA S PERFORMANCE HEREUNDER OR ANY OF THESE TERMS AND CONDITIONS HOWEVER ARISING OR CAUSED AND ON ANY THEORY OF LIABILITY WHETHER IN CONTRACT TORT INCLUDING NEGLIGENCE STRICT LIABILITY OR OTHERWISE ILLUMINA S TOTAL AND CUMULATIVE LIABILITY TO PURCHASER OR ANY THIRD PAR
68. er Gently pipette the entire volume up and down 10 times to mix thoroughly Incubate the plate at room temperature for 2 minutes Place the plate on the magnetic stand for 5 minutes or until the liquid is clear Transfer 30 ul of supernatant from each well of the PAP plate to the corresponding well of the new MIDI plate labeled with the FSP barcode SAFE STOPPING POINT If you do not plan to proceed immediately to Validate Final Product on page 66 you can safely stop the protocol here If you are stopping seal the FSP plate with a Microseal B adhesive seal and store at 25 C to 15 C for up to 7 days TruSeq Synthetic Long Read DNA Library Prep Guide 6 5 uono l s 9ZIS Protocol Validate Final Product Perform the following procedures for quality control analysis on your sample library and quantification of the final library Consumables Item Quantity Storage Supplied By TruSeq Synthetic Long Read DNA Barcode Kit contents Resuspension Buffer RSB 1 tube 2 6 Illumina KAPA Library Quantification 1 As specified by User Kit Illumina Universal manufacturer High Sensitivity DNA Kit 1 As specified by User manufacturer Qubit dsDNA HS Assay Kit 1 As specified by User manufacturer Preparation Remove the Resuspension Buffer from 2 C to 8 C storage and bring it to room temperature Remove the FSP plate from 25 C to 15 C storage if it was stored at the conclusion of Size Selection on page 63 Let
69. fication Long Fragment Plate qPCR Master Mix qPCR Long amp Primer Mix qPCR Standard Resuspension Buffer Sample Neutralization Buffer Sample Purification Beads Tagment DNA Enzyme Part 15047264 Rev B Kit Contents Check to make sure that you have all of the reagents identified in this section before starting the protocol Table 3 TruSeq Synthetic Long Read DNA Library Prep Kits and Accessories Name Catalog TruSeq Synthetic Long Read DNA Library Prep Kit 4 Samples FC 126 1001 TruSeq Synthetic Long Read DNA Barcode Kit 1 Sample FC 126 1002 TruSeq Synthetic Long Read DNA Barcode Kit 4 Samples FC 126 1003 TruSeq Synthetic Long Read DNA Accessory Kit FC 126 1004 TruSeg Synthetic Long Read DNA Library Prep Kit 4 Samples The TruSeq Synthetic Long Read DNA Library Prep Kit contains one Box A and one Box B Library Prep Kit Box A Store at 2 C to 8 C This box is shipped at room temperature As soon as you receive it store the component at 2 C to 8 C This box also contains plate barcode labels TruSeq Synthetic Long Read DNA Library Prep Kit 4 Samples Box A part 15048200 Quantity Reagent Part Description 1 SPB 15047458 Sample Purification Beads Library Prep Kit Box B Store at 25 C to 15 C This box is shipped on dry ice As soon as you receive it store the components at 25 C to 15 C TruSeq Synthetic Long Read DNA Library Prep Guide v 5 s u9 U09 UM Supporting Information Figu
70. for the preparation of PCR Quantitation on page 30 NOTE Perform these procedures according to NanoDrop manufacturer instructions User supplied Consumables Item Quantity Storage Dimethyl sulfoxide DMSO asneeded 15 C to 30 C Lab tissue low lint asneeded 15 C to 30 C PCR grade water asneeded 15 C to 30 C 100x SYBR Green stock Tul 25 to 1522 from qPCR Quantitation on page 30 preparation Preparation Remove 100x SYBR Green from 25 C to 15 C storage and thaw to room temperature 4 When removing 100 SYBR Green from storage thaw it completely then mix thoroughly while protecting it from light Measure Absorbance 1 Open the Nanodrop ND1000 software and select UV Vis from the method panel 2 Initialize the NanoDrop instrument 3 Blank the NanoDrop instrument with 100 DMSO 4 Adjust the NanoDrop instrument settings For the NanoDrop ND 1000 do not select Normalize For the NanoDrop ND 2000 do not check Baseline correction 9 8 Part 15047264 Rev B Using 1 ul 100x SYBR Green stock measure its absorbance at wavelengths 480 nm 490 nm 494 nm 500 nm and 510 nm and record the values Wipe out the sample on the stage with a low lint lab tissue and then clean the stage with a water wetted low lint lab tissue Clean the stage again with a dry low lint lab tissue Repeat step 5 6 two times to collect two additional replicate readings of the 100x SYBR Green stock Adjust Concentratio
71. h well of the plate Gently pipette the entire volume up and down 10 times to mix thoroughly NOTE Perform steps 3 9 according to manufacturer instructions in the BluePippin Quick Guide BLF7510 marker S1 Calibrate the optics Inspect the gel cassette Prepare the cassette for loading Load the DNA marker S1 into well 1 Part 15047264 Rev B 7 Transfer 40 ul from each well of the plate to wells 2 5 8 Close the lid then select and run the HF 7 11kb Lane 1 S1 Marker program 9 Allow the samples to remain on the cassette after the run for 16 18 hours Make sure that the wells are sealed to prevent evaporation x CAUTION Ilumina recommends overnight elution for maximal recovery of DNA Illumina has validated the longer elution time and it has been confirmed by Sage Science as a safe protocol 10 Transfer each sample from the cassette to a new microcentrifuge tube labeled size selected sample name 11 Proceed to Validate Library on page 29 SAFE STOPPING POINT If you do not plan to proceed immediately to Validate Library you can safely stop the protocol here If you are stopping cap the size selected sample name tube and store at 2 C to 8 C for up to 1 month Avoid a freeze thaw cycle TruSeq Synthetic Long Read DNA Library Prep Guide 97 UO0I 99 9S zis ulddid niq Supporting Information Calibrate Diluted SYBR Green This process measures the absorbance of 100x diluted SYBR Green on a NanoDrop instrument
72. ibrary on an Agilent Technologies 2100 Bioanalyzer using a High Sensitivity DNA chip The peak partially overlaps with 10 kb upper marker Figure 5 Example TruSeq Synthetic Long Read DNA Library Distribution TruSeq Synthetic Long Read DNA Library Prep Guide 2 9 Mesq Protocol QPCR Quantitation This process quantifies the long DNA fragments to make sure that the appropriate amount of DNA is used for the Long Range PCR and subsequent Tagmentation procedures Consumables 30 Item TruSeq Synthetic Long Read DNA Library Prep Kit contents Quantity Storage Supplied By MasterAmp Extra Long 0 4 ul per reaction 20 Cito 15 Illumina DNA Polymerase Mix e qPCR Long amp Primer Mix 2 ul per reaction 25 C to 15 C Illumina QPM e qPCR Master Mix QMM 11 6 ul per reaction 25 C to 15 C Ilumina e qPCR Standard QST 5 ul per standard 25 C to 15 C Illumina curve Resuspension Buffer RSB 1 tube 2 6 WO IAC Illumina e QLP Quantification Long 1 label per plate 15 C to 30 C Illumina Fragment Plate barcode label Dimethyl sulfoxide DMSO 1ml ISAC to BOC User Ice bucket As needed 25 C to 15 C User PCR grade water 1ml soe User RNase DNase free eight tube 1 15 C to 30 C User strip with caps Microcentrifuge tubes 1 per sample 3 T do SUE User qPCR plate and seal 1 15 C to 30 C User Part 15047264 Rev B Item Quantity Storage Supplied By ROX Reference Dye
73. illum na TruSeq Synthetic Long Read DNA Library Prep Guide ACGAAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATATTAACGTACCATTAAGAGCTACCGT AATGATAACAGTAACACACT TCTGTTAACCTTAAGATTACTTGTTGATCCACTGATTCAACGTACCGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTCTTCTGTTAACCTTAAGAT TACT TGATCCACTGAT TCAACGTACCGTAA OG RAC CGT ORT GLa ined eel ACCATTAAGAGCTACCGTCTICTGTT QATLA a FE ea Set ATCCACTGATTCAACGTACCGTAAAGATTAC TTGATCCACTGATTCAACGTACCGTAACGAA TTGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGT AAIGATAAGAGAACACACTICTGITAACCT TAAGATTACT I GATGCAGT GAT TCAAGGTAGOGTAACGAACGIATGAAT SAGAS TAAATAT TAACGIACCAT TAAGAGCTACOGTC TCTGT IARC IAAGATIACT GAT CCAU GAT GAACGIA LU yD GW ae HeLa es TTAAGAGCT TCTGT TAACCT TAAGAT TACTT GATCCACT GAT TCAACGTACCGTAAC CGTATCAAT TGAGACTAAATAT TAACGTACT TAACCTTAAGATTACTTGATCCACT GATT CAACGTACCGTAACGAACGTCTTCTGTTAACCTTAAGAT TACTTGATCCACTGAT TCAACGTACCGIAACGAACGTAT CAAT TGAGACTAACGACGAAA GAGACTAAATAT TAACGTACCAT TAAGAGCTACAACCT TAAGAT TACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGC TACCGT GCAACGACGAAAAGAAT GATAACAGTAACACACT NO Tete Gad
74. ly Discard the tip Cap the eight tube strip that contains the serial diluted qPCR Standard then centrifuge briefly This serves as the qPCR standard in the Long Range PCR procedure Dilute Sample 34 1 Add 5 ul size selected DNA from the microcentrifuge tube from step 21 of Purify Gel on page 27 to each tube labeled with the sample name 1 100 Dilution Using a 1000 ul single channel or multichannel pipette gently pipette the entire volume up and down 6 8 times to mix thoroughly Part 15047264 Rev B 2 Cap and store the size selected sample name tubes at 2 C to 8 C for up to 90 days Prepare Master Mix 1 3 Prepare a fresh dilution of 1 5x SYBR Green from 100x SYBR Green stock 3 ul 100x SYBR Green in 197 ul PCR grade water to create a dye mix If ROX is required for your qPCR instrument dilute SYBR Green and ROX dye together to make a 1 5x SYBR Green 10x ROX dye mixture y CAUTION This qPCR procedure is sensitive to the SYBR Green concentration Make sure that you have calibrated the 100x diluted SYBR Green on a NanoDrop instrument Set up a master mix in a sterile nuclease free microcentrifuge tube on ice using the following Using a 1000 ul single channel or multichannel pipette gently pipette the entire volume up and down 6 8 times to mix thoroughly Reagent 1 Sample 2 Samples 3 Samples 4 Samples qPCR Master Mix 255 ul 302 ul 336 ul 394 ul qPCR Long amp Primer Mix 44 ul 52 ul 58 ul 68 ul
75. ment qPCR system See qPCR Systems on page 87 Qubit 2 0 Fluorometer Vacuum manifold Optional for BluePippin size selection BluePippin Size Selection System Optional for SYBR Green calibration NanoDrop Spectrophotometer Supplier General lab supplier Life Technologies catalog Q32866 Promega catalog A7231 or QIAGEN part 4 19413 Sage Science catalog BLU0001 Thermo Scientific catalog ND 1000 or ND 2000 Part 15047264 Rev B qPCR Systems The following table lists the validated qPCR systems for the TruSeq Synthetic Long Read DNA Library Prep protocol Either a 96 well or 384 well qPCR system is required Equipment CFX96 Touch Real Time PCR Detection System CFX384 Touch Real Time PCR Detection System 7900HT Fast Real Time PCR System with 384 Well Block Module Mx3000P qPCR System LightCycler 480 Instrument II 384 well version Supplier Bio Rad part 185 5195 Bio Rad part 185 5484 Life Technologies part 4329001 Agilent part 401511 Roche part 05015243001 1 Illumina recommends using Manager software version 3 0 with Cq Determination mode Single Threshold Baseline Setting Baseline Subtracted Curve Fit and Apply Fluorescent Drift Correction for data analysis This can correct for abnormalities in fluorescence intensity of the standard curve caused by the instrument For software installation contact Bio Rad 2 When setting up the SDS software
76. n 1 Calculate the average absorption of the three replicates for each wavelength The maximum absorption value recorded should be at one wavelength 490 510 nm The ideal AbS4o4 3 nm Of 100x SYBR Green stock is 0 5 0 6 which indicates that the concentration is 100x If the maximum absorbance reading is in the 0 5 0 6 range clean the instrument then return to the preparation of PCR Quantitation on page 30 If the maximum absorbance reading is out of the 0 5 0 6 range calculate the real concentration of the 100x SYBR Green stock using the following equation 0 55 maximum Abs 100 real concentration of SYBR For example if the maximum Abs of the 100x SYBR Green is 0 8 the real concentration of 100x SYBR Green is 145 5x Adjust the concentration of the 100x SYBR Green stock to 100x based on the calculation in step 3 For concentrations gt 100x dilute 100x SYBR Green stock with DMSO For concentrations lt 100x make new 100x SYBR Green stock from SYBR Green 10 000x concentrate as follows a Mix thawed SYBR Green 10 000x thoroughly b Add 5 pl SYBR Green to 495 ul of DMSO to dilute Repeat Measure Absorbance on page 98 to measure the adjusted or new 100x SYBR Green stock until the maximum absorption is 0 5 0 6 TruSeq Synthetic Long Read DNA Library Prep Guide 9 9 13910 HAAS PSI SI2AQHUED 1 O O Part 15047264 Rev B Index 1 1 Kb DNA Extension Ladder 22 2 2 Log DNA Ladder 41 2 propanol 22 A acronyms 73
77. n Date Description of Change 15047264 B September Updated Additional Resources to remove web navigation 2014 instructions and written urls Removed use of plate name e g LFP plate except for first instance and last instance in each procedure Modified the Purify Ligation Products and Size Selection protocol to use the E Gel NGS 0 8 Agarose E Gel iBase Power System and run the E Gel 0 8 2 program e Removed the following Consumables and Equipment E Gel CloneWell 0 8 SYBR Safe gels PCR Tube Plate 384 well Prism Electrophoresis power supply Gel Opener e Added a BluePippin Size Selection protocol option and the necessary Consumables and Equipment Corrected the Collection Plate and Fragmentation Plate part numbers in Kit Contents Clarified thermal cycler and qPCR instrument requirements Updated SDS link to support illumina com sds html 15047264 A June 2014 Initial Release TruSeq Synthetic Long Read DNA Library Prep Guide VI Part 15047264 Rev B Table of Contents Chapter 1 Overview Introduction Chapter 2 Protocol Introduction Fragment DNA Perform End Repair Adenylate 3 Ends Validate Library QPCR Quantitation Long Range PCR Tagmentation Indexing PCR Size Selection Appendix A Supporting Information 0 Introduction Acronyms Kit Contents TruSeq Synthetic Long Read DNA Library Prep Guide DNA Input Recommend
78. nd expel the extracted gel band from the tool into the tube with a quick squeeze Weigh the tube containing the gel slice Subtract the weight of the empty tube to determine weight of the gel slice in milligrams mg Use the following table as an example for tracking and calculating the weight of the gel Sample Tube Weight Empty Tube Weight Gel Slice Weight mg 1 2 3 4 For each sample add X ul QIAGEN Buffer QG with X equaling three times the mg weight of the gel slice to the tube containing the gel slice For example if the gel slice weights 100 mg add 300 ul QIAGEN Buffer QG to the tube TruSeq Synthetic Long Read DNA Library Prep Guide P 2 UOH l S ZIS pue sionpoJd uoneSn Alina Protocol 10 11 12 13 14 15 16 17 18 19 20 21 22 4 28 Place the tube containing the gel and QIAGEN Buffer QG mixture on the pre heated microheating system or water bath Close the lid and incubate at 50 C for 10 minutes to melt the gel Gently flick the tube periodically until the gel is fully melted Add 1 ul Isopropanol x the mg weight of the gel slice to the gel and QIAGEN Buffer QG mixture For example if the gel slice weighs 100 mg then add 100 ul Isopropanol to the mixture Add the dissolved gel QIAGEN Buffer QG and Isopropanol mixture to a QlAquick column Centrifuge the QlAquick column to 13 000 rpm for 1 minute Remove and discard the eluate from the QlAquick column Add 750 ul PE buffer with e
79. o 15 C storage and thaw at room temperature Remove the LAP plate from 2 C to 8 C storage if it was stored at the conclusion of Long Range PCR on page 40 Remove the Fragmentation plate from the kit box TruSeq Synthetic Long Read DNA Library Prep Guide 5 1 uolyejuaube Protocol Pre program the thermal cycler with the following program and save as Tag Choose the thermal cycler pre heat lid option and set to 100 C 55 C for 15 minutes 4 C for 5 minutes 72 C for 4 minutes Hold at 4 C Procedure 1 Centrifuge the LAP plate at 500 x g for 1 minute 2 Remove the seal from the LAP plate then place the Fragmentation plate on top of LAP plate Line up the keyed corners and make sure that the wells of the Fragmentation plate are centered in the wells of the LAP plate Figure 12 Fragmentation plate on LAP A Fragmentation plate B LAP Part 15047264 Rev B e N OA OFF q 10 11 12 13 Add 36 ul Tagment DNA Enzyme and 1464 ul Fragmentation Pre Mix to a microcentrifuge tube Invert the tube 10 times to mix thoroughly then centrifuge briefly Transfer 180 ul of the mixture to each well of an eight tube strip Set a 200 ul electronic eight channel pipette to 144 ul and 3 ul per dispense Add 3 ul Tagment DNA Enzyme and Fragmentation Pre Mix to each well of the Fragmentation plate that is on top of the LAP plate Make sure that each well contains liquid Centrifuge the stacked Fragmentation plate and LAP pla
80. on 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 Hardware includes Illumina provided installation in which case the warranty period begins on the date of installation or 30 days after the date it was delivered whichever occurs first Base Hardware Warranty Upgraded Components means Illumina provided components modifications or enhancements to Hardware that was previously acquired by Purchaser Illumina warrants that Upgraded Components will conform to their Specifications for a period of 90 days from the date the Upgraded Components are installed Upgraded Components do not extend the warranty for the Hardware unless the upgrade was conducted by Illumina at Illumina s facilities in which case the upgraded Hardware shipped to Pu
81. onally equivalent reconditioned or new Hardware or components if only a component of Hardware is non conforming If the Hardware is replaced in its entirety the warranty period for the replacement is 90 days from the date of shipment or the remaining period on the original Hardware warranty whichever is shorter If only a component is being repaired or replaced the warranty period for such component is 90 days from the date of shipment or the remaining period on the original Hardware warranty whichever ends later The preceding states Purchaser s sole remedy and Illumina s sole obligations under the warranty provided hereunder Third Party Goods and Warranty Illumina has no warranty obligations with respect to any goods originating from a third party and supplied to Purchaser hereunder Third party goods are those that are labeled or branded with a third party s name The warranty for third party goods if any is provided by the original manufacturer Upon written request Illumina will attempt to pass through any such warranty to Purchaser 9 Indemnification a Infringement Indemnification by Illumina Subject to these terms and conditions including without limitation the Exclusions to Illumina s Indemnification Obligations Section 9 b below the Conditions to Indemnification Obligations Section 9 d below Illumina shall i defend indemnify and hold harmless Purchaser against any third party claim or action alleging that this Product
82. ons 50 ul input DNA at 10 ng ul is required to prepare one sample library The ultimate success or failure of library preparation strongly depends on using an accurately quantified amount of input DNA Use multiple methods of quantification to verify results Illumina recommends using fluorometric based methods for quantification such as Qubit or PicoGreen to provide accurate quantification of dsDNA UV spectrophotometric based methods such as Nanodrop measure any nucleotides present in the sample including RNA dsDNA ssDNA and free nucleotides This can give an inaccurate measurement of gDNA DNA quantification methods that rely on intercalating fluorescent dyes measure only double stranded DNA and are less subject to the presence of excess nucleic acids These methods require the preparation of calibration curves and are highly sensitive to pipetting error Make sure that pipettes are correctly calibrated and are not used at the volume extremes of their performance specifications Assessing DNA Quality Genomic DNA integrity is critical for the success of TruSeq Synthetic Long Read DNA Library Prep The DNA must be phenol free with a size gt 40 kb Illumina recommends a gDNA quality check using an agarose gel or other instrument before proceeding with the protocol To assess quality on agarose gel Illumina recommends running 100 500 ng of gDNA on a 0 8 Agarose gel with 200 ng of 1 kb DNA extension ladder Compare the results to the Fig
83. rchaser comes with a Base Hardware Warranty 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 IV Part 15047264 Rev B by Illumina unless the Product s Documentation or Specifications expressly state such third party s good is for use with the Product 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 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 replaced with functi
84. rcode label to a new 96 well PCR plate Apply an LFP2 barcode label to a new 96 well PCR plate Centrifuge the thawed End Repair Mix tube at 600 x g for 5 seconds Add 30 ul fragmented DNA sample from each gDNA 4200 x g tube to a separate well of the new PCR plate labeled with the LFP barcode E NOTE Place the gDNA 4200 x g tubes in 25 C to 15 C storage for use later in the protocol Add 20 ul End Repair Mix to each sample well of the plate Set a 200 ul pipette to 40 ul and then gently pipette the entire volume up and down 10 times to mix thoroughly Seal the plate with a Microseal B adhesive seal then centrifuge the plate at 280 x g for 1 minute Return the End Repair Mix tube to 25 C to 15 C storage TruSeq Synthetic Long Read DNA Library Prep Guide 4 3 pug WiOLed Protocol Incubate LFP 1 Place the sealed plate on the pre programmed thermal cycler Close the lid then select and run the ERP program a Choose the thermal cycler pre heat lid option and set to 100 C b 30 C for 30 minutes Hold at 4 C 2 Remove the plate from the thermal eycler vvhen the program reaches 49C 3 Centrifuge the plate at 280 x g for 1 minute Clean Up LFP 1 Remove the adhesive seal from the plate 2 Vortex the Sample Purification Beads until they are vvell dispersed 3 Add 80 ul well mixed Sample Purification Beads to each well of the plate containing 50 ul of the end repaired sample Gently pipette the entire
85. rding to manufacturer instructions Centrifuge the g TUBE with the blue cap up to 4200 x g for 1 minute with a balance Flip the g TUBE over so that the blue cap is down and centrifuge the tube one more time to 4200 x g for 1 minute with a balance Immediately remove the g TUBE from the centrifuge Use a g TUBE cap holder to transfer all of the fragmented DNA from the blue cap to the microcentrifuge tube labeled gDNA 4200 x g along with the experiment date SAFE STOPPING POINT If you do not plan to proceed immediately to Perform End Repair on page 12 you can safely stop the protocol here If you are stopping store the gDNA 4200 x g tube at 2 C to 8 C for up to 30 days TruSeq Synthetic Long Read DNA Library Prep Guide 1 1 VNQ Juauwbely Protocol Perform End Repair 12 This process converts the overhangs resulting from fragmentation into blunt ends using End Repair Mix The 3 to 5 exonuclease activity of this mix removes the 3 overhangs and the 5 to 3 polymerase activity fills in the 5 overhangs Consumables Item TruSeq Synthetic Long Read DNA Library Prep Kit contents End Repair Mix ERP e Resuspension Buffer RSB e Sample Purification Beads SPB Barcode labels for e LFP Long Fragment Plate e LFP2 Long Fragment Plate 2 96 well PCR plates Freshly prepared 80 ethanol EtOH Ice bucket Microseal B adhesive seals J NOTE Quantity 1 tube per 4 reactions 1 tube
86. re 21 TruSeq Synthetic Long Read DNA Library Prep Kit 4 Samples Box B part 15048201 066660 69 yO Quantity Reagent Part Description 1 A Tailing Mix 1 E End Repair Mix 1 AE Long Fragment Adapter 1 Ligation Mix 1 MasterAmp Extra Long DNA Polymerase Mix 1 qPCR Master Mix 1 PCR Long amp Primer Mix 1 qPCR Standard 1 Resuspension Buffer TruSeq Synthetic Long Read DNA Barcode Kit 1 Sample The TruSeq Synthetic Long Read DNA Barcode Kit 1 Sample contains one Box A one Box B and one Box C 7 6 Part 15047264 Rev B Barcode Kit 1 Sample Box A Store at 2 C to 8 C This box is shipped at room temperature As soon as you receive it store the component at 2 C to 8 C This box also contains plate barcode labels TruSeq Synthetic Long Read DNA Barcode Kit 1 Sample Box A part 15051787 Quantity Reagent Part Description 1 SPB 15044759 Sample Purification Beads Barcode Kit Box B Store as specified This box is shipped on dry ice As soon as you receive it store the components as specified Figure 22 TruSeq Synthetic Long Read DNA Barcode Kit Box B part 2 15048203 IDP Packaged Assembly TruSeq Synthetic Long Read DNA Library Prep Guide 77 S JU9 UOD YN Supporting Information Quantity Reagent Part Description Storage Temperature 1 25 C to 15 C 1 25 C to 15 C 1 25 C to 15 C 1 25 C to 15 C 1 EPM 15046175 Long amp Primer Mix 25 C to 15
87. reactions 25 C to 15 C Illumina e Long Fragment Adapters 1 tube per 4 reactions 25 C to 15 C Illumina LAD Resuspension Buffer RSB 1 tube 2 C to 8 C Illumina e Sample Purification Beads 1 tube per 4 reactions 2 C to 8 C Illumina SPB e CLP Cleaned Long 1 label per plate AS O SAS Illumina Fragment Plate barcode label 96 well PCR plate 1 15 C to 30 C User Freshly prepared 80 ethanol 400 ul per sample T5 G to 30 E User EtOH Ice bucket As needed 25 C to 15 C User Microseal B adhesive seals 2 ISCO AE User NOTE This procedure is described using 96 vvell PCR plates hovvever RNase DNase free eight tube strips vvith caps can be used in this procedure in place of the plates Preparation Prepare an ice bucket 7 8 Part 15047264 Rev B Add LIG G 0 A WO N e N Remove the Long Fragment Adapters from 25 C to 15 C storage and thaw at room temperature Place the tube on ice iy NOTE Do not remove the Ligation Mix tube from 25 C to 15 C storage until instructed to do so in the procedures Review best practices for handling magnetic beads See Additional Resources on page 5 for information about TruSeq Synthetic Long Read DNA Library Prep best practices on the Illumina website Remove the Sample Purification Beads and Resuspension Buffer from 2 C to 8 C storage and bring them to room temperature Pre program the thermal cycler with the following program and save as LIG Choose the
88. rifuge the stacked IDP and LAP plates to 500 x g for 1 minute Make sure that the LAP plate is on the bottom Place the stacked IDP and LAP plates on the benchtop with the LAP plate on the bottom Carefully remove the IDP plate from the LAP plate and place it the top side facing up on the benchtop i Make sure that all wells of the IDP plate are empty Transfer any remaining supernatant to the corresponding well of the LAP plate using a single channel pipette Mix the LAP plate thoroughly as follows a Seal the plate with a Microseal B adhesive seal b Shake the plate on a microplate shaker at 1600 rpm for 30 seconds Centrifuge the plate at 500 x g for 1 minute Part 15047264 Rev B 14 Place the sealed plate on the thermal cycler and place a compression mat on top of the plate Close the lid then select and run the PostTagAmp program The total volume of the mixture is 13 ul a Choose the thermal cycler pre heat lid option and set to 100 C b 94 C for 1 minute c 10 cycles of 94 C for 15 seconds 65 C for 4 minutes d Hold at 4 C for up to one hour 15 Remove the LAP plate from the thermal cycler SAFE STOPPING POINT 1 If you do not plan to proceed immediately to Pool and Concentrate on page 58 you can safely stop the protocol here If you are stopping store the LAP plate at 25 C to 15 C for up to 7 days or at 2 C to 8 C for up to 24 hours TruSeq Synthetic Long Read DNA Library Prep Guide 5
89. rkflow pre program your thermal cyclers as follows Program Name Thermal cycler Program 42 Phasing15 384 well Choose the thermal cycler pre heat lid option and set to 95 C 100 C e 94 C for 1 minute 15 cycles of e 94 C for 30 seconds 65 C for 30 seconds e 68 C for 10 minutes 68 C for 10 minutes Hold at 4 C Phasing20QC 96 well Choose the thermal cycler pre heat lid option and set to 95 C 100 C e 94 C for 1 minute 20 cycles of e 94 C for 30 seconds 65 C for 30 seconds 68 C for 10 minutes 68 C for 10 minutes Hold at 4 C Part 15047264 Rev B For the Long Read workflow pre program your thermal cyclers as follows Program Name Thermal cycler Program LongRead21 384 well Choose the thermal cycler pre heat lid option and set to 95 C 100 C 94 C for 1 minute e 21 cycles of e 94 C for 30 seconds 65 C for 30 seconds e 68 C for 10 minutes 68 C for 10 minutes e Hold at 4 C LongRead26QC 96 well Choose the thermal cycler pre heat lid option and set to 95 C 100 C e 94 C for 1 minute 26 cycles of e 94 C for 30 seconds 65 C for 30 seconds 68 C for 10 minutes 68 C for 10 minutes e Hold at 4 C Label five nevv microcentrifuge tubes vvith a smudge resistant pen as follovvs GST1 GST2 GST3 GST4 2 Log Ladder Apply a LAP barcode label to a new 384 well PCR plate Dilute Template 1 De
90. spense for a total of 24 dispenses Add 5 ul PCR master mix to each well of the new 384 well PCR plate labeled with the LAP barcode Repeat steps 4 and 5 one time Make sure that each well contains 5 ul PCR master mix Quickly seal the plate with a Microseal B adhesive seal then centrifuge the plate at 500 x g for 1 minute Cap the PCR master mix eight tube strip and keep the strip on ice Part 15047264 Rev B Long Amp Plate 1 Place the sealed plate on the 384 well thermal cycler and place a compression mat on top of the plate Close the lid then select and run the Phasing15 or LongRead21 program depending on the workflow Workflow Phasing Long Read Program Name Phasing15 LongRead21 Program Choose the thermal Choose the thermal cycler pre heat lid cycler pre heat lid option and set to option and set to 95 C 100 C 95 C 100 C e 94 C for 1 minute e 94 C for 1 minute e 15 cycles of e 21 cycles of 94 C for 30 e 94 C for 30 seconds seconds 65 C for 30 e 65 C for 30 seconds seconds e 68 C for 10 e 68 C for 10 minutes minutes 68 C for 10 minutes 68 C for 10 minutes Hold at 4 C Hold at 4 C Hours to 3 4 5 Complete 2 While the thermal cycler is running proceed to Long Amp Quality Control Long Amp Quality Control 1 Add 50 ul PCR master mix from Prepare PCR Master Mix to one well of a new PCR plate or an eight tube strip 2 Seal the plate with a Microseal B adhesive seal or
91. t until it is immersed in the solution Gently pipette the entire volume up and down 10 times to mix thoroughly Incubate the plate at room temperature for 2 minutes Place the plate on the magnetic stand for 5 minutes or until the liquid is clear Transfer 20 ul of the supernatant from each well of the LFP2 plate to the corresponding well of the new PCR plate labeled with the CLP barcode Take care not to disturb the beads SAFE STOPPING POINT If you do not plan to proceed immediately to Purify Ligation Products and Size Selection on page 22 you can safely stop the protocol here If you are stopping seal the CLP plate with a Microseal B adhesive seal and store at 2 C to 8 C overnight TruSeq Synthetic Long Read DNA Library Prep Guide 2 4 s1 depy o1e6rq Protocol Purify Ligation Products and Size Selection 22 This process purifies the products of the ligation reaction on a gel and removes unligated adapters as well as any adapters that might have ligated to one another Long adapter ligated fragments of DNA of 8 10 kb in size are selected for Long Range PCR and subsequent Tagmentation procedures NOTE TruSeq Synthetic Long Read DNA size selection is performed using agarose gel electrophoresis However an alternative method using the BluePippin System can be performed in place of the procedures in this section To perform the alternative method see BluePippin Size Selection on page 95 Consumables Tem Tr
92. termine if the 1 100 diluted library template is of sufficient quantity for Long Range PCR For the Phasing workflow 75 fg library is required per well A total of 37 500 fg library per plate For the Long Read workflow 3 fg library is required per well A total of 1500 fg library per plate 2 If there is not enough library template to make the dilution use the undiluted template from step 2 of Dilute Sample on page 34 TruSeq Synthetic Long Read DNA Library Prep Guide 43 H d ebuey 6u07 Protocol 3 Dilute the library template with Resuspension Buffer to the following concentration with a total volume of 750 ul Use the table as an example for tracking and calculating the dilution For the Phasing workflow dilute the library template to 50 fg ul For the Long Read workflow dilute the library template to 2 fg ul 1 100 Diluted Library Diluted Library Resuspension Buffer Total Volume Sample fg ul ul ul ul WO Prepare PCR Master Mix 44 Set up a PCR master mix in a sterile nuclease free 15 ml conical tube on ice using the following Reagent Volume ul Diluted template 750 Long amp Master Mix 1450 Long amp Primer Mix 250 MasterAmp Extra long DNA Polymerase Mix 50 Total Volume 2500 Cap the tube and gently invert the tube several times to mix Aliquot 280 ul PCR master mix into each well of an eight tube strip Set a 200 ul electronic eight channel pipette to 120 ul and 5 ul per di
93. tes to 500 x g for 1 minute Make sure that the LAP plate is on the bottom Place the stacked Fragmentation plate and LAP plates on the benchtop with the LAP plate on the bottom Carefully remove the Fragmentation plate from the LAP plate and discard the Fragmentation plate Mix the LAP plate thoroughly as follows a Seal the plate with a Microseal B adhesive seal b Shake the plate on a microplate shaker at 1600 rpm for 30 seconds Centrifuge the plate at 500 x g for 1 minute Place the sealed plate on the thermal cycler and place a compression mat on top of the plate Close the lid and then select and run the Tag program The total volume of the mixture is 8 ul a Choose the thermal cycler pre heat lid option and set to 100 C b 55 C for 15 minutes 4 C for 5 minutes d 72 C for 4 minutes e Hold at 4 C 14 Remove the LAP plate from the thermal cycler TruSeq Synthetic Long Read DNA Library Prep Guide 5 3 uolyejuaube Protocol Indexing PCR This process amplifies tagmented DNA by PCR A unique index and the P5 and P7 adapters are added to the tagmented DNA in each well of the 384 well plate The P5 and P7 adapters are required for cluster generation and sequencing Consumables Item Quantity Storage Supplied By TruSeq Synthetic Long Read DNA Barcode Kit contents Indexing Plate IDP 1 25 C to 15 C Ilumina Optional TruSeq Synthetic Long Read DNA Accessory Kit contents e Alignment ring
94. thanol added to the QlAquick column Centrifuge the QlAquick column to 13 000 rpm for 1 minute Remove and discard the supernatant from the QlAquick column Centrifuge the QlAquick column to 13 000 rpm for 1 minute Remove and discard the supernatant from the QlAquick column Remove the QlAquick column from the collection tube and place it in the new microcentrifuge tube labeled size selected sample name Add 52 ul Resuspension Buffer to the QIAquick column in the microcentrifuge tube Incubate the microcentrifuge tube at room temperature for 1 minute Centrifuge the QlAquick column in the microcentrifuge tube at 13 000 rpm for 1 minute Discard the QlAquick column SAFE STOPPING POINT If you do not plan to proceed immediately to Validate Library on page 29 you can safely stop the protocol here If you are stopping cap the size selected sample name tube and store at 2 C to 8 C for up to 3 months Avoid a freeze thaw cycle Part 15047264 Rev B Validate Library Perform the following procedures for quality control analysis and quantification of the long DNA fragments Quantify Libraries Quantify 2 ul of the library using the Qubit dsDNA HS Assay Kit The library should yield gt 0 05 ng ul NOTE For information on how to perform the Qubit dsDNA HS Assay see manufacturer instructions Optional Quality Control To verify the size of your fragments check the template size distribution Run 1 ul of the DNA l
95. the FSP plate thaw at room temperature Centrifuge the FSP plate at 280 x g for 1 minute Remove the adhesive seal from the FSP plate Quantify Libraries To achieve the highest quality data on Illumina sequencing platforms it is important to create optimum cluster densities across every lane of the flow cell Optimizing cluster densities requires accurate quantitation of DNA library templates Illumina recommends that you quantify your libraries by qPCR 6 6 Part 15047264 Rev B L NOTE TruSeq Synthetic Long Read DNA Library Prep library quantitation has been validated using the KAPA Library Quantification Kit specified in the Consumables and Equipment on page 82 Follow the KAPA instructions with the KAPA standard Follow qPCR instructions included in the KAPA Library Quantification Kits for Illumina sequencing platforms Technical Data Sheet using the KAPA standard www kapabiosystems com with the following modification NOTE You can download the KAPA Library Quantification Kits for Illumina sequencing platforms Technical Data Sheet from the Kapa Biosystems website www kapabiosystems com Perform a size adjustment calculation to account for the difference in size between the average fragment length of the library and the KAPA DNA Standard 452 bp Determine the average fragment length of the library between 200 2000 bp using an Agilent Technologies 2100 Bioanalyzer or equivalent Use this average fragment length for the size a
96. uSeq Synthetic Long Read DNA Library Prep Kit contents e Resuspension Buffer RSB 1 Kb DNA Extension Ladder 2 propanol Isopropanol E Gel NGS 0 8 Agarose Fragmented gDNA 4200 x g from Fragment DNA on page 10 Lab pen Microcentrifuge tubes QIAquick Gel Extraction Kit Ruler X tracta Gel Extraction Tool Quantity 1 tube 0 5 ul per sample 1 ul x the mg weight of each gel slice 1 per sample 5 ul per sample 1 2 per sample 2 1 1 1 per sample Storage AC O one 15 C to 30 C IBC ho SUE 15 C to 30 C 5 0 15C to 30 C AE tos Use 15 C to 30 C IC G C 15C to 30 C Supplied By Illumina User User User E ser ser ser ser ser C MN Mel C ser Part 15047264 Rev B Preparation Remove the Resuspension Buffer from 2 C to 8 C storage and bring it to room temperature Remove the CLP plate from 2 C to 8 C storage if it was stored at the conclusion of Clean Up LFP2 on page 20 Let the CLP plate stand to bring it to room temperature Centrifuge the CLP plate at 280 x g for 1 minute Remove the adhesive seal from the CLP plate Prepare E Gel NGS 0 8 Agarose by removing the comb One gel is recommended per sample to avoid cross contamination Pre heat the microheating system or water bath to 50 C Label one new microcentrifuge tube for each sample with the name of the sample using a smudge resistant pen Weigh each microc
97. until all of the liquid has passed through the Zymo Spin V column and into the vacuum manifold Add the remaining master mix to the Zymo Spin V column Run the master mix through the vacuum until all of the liquid has passed through the Zymo Spin V column and into the vacuum manifold Add 4 ml Zymo DNA Wash Buffer with ethanol added to the Zymo Spin V column to wash the sample while it is on the vacuum Remove the Zymo Spin V column from the vacuum manifold and unattach the reagent reservoir from the column Discard reagent reservoir TruSeq Synthetic Long Read DNA Library Prep Guide 61 2 pue OOd Protocol 16 Centrifuge Zymo Spin V column at 11 000 x g for 1 minute in a microcentrifuge tube to remove any residual Zymo DNA Wash Buffer 17 Place the Zymo Spin V column into a new microcentrifuge tube then add 160 ul Resuspension Buffer to the column Figure 18 Place Zymo Spin V Into Microcentrifuge Tube m V 18 Centrifuge the microcentrifuge tube at 10 000 x g for 1 minute to collect the eluate 19 Transfer 150 ul from the microcentrifuge tube to a single well of the new MIDI plate labeled with the PAP barcode 20 Transfer 5 ul from the microcentrifuge tube to the new eight tube strip labeled QC1 Pre Size Selection T SAFE STOPPING POINT If you do not plan to proceed immediately to Size Selection on page 63 you can safely stop the protocol here If you
98. ure 1 examples of 0 8 CloneWell gels run for 30 minutes TruSeq Synthetic Long Read DNA Library Prep Guide 3 SUOITEPUSLULUOD8Y 110 YNG Overview Table 1 Quality Assessment Guidelines Pass gDNA gel QC All gDNA result migrates in a discreet band gt 40 kb Success in gt 95 TruSeq Synthetic Long Read DNA Library Prep Mode of failure N A Recommendation N A for a second library prep attempt Figure 1 Examples of DNA Quality A Pass B Intermediate C Fail Intermediate Some gDNA migrates in a discreet band gt 40 kb some gDNA migrates as a smear lt 40 kb 60 Insufficient yield in Long Fragment qPCR quantitation lt 20 of gDNA samples succeed in second attempt Fail All gDNA migrates as a smear lt 40 kb 0 Insufficient yield in Long Fragment qPCR quantitation 0 of gDNA samples succeed in second attempt Part 15047264 Rev B Additional Resources The following documentation is available for download from the Illumina website Resource TruSeq Synthetic Long Read Library Prep Experienced User Card and Lab Tracking Form part 15047265 Illumina Experiment Manager Guide part 15031335 and IEM TruSeq Synthetic Long Read DNA Quick Reference Card part 15056316 BaseSpace User Guide part 15044182 Description Provides protocol instructions but with less detail than what is provided in this user guide New or less experienced users are advise
99. vernight TruSeq Synthetic Long Read DNA Library Prep Guide High Sensitivity DNA Kit KAPA Library Quantification Kit RSB MOI ION day eq Protocol Fragment DNA 10 This process describes how to optimally fragment the gDNA using a g TUBE for phasing and long read workflows Consumables Item Quantity TruSeq Synthetic Long Read DNA Library Prep Kit contents Resuspension Buffer RSB 1 tube gDNA samples 500 ng at 10 ng ul per sample g TUBE 1 Microcentrifuge tubes 2 Qubit dsDNA BR or HS assay 1 kit Preparation Review DNA Input Recommendations on page 3 Storage 25 C to 15 C PLE WO ENE after initial thaw 25 C to 15 C b O SUE 15 C to 30 C As specified by manufacturer Supplied By Illumina User User User User Remove the Resuspension Buffer from 25 C to 15 C storage and thaw it at room temperature NOTE The Resuspension Buffer can be stored at 2 C to 8 C after the initial thaw Label a new microcentrifuge tube gDNA 4200 x g and the experiment date with a smudge resistant pen Part 15047264 Rev B Procedure Quantify the gDNA sample using the Qubit dsDNA BR or HS assay kit Normalize the gDNA sample with Resuspension Buffer to a final volume of 50 ul at 10 ng ul in a new microcentrifuge tube Transfer 50 ul normalized gDNA to a g TUBE 2 Steps 4 7 must be performed within 15 minutes of the gDNA being added to the g TUBE acco
100. volume up and down 10 times to mix thoroughly 4 Incubate the plate at room temperature for 5 minutes 5 Place the plate on the magnetic stand for 5 minutes or until the liquid is clear 6 Using a 200 ul single channel or multichannel pipette set to 127 5 ul remove and discard 127 5 ul of supernatant from each well of the plate NOTE Leave the plate on the magnetic stand while performing the following 80 EtOH wash steps 7 9 7 With the plate on the magnetic stand add 200 ul freshly prepared 80 EtOH to each well without disturbing the beads 8 Incubate the plate at room temperature for 30 seconds and then remove and discard all of the supernatant from each well Take care not to disturb the beads 9 Repeat steps 7 and 8 one time for a total of two 80 EtOH washes 10 Remove and discard any remaining EtOH from each well of the plate with a 10 ul 14 pipette Part 15047264 Rev B 11 Let the plate stand at room temperature for 5 minutes to dry and then remove the plate from the magnetic stand 12 Add 20 ul Resuspension Buffer to each well of the plate Gently pipette the entire volume up and down 10 times to mix thoroughly 13 Incubate the plate at room temperature for 2 minutes 14 Place the plate on the magnetic stand for 5 minutes or until the liquid is clear 15 Transfer 17 5 ul of supernatant from each well of the LFP plate to the corresponding well of the new PCR plate labeled with the LFP2 plate barcode 1 SAF

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