DNA Library Kit - Swift Biosciences
Contents
1. 37 C until all samples are mixed and loaded 2 On ice make the Adaptase Reaction Mix with the following amounts of each reagent Volume 1 reaction Low EDTA TE 12 5 ul Reagent Gi A Add the reagents in the Reagent G2 specified order Reagent G3 2 5 ul Enzyme GA 3 Mix the Adaptase Reaction Mix well and then add 30 ul to each PCR tube containing 10 ul of denatured DNA Place each sample in the thermocycler and run the program Extension 1 Load the Extension Thermocycler Program on the thermocycler and pause it at the first step 98 C until all samples are mixed and loaded 2 On ice make an Extension Reaction Mix with the following amounts of each reagent Add the reagents in the Volume 1 reaction Reagent Y1 Enzyme Y2 43 ul specified order 3 Mix the Extension Reaction Mix well and then add 46 ul to each PCR tube containing a 40 ul Adaptase Reaction Mix by pipetting Place in the thermocycler and run the program SPRI Step 1 Clean up the Extension Reaction using SPRIselect beads for protocol see Appendix and freshly prepared 80 ethanol refer to Page 6 Insert Size Sample Volume SPRI Volume Elution Volume 100 bp 86 ul 120 ul ratio 1 4 20 ul 200 bp 86 ul 69 ul ratio 0 8 20 ul SAFE STOPPING K POINT Store eluate at 4 C until ready to proceed 10 Ligation 1 Load the Ligation Thermocycler Program on the thermocycler and pause it at the first step 25 C until al2. SPRI Volume and Elution Volume as indicated in the table for each step 1 Invert or briefly vortex beads to homogenize the suspension before use 2 For samples with total volumes Sample Volume SPRI Volume greater than 180 ul transfer each Sample Volume sample to a 1 5 ml tube Add SPRI Volume beads to each sample Mix by pipetting 10 times or until homogenous Ensure no bead sample suspension droplets are left on the sides of the tube 3 Incubate the samples for 5 minutes at room temperature 4 Pulse spin the samples in a microfuge Place the sample tubes on a magnetic rack until the solution clears and a pellet is formed 2 minutes 5 Remove and discard the supernatant without disturbing the pellet 6 Add 180 ul of freshly prepared ethanol solution refer to Page 6 to the pellet while it is still on the magnet Use care not to disturb the pellet Incubate for 30 seconds and then carefully remove the ethanol solution 7 Repeat step 6 once for a second wash with the ethanol solution 8 Pulse spin the samples in a microfuge place back onto the magnet and remove any residual ethanol solution from the bottom of the tube 9 Air dry the pellet watching the pellet to avoid cracking or over drying 10 Add Elution Volume of Low EDTA TE to resuspend the pellet mixing well by pipetting up and down until homogenous If droplets of the resuspension are on the side of the tube pulse spin the tube in a microf
3. and whether or not Swift is advised of the possibility of such damages including without limitation damages arising from or related to loss of use loss of data downtime procurement of substitute products or services or for loss of revenue profits goodwill or business or other financial loss The total liability of Swift arising under or in connection with the purchase of the products including for any breach of contractual obligations and or any misrepresentation misstatement or tortious act or omission including without limitation negligence and liability for infringement of any third party intellectual property rights shall be limited to damages in an amount equal to the amount paid to Swift under the purchase agreement The exclusion of liability shall apply only to the extent not prohibited by applicable law 15 0275 04 15
4. is more dilute than 5 ng Input DNA is too dilute Concentrate DNA with column purification kit of pure DNA in a 10 ul volume p SpeedVac or other method DNA does not fragment properly broad or lop sided high molecular weight i fragmentation bee Fragmentation device ee sonication profile of l Ensure fragmentation device is functioning malfunction a fragmented DNA within manufacturer s parameters Incomplete resuspension of SO OSN beads after ethanol wash Over drying of beads ONNUR PIPETY ie AGANE tie peace 1 i break up clumps for complete resuspension during SPRI steps Isopropanol purification bead cleanup column pU ISA purification or other method before or If you experience problems with your library prep please contact us Email technicalsupport swiftbiosci com Phone 734 330 2568 9 00AM 5 00PM ET Monday through Friday 14 Indexed Adapter Sequences When you reach the Ligation step in the protocol you may use a unique barcoded adapter in place of non barcoded Reagent B2 to label each library Libraries made with unique barcoded adapters may be multiplexed during emulsion PCR and co sequenced on the same Ion Torrent sequencing chip CONTENTS Twenty unique barcoded adapters Barcode 1 B2 through Barcode 20 B2 provided at the same concentration as the non barcoded Reagent B2 provided in DL ION1 10 50 and which should be used as a direct replacement for non barcoded
5. ng to 5 ug The technology underlying Accel NGS does not require intact double stranded DNA making it compatible with single stranded denatured or nicked samples The unique sequential adaptation process also reduces adapter dimer formation to further maximize sequencing output The kit has been validated for use on the Ion Torrent PGM and Proton instruments The Accel NGS DNA Library Kit for Ion Torrent is suitable for the following sample types gt Single stranded samples gt Double stranded samples heat denatured gt Nicked DNA samples inquire for recommended changes to bead based clean ups gt Samples with a mixture of single stranded and double stranded DNA gt First strand cDNA The Accel NGS DNA Library Kit for Ion Torrent is suitable for the following applications gt Whole Genome Sequencing genomic DNA and Whole Genome Amplification samples gt Amplicon Sequencing long range PCR fragments and multiplex PCR gt ChIP Seq Our Technical Support team may be reached at TechnicalSupport Swiftbiosci com or by calling 734 330 2568 and pressing 2 when prompted Before You Start gt Upon receipt store the kit at 20 C gt Please read this manual carefully before starting Kit Contents Kits contain enough reagents for the preparation of either 10 or 50 libraries 10 excess volume provided Adaptase Reagent G1 44 ul 220 ul Extension Reagent Y1 33 ul 165 ul Reagents Reagent G2 88 pl 440 ul Reagents Enzyme
6. using 0 5 Sodium Hypochlorite 10 Bleach 2 Use specialty barrier pipette tips that seal when exposed to potential contaminants Prepare the Library A For best results please follow these suggestions To maximize efficient use of enzyme reagents remove enzyme tubes from 20 C storage and place on ice NOT in a cryocooler for at least 10 minutes to allow reagents to reach 4 C prior to pipetting Attempting to pipette enzymes at 20 C may result in a shortage of enzyme reagents gt After thawing reagents invert or briefly vortex except enzymes to mix them well Spin down tubes before opening gt If you have a limiting amount of starting input material fragment your sample to the smallest acceptable size in order to maximize yield as indicated in the table on Page 7 This is due to the impact of fragmentation and size selection on yield gt For heavily damaged samples it is important to use a quality control metric to analyze DNA integrity and purity If you have questions related to sample quality please contact us gt If preparing multiple libraries at once assemble reagent master mixes for each step and scale volumes as appropriate using 5 excess volume to compensate for pipetting loss Multiplexing samples for sequencing on the same chip requires the Accel NGS Barcoding Kit for Ion Torrent Cat No BC ION1 10 50 sold separately gt Always add enzymes last to master mixes immediately before adding to samp
7. For Research Use Only Swift Biosciences Inc All rights reserved Cat No DL ION1 10 50 Version 042915 WY Accel NGS DNA Library Kit for the Ion Torrent platform PCR Free NGS Library Preparation Instruction Manual Notice to Purchaser Limited License This product is for research use only and is licensed to the user under Swift Biosciences intellectual property only for the purchaser s internal purposes Trademarks Used in this Manual Accel NGS and the Swift logo are trademarks of Swift Biosciences Inc Ion Torrent PGM and Ion Proton are registered trademarks of Ion Torrent Systems Inc now part of Life Technologies Corp Agencourt SPRIselect and SPRIPlate are registered trademarks of Beckman Coulter Inc Bioanalyzer is a registered trademark of Agilent Technologies Inc Covaris is a registered trademark of Covaris Inc DynaMag is a registered trademark of Life Technologies Corp NanoDrop is a registered trademark of Thermo Fisher Scientific Inc Qubit is a registered trademark of Life Technologies Corp SpeedVac is a registered trademark of Thermo Fisher Scientific Inc TaqMan is a registered trademark of Roche Molecular Systems Inc Zymo DCC is a registered trademark of Zymo Research Corp A ee ee SS EEE Se Contents TATOU OM pe ee ee ee 3 PIOLOCON OVE NICWN cee aeeacteeeeeccscestbaccecsetactenceeaetenstecececeteestcarceceteasenncceaseeacaeusesacaeese
8. Reagent B2 at the 10 u I volume indicated in the Ligation step in the library preparation manual Adapter 50A 10B 50B Barcode 1 62 cracca 0w f sow S TS Barcode 2 B2 TAAGGAGAAC 10m som lt i Barcode 3 B2 AAGAGGATTC 109 sow f i Barcode 4 B2 TACCAAGATC 109 sow f o i Barcode 5 82 CAGAAGGAAC 109 sow f o ooo Barcode 6 82 CTacaacric 10w sow f f o ooo Barcode 7 82 Trcereartc tom sow f f oo Barcode 8 82 TTCCGATAAC 109 sow f ooo B rcode9 82 Teaccccmc 109 sow f f ooo Barcode 10 62 cTcaccomac 10w sow f o ooo Barcode 11 2 TecTcemarc Barcode 12 62 tacete Barcode 13 82 TCTAACGGAC O io i om Barcode 1462 Trecate C io o Barcode 15 62 TetaGacere Barcode 1682 TCTGGATGAC Barcode 17 62 _Terartcere Barcode 18 62 AGGCAATTGC Barcode 1982 TTAGTCGGAC Barcode 202 CAGATCCATC These sequences are identical to the IonXpress_001 through IonXpress_020 barcodes Barcode 1 B2 Lot 0358130582 Accel NGS Store at 20 C The number on the product tube label indicates which barcoded adapter is provided in the tube During library prep make sure to note which barcoded adapter you are using with your sample and do not use the same barcoded adapter on two different samples you plan to multiplex together 15 General Warranty Swift Biosciences Inc Swift warrants that it
9. Y2 460 ul 2 2 ml Reagent G3 27 5 ul 137 5 ul Ligation Buffer B1 44 ul 220 ul Enzyme G4 33 ul 165 ul Reagents Non barcoded Reagent B2 110 pl 9590 pl Buffer Low EDTA TE 2 ml 5 ml Enzyme B3 33 ul 165 ul A barcoded adapter may be used in place of non barcoded Reagent B2 when multiplexing This requires the Accel NGS Barcoding Kit for Ion Torrent BC ION1 10 50 see Appendix Required materials not supplied gt If multiplexing is desired a compatible Accel NGS Barcoding Kit for Ion Torrent Reagent B2 see Appendix Barcoding Kit for Ion Torrent Barcodes 1 10 Set A Cat No BC ION1 10A Barcoding Kit for Ion Torrent Barcodes 11 20 Set B Catalog No BC ION1 10B Barcoding Kit for Ion Torrent Barcodes 1 10 Set A Catalog No BC ION1 50A Barcoding Kit for Ion Torrent Barcodes 11 20 Set B Catalog No BC ION1 50B gt SPRIselect Beckman Coulter Catalog B23317 B23318 B23319 gt Invitrogen DynaMag Agencourt SPRIPlate or similar magnetic rack for bead cleanups gt qPCR based library quantification kit such as KAPA Biosystems Catalog KK4827 Life Technologies Catalog 4468802 gt NanoDrop Qubit or other device for determining DNA concentration gt Fragmentation device and associated reagents for DNA shearing e g Covaris gt Microcentrifuge gt Programmable thermocycler gt 0 2 ml PCR tubes gt 1 5 ml microfuge tubes gt Aerosol resistant tips and P2 through P1000 range pipettes gt 200 proof absol
10. accecueeacteneteaseeneseeneceetrestcs 5 Notes On Starting Input Material ssiccssesracevestvaveieeniaysienelaesines teansttenieecaneeraes ieee eee een 6 Prepare the Library vawacen becouse dca cucutessumahenewnnieacscamescncataceucasenencuieseusanewamentedaumebeneqnmimecaqumencnameeedmawanenteneneinent 6 DNA Fragmin g203220 c2ese2cscceanseenensncescsaeceesctciaecscseeceecesossicaciciesesaiesnseencesacencieietcscieccecectesnsessieeeiesceeea 8 BSa ni a a PA E E E E E E weNeNnRuenesnE 8 PAs S PP A E ge gw E E ene E te E T 9 EN S OI Nese cect ce E A EEEE E E E E een eee ee 9 no Eo a E oO eo E oO oOo T E oO oO ee E E oe ee oe ee oe eee eee eee 9 HOON e 10 RRE STOD 2 aer 10 Expected RESUS vsiorrrerieres Caner ENEE EEEE EEEE EEEE EEEE eee EEEE EAEE ees 11 Appendix ssssesssssnsrsnrsrnrrrnrrnrnrnrrnrnrnernrnnnornrnrnnrntnnr rnrn n sen dewucawe dew tuans cinuacwesiwiesuu dandeeandensuanngeweusaucieneans 12 Helpful Information and TrouDlGShOOting cccccscccseceeeeeeeeeeeeeeeeeeeeeeaeeeeeeaeeeeeea eens eaeeesaeaeeeeaeaneeseeaneegags 12 General Warranty cccscscccsscseceseenececneneeesneneeesneneeesneneeeseenseseenneeseeaneeseeareeseearseseearseseearseseearsaseeatsaseeatss 15 Epiese ngom NVI acs sda E E A T A E E 15 Introduction The Accel NGS DNA Library Kit for Ion Torrent is designed to enable you to make high complexity PCR free Next Generation Sequencing NGS libraries from single or double stranded DNA with input ranging from 5
11. h the use of any product including without limitation any claim of inaccurate invalid or incomplete results whether arising from a statute or otherwise in law or from a course of performance dealing or usage of trade Limitation of Liability Swift Biosciences Inc Swift shall have no liability under the warranties cited above with respect to any defect in the products arising from i specifications or materials supplied by the buyer ii wilful damage or negligence of the buyer or its employees or agents iii abnormal working conditions at the buyer s premises iv failure to follow Swift s use restrictions or instructions whether oral or in writing v misuse or alteration of the products without Swift s approval or vi if the buyer is in breach of its payment obligations in regards to purchasing the products To the fullest extent allowed by law in no event shall Swift be liable whether in contract tort strict liability negligence warranty or under any statute or on any other basis for any special incidental indirect exemplary punitive multiple or consequential damages sustained by the buyer or any other person or entity arising out of or caused by product Swift s performance or failure to perform its obligations relating to the purchase of product or performance of services Swift s breach of these terms the possession or use of any product or the performance by Swift of any services whether or not foreseeable
12. ibrary is now ready for quantification SAFE s AANA which should be performed by qPCR K POINT Note when preparing PCR free libraries it is important to specifically quantify functionally adapted molecules prior to using the OneTouch 2 Since PCR free libraries are unenriched for functional library molecules a Bioanalyzer trace will also reflect unused adapters and partially adapted library molecules Hence quantifying by this method inaccurately estimates the number of library molecules Using a qPCR based method such as the Ion Library Quantitation Kit Life Technologies Cat No 4468802 or KAPA s Library Quantification Kit for Ion Torrent Cat No KK4827 is recommended since it will lead to more accurate loading of the correct number of functional library molecules into the emulsion PCR 11 Expected Results Expected Dilution Factors for Input Quantities Regardless of Insert Size Input Quantity Expected Dilution Factor 5 ng gt 1 Example Library Size Distribution by Agilent Bioanalyzer for a 150 bp Insert Size Library Prepared from E colrDNA 35 100 150 200 300 400 500 600 1000 2000 10380 bp With lower input quantities library concentration may be too low to visualize by Bioanalyzer without PCR even when using the High Sensitivity chip E E Ct Ee M eee 12 Appendix SPRiselect Clean Up Protocol Please use the following protocol for each SPRI Step substituting in the correct Sample Volume
13. l Samples are mixed and loaded 2 Make a Ligation Reaction Mix with the following amounts of each reagent Keep on ice When barcoding multiple samples use a barcoded Reagent B2 Accel NGS Barcoding Kit for Ion Torrent see Appendix If Multiplexing Make the Ligation Reaction Mix below with all components except non barcoded Reagent B2 Add 10 ul of the appropriate barcoded Reagent B2 BC ION1 10 50 directly to each 20 ul eluate and then add 10 ul of pre mixed Ligation Reaction Mix to each sample The final reaction volume for each sample is 40 ul If Not Multiplexing Make the Ligation Reaction Mix below including 10 ul per reaction of non barcoded Reagent B2 Add 20 ul of the Ligase Reaction Mix to each PCR tube containing a 20 ul eluate The final reaction volume for each sample is 40 ul Volume 1 reaction Add the reagents in the us Reagent B2 10 ul Enzyme B3 Low EDTA TE 3 Mix the reaction well Place each sample in the thermocycler and run the program After the incubation Add 10 ul of Low EDTA TE buffer to the completed 40 ul reaction and proceed to SPRI Step 2 SPRI Step 2 Clean up the Ligation Reaction using SPRIselect beads for protocol see Appendix and freshly prepared 80 ethanol refer to Page 6 specified order Insert Size Sample Volume SPRI Volume Elution Volume 100 bp 50 ul 70 ul ratio 1 4 20 ul 200 bp 50 ul 40 ul ratio 0 8 20 ul Store freshly prepared libraries at 4 C Your l
14. les gt Before starting prepare a fresh 80 ethanol solution Approximately 720 ul will be used per sample formula is for proper volumetric addition and excess gt All loading calculations for PCR free libraries must be based on quantification by qPCR not Bioanalyzer in order to accurately load the OneTouch 2 For these reasons we recommend using the KAPA Library Quantification Kit Illumina Universal Cat No KK4824 to quantify your libraries gt Pre program a thermocycler with the following programs to expedite the workflow Denaturation Thermocycler Program Extension Thermocycler Program 95 C for 2 minutes 98 C for 1 minute 4 C hold 65 C for 5 minutes 72 C for 1 minute 4 C hold Adaptase Thermocycler Program Ligation Thermocycler Program 37 C for 10 minutes 25 C for 15 minutes 95 C for 2 minutes for enzyme inactivation 4 C hold 4 C hold DNA Fragmentation 1 Determine DNA concentration and purity using a NanoDrop with A260 280 ratio Qubit or similar method Accurate determination of DNA input amount and purity is critical for Accel NGS performance However it is not necessary to specifically quantify dsDNA as Accel NGS is compatible with both ssDNA and dsDNA 2 Fragment the DNA Multiple fragmentation methods are available this kit was validated on Covaris fragmented DNA in the appropriate size range Do not denature double stranded samples prior to fragmentation When possible fragme
15. nt double stranded DNA as yield of desired insert size will be more consistent Fragmentation of single stranded DNA may result in shorter fragments and lower library yield Depending on the method used optimization may be required Please note that single stranded DNA cannot be visualized by Bioanalyzer Either other means are required or finished libraries can be assessed by Bioanalyzer Starting Material 5ng 5 ug 100 bases 100 base read 10 ng 5 ug 200 bases 200 base read Other insert sizes are possible by adjusting SPRISelect bead ratios Optional Concentration Step If you have performed enzymatic reactions including enzymatic fragmentation OR your fragmented DNA concentration is too low to provide sufficient quantity in the 10 ul DNA starting volume specified in the Adaptase step concentrate with Zymo DCC or other method and elute in 10 ul of the Low EDTA TE buffer supplied Otherwise skip to the Denaturation Step Denaturation 1 Use the Denaturation Thermocycler Program on the thermocycler and pause it at the first step to pre heat to 95 C until all samples are ready to be loaded 2 Add 10 ul of fragmented DNA to a 0 2 ml PCR tube 3 Place each sample in the thermocycler and run the program to denature the DNA Place on ice immediately for 2 minutes Proceed immediately to the Adaptase step Adaptase 1 Use the Adaptase Thermocycler Program on the thermocycler and pause it at the first step to pre heat to
16. s products meet Swift s specifications at the time of delivery Any sample or model used in connection with Swift s product literature is for illustrative purposes only and does not constitute a warranty that the products will conform to the sample or model To the maximum extent permitted by applicable law Swift hereby expressly disclaims and the buyer hereby expressly waives any warranty regarding results obtained through the use of the products including without limitation any claim of inaccurate invalid or incomplete results All other warranties representations terms and conditions statutory express implied or otherwise as to quality condition description merchantability fitness for purpose or non infringement except for the implied warranty of title are hereby expressly excluded All warranty claims on products must be made in writing within ninety 90 days of receipt of the products Swift s sole liability and the buyer s exclusive remedy for a breach of this warranty is limited to replacement or refund at the sole option of Swift The warranties identified in this paragraph are Swift s sole and exclusive warranties with respect to the products and are in lieu of all other warranties statutory express or implied all of which other warranties are expressly disclaimed including without limitation any implied warranty of merchantability fitness for a particular purpose non infringement or regarding results obtained throug
17. uge to collect contents After at least 2 minutes place the tube on the magnet Transfer the entire eluate to a new 0 2 ml PCR tube Ensure that eluate does not contain magnetic beads indicated by brown coloration in eluate If magnetic beads are present pipette eluate into a new tube place on magnet and transfer eluate again Helpful Information and Troubleshooting Data Analysis and Informatics Considerations Swift s Adaptase technology adds a homopolymer tail with an average read length of 6 12 nucleotides to the 3 end of each fragment during the addition of the first adapter molecule Therefore it is normal and expected to observe such tails at the end of the read Alignment to reference genomes does not require trimming of the tails Quality control software such as FastQC Babraham Bioinformatics may raise Per base sequence content or Per base GC content flags These flags are expected due the homopolymer tail At low input an Overrepresented sequence flag may be raised due to adapter dimer contamination Please contact technical support with further questions 13 Troubleshooting Common Problems Possible Cause Suggested Remedy PCR free protocol allows Quantify the library by qPCR for emPCR the short oligonucleotides short oligonucleotides are not dimers and will through the final SPRI i not affect sequencing cleanup and into the product Library shows Bioanalyzer peaks at 30 45 bp Input
18. ute ethanol molecular biology grade gt Nuclease free water molecular biology grade I ee ee ee Eee Protocol Overview ssDNA Fragment pO Adaptase N adapter 1 tail Extension o CV SPRI adapter 2 Ligation 00 oy E SPRI Library The protocol uses a unique sequential adaptation process to attach adapters to the ends of single stranded DNA fragments The Adaptase step is a highly efficient proprietary reaction that simultaneously performs end repair tailing of 3 ends and ligation of the first adapter to 3 ends The Extension step is used to facilitate ligation of the second adapter The synthesized strand lacks full length adapters and does not get sequenced Bead based SPRI cleanups are used to remove oligonucleotides and small fragments and to change enzymatic buffer composition The Ligation reaction is used to add the second adapter to the 5 ends Libraries can be prepared from as low as 5 ng input material The method is ideal for samples containing single stranded or nicked DNA as well as first strand cDNA Notes on Starting Input Material gt Please consider genome complexity and sample quality when choosing input DNA quantity Although libraries may be successfully prepared from ultra low inputs reduced representation of genome complexity may occur gt To reduce the risk of DNA and library contamination particularly at low input 1 Clean lab areas
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