Accel-NGS Methyl-Seq DNA Library Kit for Illumina Platforms

Transcription

1 Instruction Manual Accel-NGS Methyl-Seq DNA Library Kit for Illumina Platforms NGS Prep with Bisulfite-Converted DNA Capability Cat. No. DL-ILMMS-12

2 Contents Introduction... 1 Experienced User Protocol... 2 Before You Start... 4 Protocol Overview... 5 Prepare the Library... 6 DNA Fragmentation... 6 Optional Concentration Step... 6 Bisulfite Conversion... 7 Denaturation... 7 Adaptase... 7 Extension... 7 SPRI Step Ligation... 8 SPRI Step Indexing PCR... 9 Post-PCR SPRI Step... 9 Expected Results Appendix SPRIselect Clean-Up Protocol Reduced Representation Bisulfite Sequencing (RRBS) Helpful Information and Troubleshooting Data Analysis and Informatics Troubleshooting Common Problems Indexed Adapter Sequences General Warranty Limitation of Liability Notes

3 Introduction The Accel-NGS Methyl-Seq DNA Library Kit for Illumina platforms enables the preparation of high complexity Next Generation Sequencing (NGS) libraries from bisulfite-converted DNA. Libraries can be made from as low as 100 pg of starting material prior to bisulfite conversion. The technology powering Accel-NGS Methyl-Seq is compatible with singlestranded DNA, making it an ideal choice for NGS library prep for DNA fragments damaged and denatured by bisulfite conversion. This single-strand compatibility overcomes the library loss associated with methylated adapter ligation followed by bisulfite conversion, enabling lower input quantities and fewer PCR cycles. The Accel-NGS Methyl-Seq Kit utilizes Illumina-compatible adapter sequences and has been validated for human whole genome bisulfite sequencing on HiSeq platforms and reduced representation bisulfite sequencing samples on the MiSeq platform. Swift Biosciences has validated and recommends the kit using the EZ DNA Methylation-Gold Kit (Zymo Research) for bisulfite conversion. The Accel-NGS Methyl-Seq DNA Library Kit is suitable for the following sample types and applications: Applications Whole Genome Bisulfite Sequencing (WGBS) Reduced Representation Bisulfite Sequencing (RRBS)* (see Appendix) Hybridization capture using NimbleGen SeqCap Epi Enrichment System* Bisulfite-converted DNA enriched by MeDIP, ChIP or other methods* *Enrichment methods generally require higher input quantities; the input quantities specified herein refer to the input quantity of either whole genomic or enriched bisulfite-treated DNA recommended for library preparation. Inquire for compatible indexing primers for use with the NimbleGen SeqCap Epi hybridization reaction. Our Technical Support team may be reached at or by calling and pressing 2 when prompted. 1

4 Experienced User Protocol 1. DNA Fragmentation (page 8) 2. Bisulfite Conversion (page 9) 3. Denaturation (page 9) Add 15 μl of fragmented DNA to a 0.2 ml PCR tube Thermocycler Conditions: 95 C for 2 minutes, lid heating ON Immediately place on ice for 2 minutes 4. Adaptase (page 9) Low EDTA TE 11.5 μl Thermocycler Conditions: Buffer G1 4 μl 37 C for 15 minutes, lid heating ON Reagent G2 4 μl 95 C for 2 minutes, lid heating ON Reagent G3 2.5 μl 4 C hold Enzyme G4 1 μl Enzyme G5 1 μl Enzyme G6 1 μl Reaction Mix 25 μl Sample 15 μl Total 40 μl 5. Extension (page 9) Reagent Y1 2 μl Thermocycler Conditions: Enzyme Y2* 42 μl 98 C for 1 minute, lid heating ON Reaction Mix 44 μl 62 C for 2 minutes, lid heating ON Adaptase Reaction 40 μl 65 C for 5 minutes, lid heating ON Total 84 μl 4 C hold *Enzyme Y2 should be mixed prior to use. 6. SPRI Step 1 (page 10) Input Sample SPRI Elution 10 ng, 350 bp 84 µl 101 µl (ratio: 1.2) 15 µl 165 bp (cfdna) 84 µl 101 µl (ratio: 1.2) 15 µl Input Sample SPRI Elution 1 st SPRI 84 µl 101 µl (ratio: 1.2) 50 µl < 10 ng, 350 bp 2 nd SPRI 50 µl 60 µl (ratio: 1.2) 15 µl 2

5 7. Ligation (page 10) Buffer B1 3 µl Thermocycler Conditions: Reagent B2 10 µl 25 C for 15 minutes, lid heating ON Enzyme B3 2 µl 4 C hold Reaction Mix 15 μl SPRI 1 Eluate 15 μl Total 30 μl Ligation Reaction 30 µl Low EDTA TE 20 µl Total 50 µl 8. SPRI Step 2 (page 10) Input Sample SPRI Elution 350 bp 50 µl 50 µl (ratio: 1.0) 20 µl 165 bp (cfdna) 50 µl 60 µl (ratio: 1.2) 20 µl 9. Indexing PCR (page 11) Low EDTA TE 10 μl Thermocycler Conditions: Buffer R1 10 µl 98 C for 30 seconds, lid heating ON Reagent R2 4 µl PCR Cycles: Enzyme R3 1 µl 98 C for 10 seconds, lid heating ON Reaction Mix 25 µl 60 C for 30 seconds, lid heating ON SPRI 2 Eluate 20 µl 68 C for 60 seconds, lid heating ON Index D50X* 2.5 µl Hold at 4 C then proceed immediately Index D70X* 2.5 µl to SPRI Step Total 50 µl * Dual indexed adapter primers must be added to each sample individually. Input Insert Size PCR Cycles 100 ng 350 bp 4 10 ng 350 bp 7 1 ng 350 bp pg 350 bp 14 5 ng 165 bp (cfdna) Post-PCR SPRI (page 11) Input Sample SPRI Elution 350 bp 50 µl 42.5 µl (ratio: 0.85) 20 µl 165 bp (cfdna) 50 µl 40 µl (ratio: 0.8) 20 µl 3

6 Before You Start Upon receipt, store the kit at -20 ºC. Please read this manual carefully before starting. Accel-NGS Methyl-Seq has been validated for an input range of 100 pg-100 ng. If you wish to use inputs outside of this range, please contact Technical Support for recommended modifications to the standard workflow. Kit Contents: Kits contain enough reagents for the preparation of 12 libraries (10% excess volume provided). Kit 12 Reaction 12 Reaction Adaptase Buffer G1 53 µl Ligation Buffer B1 40 µl Reagents Reagent G2 53 µl Reagents Reagent B2 132 µl Reagent G3 33 µl Enzyme B3 26 µl Enzyme G4 13 µl PCR Buffer R1 132 µl Enzyme G5 13 µl Reagents Reagent R2 26 µl Enzyme G6 13 µl Index 50X* 12 µl Index 70X* 9 µl Extension Reagent Y1 27 µl Enzyme R3 13 µl Reagents Enzyme Y2 568 µl Buffer Low EDTA TE 2 ml *The dual indexed adapter primers are provided separately in the Accel-NGS Methyl-Seq Indexing Kit for Illumina platforms (see Appendix). Required Materials Not Supplied: A compatible Accel-NGS Methyl-Seq Indexing Kit (see Appendix) Bisulfite conversion kit which does not include a nucleic acid carrier (see page 9), e.g. Zymo Research EZ DNA Methylation-Gold Kit (Cat. No. D5005/D5006) Magnetic beads for SPRI steps, e.g. SPRIselect beads (Beckman Coulter, Cat. No. B23317/B23318/B23319) Magnetic rack for SPRI steps, e.g. Invitrogen DynaMag qpcr-based library quantification kit NanoDrop, Qubit or other device for determining DNA concentration Method for fragmentation of input DNA by mechanical shearing or enzymatic shearing Microfuge Programmable thermocycler 0.2 ml PCR tubes 1.5 ml microfuge tubes Aerosol-resistant tips and 2 µl-1000 µl pipettes 200-proof/absolute ethanol (molecular biology grade) Nuclease-free water (molecular biology grade) 4

7 Protocol Overview The Accel-NGS Methyl-Seq protocol sequentially attaches adapters to 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 incorporate truncated adapter 1 by a primer extension reaction. The Ligation step is used to add the truncated second adapter to the bottom strand only. The Indexing PCR step incorporates full length, dual indexed adapters. Bead-based SPRI clean-ups are used to remove both oligonucleotides and small fragments, as well as to change enzymatic buffer composition. 5

8 Prepare the Library For best results, please follow these suggestions: To reduce the risk of DNA and library contamination, particularly at low input: Physically separate the laboratory space, equipment, and supplies where pre-pcr and post-pcr processes are performed. Clean lab areas using 0.5% sodium hypochlorite (10% bleach). Use barrier pipette tips. 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. After thawing reagents, invert or briefly vortex (except enzymes) to mix them well. Spin tubes in a microfuge to collect contents prior to opening. Before starting, prepare a fresh 80% ethanol solution using 200-proof/absolute ethanol and nuclease-free water (approximately 5 ml will be used per sample). Pre-program a thermocycler with the thermocycler programs to expedite the workflow. Assemble reagent master mixes for the Adaptase, Extension, Ligation and Indexing PCR steps ON ICE and scale volumes as appropriate, using 5% excess volume to compensate for pipetting loss. Add the reagents in the specified order. Libraries prepared with Accel-NGS Methyl-Seq should be sequenced as follows: MiSeq: Using the most recent software and a 10% spike in of PhiX or a balanced, high complexity library (see Appendix). HiSeq 2500: Using the most recent software, a 10% spike in of PhiX or a balanced, high complexity library, and a control lane containing a balanced, high-complexity sample (see Appendix). NextSeq 500: This instrument is sensitive to low complexity libraries, such as those produced from bisulfiteconverted DNA. Contact Illumina technical support for recommendations. DNA Fragmentation: 1. For cfdna, consult the Assessment of Concentration and Integrity of Cell-free DNA technical note to determine DNA concentration, then proceed directly to the bisulfite conversion step. For all other sample types, determine DNA concentration and purity using a fluorometric method, such as a Qubit, or similar method. However, it is not necessary to specifically quantify dsdna, as Accel-NGS Methyl-Seq is compatible with both ssdna and dsdna. As mentioned in the bisulfite conversion step below, bisulfite treatment leads to kit-dependent DNA loss. Input quantities referenced in this manual refer to total DNA quantified prior to DNA Fragmentation. 2. Fragment the DNA to 350 bp* prior to bisulfite conversion. Multiple fragmentation methods are available; this kit was validated on Covaris -fragmented DNA in the appropriate size range. Depending on the method used, optimization may be required. *Other insert sizes are possible but may require SPRIselect bead ratio adjustment. 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 20 µl DNA starting volume specified in the EZ DNA Methylation-Gold Kit, concentrate with Zymo Research DNA Clean & Concentrator or other method and elute in 15 µl of the Low EDTA TE buffer supplied. If using a different bisulfite conversion kit, follow recommendations for its starting volume. Otherwise, skip to the Bisulfite Conversion Step. 6

9 Bisulfite Conversion: Accel-NGS Methyl-Seq has been validated using the EZ DNA Methylation-Gold Kit following the manufacturer s instructions. Accel-NGS Methyl-Seq has not been validated with the QIAGEN EpiTect Bisulfite kit or other conversion kits that contain nucleic acid carriers which may interfere with the Accel-NGS Methyl-Seq protocol. Input quantities and PCR cycling recommendations are based on 50% recovery of input DNA from the EZ DNA Methylation-Gold Kit. If using another bisulfite conversion kit, become familiar with percent DNA recovery for the kit used in order to ensure sufficient bisulfite converted DNA quantity for library synthesis. We recommend quantifying the amount of DNA recovered using a NanoDrop on the RNA setting after the bisulfite conversion process to become familiar with the amount of DNA loss. Low input quantities (less than 100 ng) may not be detectable on the NanoDrop. It is also important to note that DNA will be single-stranded following bisulfite conversion. 1. The first step of the Accel-NGS Methyl-Seq library prep requires a maximum DNA volume of 15 µl, please take note of the volume for final elution of bisulfite-converted DNA to prevent sample over-dilution. For cfdna samples, quantification post-bisulfite conversion is not required. Proceed with the entire bisulfite-converted cfdna sample into the denaturation step below. Denaturation: 1. Add 15 μl of fragmented DNA to a 0.2 ml PCR tube. 2. Run the Denaturation Thermocycler Program. After the end of the program, immediately place the samples on ice for 2 minutes before proceeding to the Adaptase step. Denaturation Thermocycler Program 95 C for 2 minutes, lid heating ON Immediately place on ice for 2 minutes Adaptase: 1. Add 25 µl of the pre-mixed Adaptase Reaction Mix to the denatured DNA sample and mix the reaction well. Run the Adaptase Thermocycler Program. Reagent Low EDTA TE Buffer G1 Reagent G2 Reagent G3 Enzyme G4 Enzyme G5 Enzyme G6 Volume Per Reaction 11.5 μl 4 μl 4 μl 2.5 μl 1 μl 1 μl 1 μl Adaptase Thermocycler Program 37 C for 15 minutes, lid heating ON 95 C for 2 minutes, lid heating ON 4 C hold Extension: 1. Add 44 μl of the pre-mixed Extension Reaction Mix to each PCR tube containing 40 µl of the Adaptase Reaction and mix the reaction well. Run the Extension Thermocycler Program. Enzyme Y2 should be mixed prior to use. Reagent Volume Per Extension Thermocycler program Reaction Reagent Y1 2 μl 98 C for 1 minute, lid heating ON Enzyme Y2 42 μl 62 C for 2 minutes, lid heating ON 68 C for 5 minutes, lid heating ON 4 C hold 7

10 SPRI Step 1: 1. For 350 bp inputs 10 ng or cfdna, clean up the Extension Reaction using SPRIselect beads (refer to Appendix) and freshly prepared 80% ethanol: Insert Size Sample Volume SPRI Volume Elution Volume 10 ng, 350 bp 84 µl 101 µl (ratio: 1.2) 15 µl 165 bp (cfdna) 84 µl 101 µl (ratio: 1.2) 15 µl 2. For 350 bp inputs <10 ng, clean up the Extension Reaction with two consecutive cleanups using SPRIselect beads (refer to Appendix) and freshly prepared 80% ethanol: Insert Size Sample Volume SPRI Volume Elution Volume 1 st SPRI 84 µl 101 µl (ratio: 1.2) 50 µl < 10 ng, 350 bp 2 nd SPRI 50 µl 60 µl (ratio: 1.2) 15 µl Store eluate at 4 C until ready to proceed. Ligation: 1. Add 15 µl of the pre-mixed Ligation I Reaction Mix to each PCR tube containing 15 µl of the SPRI Step 1 eluate and mix the reaction well. Run the Ligation Thermocycler Program. 2. After the incubation, add 20 µl of Low EDTA TE buffer to the 30 µl reaction in preparation for SPRI Step 2. Reagent Volume Per Ligation Thermocycler Program Reaction Buffer B1 3 μl 25 C for 15 minutes, lid heating ON Reagent B2 10 μl 4 C hold Enzyme B3 2 μl Low EDTA TE* 20 µl *Add Low EDTA TE buffer after the incubation. SPRI Step 2: 1. Clean up the Ligation Reaction using SPRIselect beads (refer to Appendix) and freshly prepared 80% ethanol: Insert Size Sample Volume SPRI Volume Elution Volume 350 bp 50 µl 50 µl (ratio: 1.0) 20 µl 165 bp (cfdna) 50 µl 60 µl (ratio: 1.2) 20 µl Store eluate at 4 C until ready to proceed. 8

11 Indexing PCR: 1. Add 25 µl of the pre-mixed Indexing PCR Reaction Mix, 2.5 µl of D50X indexing primer, and 2.5 µl of D70X indexing primer to each PCR tube containing 20 µl of eluted library. Mix by pipetting. Reagent Low EDTA TE Buffer R1 Reagent R2 Enzyme R3 Volume Per Reaction 10 μl 10 μl 4 μl 1 μl Index D50X* 2.5 μl Index D70X* 2.5 μl *Dual indexed adapter primers must be added to each sample individually. 2. Run the Indexing PCR Thermocycler Program. Indexing PCR Thermocycler Program 98 C for 30 seconds, lid heating ON Input Insert Size PCR Cycles PCR Cycles: 100 ng 350 bp 4 98 C for 10 seconds, lid heating ON 10 ng 350 bp 7 60 C for 30 seconds, lid heating ON 1 ng 350 bp C for 60 seconds, lid heating ON 100 pg 350 bp 14 Hold at 4 C 5 ng 165 bp (cfdna) 7 Input quantities referenced in this manual refer to total DNA quantified prior to DNA Fragmentation. The number of cycles required to produce enough library for sequencing will depend on input quantity and quality. In the case of low quality samples including FFPE, the number of cycles required may vary based on the quality of the sample and amount of usable DNA present. Approximate guidelines for high quality DNA are listed above, but the exact number of cycles required must be determined by the user. Post-PCR SPRI Step: 1. Clean up the Indexing PCR Reaction using SPRIselect beads (refer to Appendix) and freshly prepared 80% ethanol: Insert Size Sample Volume SPRI Volume Elution Volume 350 bp 50 µl 42.5 µl (ratio: 0.85) 20 µl 165 bp (cfdna) 50 µl 40 µl (ratio: 0.8) 20 µl Store freshly prepared libraries at 4 C (or long term at -20 C). The libraries are now ready for quantification. Analysis of libraries by Bioanalyzer may be performed to assess size distribution. Please note the sensitivity limits specified by Agilent, and consult the application note released by Covaris titled Analysis of DNA Fragments Using the Agilent 2100 Bioanalyzer to ensure proper analysis of library size. 9

12 Expected Results Expected Yields for Input Quantities Input Quantity Insert Size PCR Cycles Expected Yield (Average) 100 ng 350 bp nm 100 pg 350 bp nm Libraries were generated from high quality, Coriell DNA (NA12878) and yields were quantified by qpcr. Library yields may vary based on DNA recovery after bisulfite conversion and input DNA quality. Libraries from 100 ng BS-converted and Unconverted Genomic DNA after PCR It is normal to observe a size shift for converted libraries relative to unconverted libraries. This is due to further fragmentation of input DNA during the bisulfite conversion process. Representative Bioanalyzer traces of libraries produced by Accel-NGS are shown here. We recommend using the High Sensitivity Chip. 10

13 Appendix SPRIselect Clean-Up Protocol Please use the following protocol for each SPRI Step, substituting in the correct Sample Volume, 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. Transfer each Sample Volume to a 1.5 ml tube and add SPRI Volume beads to each sample. Mix by vortexing. Ensure no 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 (< 5 µl may be left behind). 6. Add 500 μl of freshly prepared 80% ethanol solution 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 and discard 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 the Elution Volume of Low EDTA TE to resuspend the pellet, mixing well by pipetting up and down until homogenous. Incubate at room temperature for 2 minutes then 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. SPRI Step 1: Insert Size Sample Volume SPRI Volume Elution Volume 10 ng, 350 bp 84 µl 101 µl (ratio: 1.2) 15 µl 165 bp (cfdna) 84 µl 101 µl (ratio: 1.2) 15 µl Insert Size Sample Volume SPRI Volume Elution Volume <10 ng, 350 bp 1 st SPRI 84 µl 101 µl (ratio: 1.2) 50 µl 2 nd SPRI 50 µl 60 µl (ratio: 1.2) 15 µl SPRI Step 2: Insert Size Sample Volume SPRI Volume Elution Volume 350 bp 50 µl 50 µl (ratio: 1.0) 20 µl 165 bp (cfdna) 50 µl 60 µl (ratio: 1.2) 20 µl Post-PCR SPRI Step: Insert Size Sample Volume SPRI Volume Elution Volume 350 bp 50 µl 42.5 µl (ratio: 0.85) 20 µl 165 bp (cfdna) 50 µl 40 µl (ratio: 0.8) 20 µl 11

14 Reduced Representation Bisulfite Sequencing (RRBS) Accel-NGS Methyl-Seq is compatible with RRBS. However, due to its use of bisulfite-converted DNA as an input, it requires a few minor changes to the traditional RRBS protocol. These changes are noted in the workflow charts below. Please contact technical support for further details if you would like to use this application. 12

15 Helpful Information and Troubleshooting Data Analysis and Informatics Swift s Adaptase technology adds a low complexity tail with an average length of 8 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 beginning of Read 2. Trimming 8-10 bases using publicly available tools like Trimmomatic (Bolger, et al Bioinformatics) or Cutadapt (Martin, et al EMBnet.journal) as a part of the normal bisulfite sequence processing eliminates the majority of these tails. This is necessary to achieve accurate methylation analysis. For HiSeq V4 chemistry with 125 base reads, we recommend trimming up to 20 bases from both ends of Read 1 and Read 2 to avoid read overlap with the low complexity tail. For more specific tail trimming recommendations please contact technical support. Additionally, bisulfite conversion lowers the complexity of the DNA sample. For this reason, it is important to follow the HiSeq 2500, MiSeq, and NextSeq sequencing recommendations on page 8. The effect of trimming can be visualized by running quality control software, such as FastQC (Babraham Bioinformatics), on trimmed and untrimmed reads. The length of the tail may be estimated by the Per base sequence content plot of the FastQC. Please contact technical support with further questions. Troubleshooting Common Problems Problem Possible Cause Suggested Remedy Incomplete resuspension of beads after ethanol wash during SPRI steps. Over-drying of beads. Continue pipetting the liquid over the beads to break up clumps for complete resuspension. Shortage of enzyme reagents. Pipetting enzymes at -20 C instead of 0-4 C. Allow enzyme reagents to equilibrate to 0-4 C for 10 minutes prior to pipetting. Low library yields. Less than 50% recovery from bisulfite conversion. Low quality sample. Quantify DNA present before and after conversion. Add more DNA into conversion or increase number of PCR cycles if recovering less than 50% Add more DNA into conversion or increase number of PCR cycles If you experience problems with your library prep, please contact us by at: technicalsupport@swiftbiosci.com, or by phone at (9:00 am-5:00 pm ET, Monday-Friday). 13

16 Indexed Adapter Sequences During the Indexing PCR step in the protocol, you must add one unique Index D50X and one unique Index D70X to each library. Libraries made with uniquely indexed adapters may be multiplexed during cluster generation and cosequenced on the same Illumina flow cell. CONTENTS: Unique dual indexed adapters (see table below) which should be used where this manual calls for addition of index in the Indexing PCR step: Adapters Sequence DI-ILMMS-12 Index D501 TATAGCCT 12 μl Index D502 ATAGAGGC 12 μl Index D503 CCTATCCT 12 μl Index D701 ATTACTCG 9 μl Index D702 TCCGGAGA 9 μl Index D703 CGCTCATT 9 μl Index D704 GAGATTCC 9 μl Index D501 Lot Store at -20 o C The number on the product tube label indicates which dual indexed adapter is provided in the tube. During library prep, make sure to note which indexed adapter you are using with your sample and do not use the same set of dual indexed adapters on two different samples you plan to multiplex together. 14

17 General Warranty Swift Biosciences, Inc. ( Swift ) warrants that its 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 through 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) willful 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 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. 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. Not for use in diagnostic procedures. 15

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19 Notes 17

20 This product is for Research Use Only. Not for use in diagnostic procedures. Swift Biosciences, Inc. 58 Parkland Plaza, Suite 100 Ann Arbor, MI , Swift Biosciences, Inc. The Swift logo, Accel-NGS logo, Accel-NGS, and Adaptase are trademarks of Swift Biosciences. Illumina, HiSeq, MiSeq and NextSeq are trademarks of Illumina, Inc. EZ DNA Methylation-Gold and DNA Clean & Concentrator are trademarks of Zymo Research. SPRI and SPRIselect are trademarks of Beckman Coulter, Inc. Nimblegen and SeqCap are trademarks of Roche NimbleGen, Inc. Covaris is a trademark of Covaris, Inc. DynaMag is a trademark of Thermo Fisher Scientific Inc. NanoDrop is a trademark of Thermo Fisher Scientific, Inc.; Qubit is a trademark of Life Technologies, Inc. EpiTect is a trademark of QIAGEN. Oligonucleotide sequences Illumina, Inc. All rights reserved , 04/15