FOR REFERENCE PURPOSES

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1 FOR REFERENCE PURPOSES This manual is for Reference Purposes Only. DO NOT use this protocol to run your assays. Periodically, optimizations and revisions are made to the kit and protocol, so it is important to always use the protocol included with the kit. NEXTflex DNA-Seq Kit for Ion Torrent Catalog # (8 reactions) Bioo Scientific Corp V15.12

2 This product is for research use only. This manual is proprietary to Bioo Scientific Corp., and intended only for customer use in connection with 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 without the prior written consent of Bioo Scientific. Periodic optimizations and revisions are made to kit components and manuals. Follow the protocol included with the kit. Bioo Scientific makes no warranty of any kind, either expressed or implied, except that the materials from which its products are made are of standard quality. There is no warranty of merchantability for this product, or of the fitness of the product for any purpose. Bioo Scientific shall not be liable for any damages, including special or consequential damages, or expense arising directly or indirectly from the use of this product. Bioo Scientific, NEXTflex, AIR, The NGS Experts, qrna, and NanoQ are trademarks or registered trademarks of Bioo Scientific. All other brands and names contained herein are the property of their respective owners.

3 NEXTflex DNA-Seq Kit for Ion Platforms GENERAL INFORMATION 2 Product Overview 2 Contents, Storage and Shelf Life 2 Required Materials Not Provided 3 Warnings and Precautions 4 Revision History 4 NEXTflex DNA SAMPLE PREPARATION PROTOCOL 5 NEXTflex DNA Sample Preparation Flow Chart 5 Starting Material 6 Reagent Preparation 6 STEP A: End-Repair 7 STEP B: Clean-Up 8 STEP C1: Adapter Ligation Multiplexed Samples 9 STEP C2: Adapter Ligation Non-multiplexed Samples 10 STEP D: Clean-Up 11 STEP E: Agarose Gel Size Selection 13 STEP F: PCR Amplification 15 STEP G: Library Quantification 17 LIBRARY VALIDATION 18 APPENDIX A 19 Oligonucleotide Sequences 19 RELATED PRODUCTS 20 THE NGS EXPERTS 1

4 GENERAL INFORMATION Product Overview The NEXTflex DNA Sequencing Kit for Ion Torrent is designed to prepare genomic DNA or ChIP-Seq libraries for sequencing on Ion Torrent sequencing platforms. The NEXTflex DNA Sequencing Kit simplifies workflow by using master mixed reagents and magnetic bead based cleanup, reducing pipetting and eliminating time consuming steps in library preparation. In addition, the availability of up to 64 unique adapter barcodes enables high-throughput applications. The chemistry of this DNA sequencing kit is compatible with all Ion chips and base pair chemistries (including 400 bp). There are five main steps involved in preparing genomic DNA for sequencing: DNA extraction, DNA fragmentation, DNA end repair, adapter ligation and PCR amplification. The NEXTflex Sequencing Kit contains the necessary material to prepare the user s purified and fragmented genomic DNA for emulsion PCR and subsequent sequencing. Contents, Storage and Shelf Life The NEXTflex DNA Sequencing Kit for Ion Torrent contains enough material to prepare 8 genomic DNA samples for sequencing on Ion Torrent platforms. The shelf life of all reagents is 12 months when stored properly. DNA Binding Buffer, DNA Wash Buffer and the Clean-Up Spin Columns can be stored at room temperature. All of the other components can be safely stored at -20 C. Kit Contents RED CAP Amount NEXTflex End Repair Buffer Mix 56 µl NEXTflex End Repair Enzyme Mix 24 µl PURPLE CAP NEXTflex Ligation Mix 252 µl NEXTflex Adapter Mix (6.25 µm) 40 µl GREEN CAP NEXTflex Primer Mix (12.5 µm) 16 µl NEXTflex 5X PCR Master Mix 80 µl ORANGE CAP 6X Loading Dye 500 µl MW Ladder Ready-to-Load 100 bp 80 µl YELLOW CAP Column Elution Buffer 1.8 ml 2

5 WHITE CAP Nuclease-free Water Resuspension Buffer 1.5 ml 1.5 ml CLEAR CAP BOTTLE DNA Binding Buffer 5X DNA Wash Buffer 5 ml 3 ml Clean-Up Spin Columns 10 Required Materials Not Provided 10 ng, 100 ng or 1 µg of fragmented genomic DNA in up to 40 µl nuclease-free water. Optional: NEXTflex DNA Barcodes for Ion Torrent 8 / 16 / 32 / 64 (Cat # , , , ) Ethanol 100% (room temperature) Ethanol 80% (room temperature) AIR DNA Fragmentation Kit (Bioo Scientific, Cat # ) / or / Covaris System (S2, E210) 96 well PCR Plate Non-skirted (Phenix Research, Cat # MPS-499) / or / similar 96 well Library Storage and Pooling Plate (Fisher Scientific, Cat # AB-0765) / or / similar Adhesive PCR Plate Seal (BioRad, Cat # MSB1001) Agencourt AMPure XP 5 ml (Beckman Coulter Genomics, Cat # A63880) Magnetic Stand -96 (Ambion, Cat # AM10027) / or / similar Heat block Thermocycler 2, 10, 20, 200 and 1000 µl pipettes / multichannel pipettes Nuclease-free barrier pipette tips Microcentrifuge 1.5 ml nuclease-free microcentrifuge tubes Low melt agarose such as Low Gelling Temperature Agarose with a melt point of 65ºC (Boston Bioproducts, Cat # P-730) / or / E-gel system 1X TAE buffer Clean razor or scalpel SYBR Gold (Invitrogen, Cat # S11494) UV transilluminator or gel documentation instrument Gel electrophoresis apparatus Electrophoresis power supply Vortex THE NGS EXPERTS 3

6 Warnings and Precautions Bioo Scientific strongly recommends that you read the following warnings and precautions. Periodically, optimizations and revisions are made to the components and manual. Therefore, it is important to follow the protocol included with the kit. If you need further assistance, you may contact your local distributor or Bioo Scientific at Do not use the kit past the expiration date. DTT in buffers may precipitate after freezing. If precipitate is seen, vortex buffer for 1-2 minutes or until the precipitate is in solution. The performance of the buffer is not affected once precipitate is in solution. Ensure pipettes are properly calibrated as library preparations are highly sensitive to pipetting error. This kit contains a single adapter mix that will produce non-barcoded libraries. Users who do not plan on multiplexing samples during sequencing should follow Step C2 and purchase NEXTflex DNA Barcodes for Ion Torrent platforms separately. Users who wish to multiplex samples for sequencing should use the NEXTflex DNA Barcodes for Ion platforms and follow Step C1. Do not heat the DNA Adapters above room temperature. Try to maintain a laboratory temperature of 20º 25ºC (68º 77ºF). DNA sample quality may vary between preparations. It is the user s responsibility to utilize high quality DNA. DNA that is heavily nicked or damaged may cause library preparation failure. Absorbance measurements at 260 nm are commonly used to quantify DNA and 260 nm / 280 nm ratios of usually indicate relatively pure DNA. Other quantification methods using fluorescent dyes may also be used. The user should be aware that contaminating RNA, nucleotides and single-stranded DNA may affect the amount of usable DNA in a sample preparation. Revision History Version Date Description of Change V12.06 June 2012 Initial product launch. V12.08 August 2012 Adapter and Primer Mix concentration has been added. V13.02 February 2013 Clean-Up steps have been optimized to increase ease of use. V13.06 June 2013 V15.02 February 2015 V15.12 December 2015 Wording has been changed to include Ion PGM and Ion Proton. Agarose Gel Size Selection Step instructions have been clarified for 200 bp and 400 bp chemistry. Incubation times during bead clean-ups have been optimized. The Adapter Ligation step now has two options: Step C1 is intended for users who wish to multiplex samples for sequencing. Step C2 is intended for users who do not plan on multiplexing samples during sequencing. The post- End Repair Clean-Up has been modified to allow for both Adapter Ligation options. Oligo sequences of Primers 1 and 2 in the Primer Mix have been optimized. The 48 reaction size kit no longer provides a non-multiplexing Adapter Ligation option. Users should purchase the appropriate NEXTflex DNA Barcodes for Ion Torrent to use during the Adapter Ligation step. 4

7 NEXTflex DNA SAMPLE PREPARATION PROTOCOL NEXTflex DNA Sample Preparation Flow Chart Figure 1: Sample flow chart with approximate times necessary for each step. GENOMIC DNA FRAGMENT END REPAIR (30 Minutes) (Optional Stop Point) P1 Adapter LIGATION (15 Minutes) (Optional Stop Point) A Adapter CTGA GACT Key Sequence P1 Adapter SIZE-SELECTION (1-2 Hours) (Optional Stop Point) A Adapter CTGA GACT P1 Adapter PCR (1 Hour) (Optional Stop Point) A Adapter GACT CTGA GACT DILUTION FACTOR DETERMINATION BIOANALYZER AND/OR qpcr DNA TEMPLATE PREPARATION THE NGS EXPERTS 5

8 Starting Material The NEXTflex DNA Sequencing Kit for Ion Torrent has been optimized and validated using genomic DNA. This kit is designed to work with 10 ng, 100 ng or 1 µg of high quality fragmented genomic DNA. Reagent Preparation 1. Briefly spin down each component to ensure material has not lodged in the cap or side of tube. Keep on ice and vortex each NEXTflex Mix just prior to use. 2. DTT in buffers may precipitate after freezing. If precipitate is seen in any mix, vortex for 1 minute or until the precipitate is in solution. The performance of the mix is not affected once precipitate is in solution. 3. Allow Agencourt AMPure XP Beads to come to room temperature and vortex the beads until liquid appears homogenous before every use. 4. Add 56 ml of 100% ethanol to the bottle of 5X DNA Wash Buffer. Check box on bottle to show ethanol has been added. 6

9 STEP A: End-Repair Materials Bioo Scientific Supplied RED CAP - NEXTflex End Repair Buffer Mix, NEXTflex End Repair Enzyme Mix WHITE CAP - Nuclease-free Water User Supplied Thermocycler 96 well PCR Plate Adhesive PCR Plate Seal Ice Fragmented DNA in 40 µl (or less) nuclease-free water 1. For each sample, combine the following reagents on ice in a nuclease-free 96 well PCR Plate: _ µl _ µl Nuclease-free Water 2. Mix well by pipetting. Fragmented DNA (10 ng, 100 ng or 1 µg) 7 µl NEXTflex End Repair Buffer Mix 3 µl NEXTflex End Repair Enzyme Mix 50 µl TOTAL 3. Apply adhesive PCR plate seal and incubate on a thermocycler for 30 minutes at 22 C. 4. Proceed to Step B: Clean-Up. THE NGS EXPERTS 7

10 STEP B: Clean-Up Materials Bioo Scientific Supplied WHITE CAP - Resuspension Buffer User Supplied Agencourt AMPure XP Magnetic Beads (room temperature) 80% Ethanol, freshly prepared (room temperature) Magnetic Stand End Repaired DNA (From STEP A) 1. Add 90 µl of AMPure XP Beads to each sample and mix well by pipetting. 2. Incubate at room temperature for 5 minutes. 3. Place the 96 well PCR Plate on the magnetic stand at room temperature for 5 minutes or until the sample appears clear. 4. Remove and discard clear supernatant taking care not to disturb beads. Some liquid may remain in wells. 5. With plate on stand, gently add 200 µl of freshly prepared 80% ethanol to each magnetic bead pellet and incubate plate at room temperature for 30 seconds. Carefully, remove ethanol by pipette. 6. Repeat previous step, for a total of 2 ethanol washes. Ensure all ethanol has been removed. 7. Remove the plate from the magnetic stand and let dry at room temperature for 3 minutes. 8. Resuspend dried beads with 14 µl Resuspension Buffer. Mix well by pipetting. Ensure beads are no longer attached to the side of the well. 9. Incubate resuspended beads at room temperature for 2 minutes. 10. Place plate on magnetic stand at room temperature for 5 minutes or until the sample appears clear. 11. Transfer 13 µl of clear sample to new well. 12. If you wish to pause your experiment, the procedure may be safely stopped at this step and samples stored at -20 C. To restart, thaw frozen samples on ice before proceeding. 13. Proceed to Step C1 if preparing barcoded samples for multiplexed sequencing. Proceed to Step C2 if preparing non-barcoded samples for non-multiplexed sequencing. 8

11 STEP C1: Adapter Ligation Multiplexed Samples Materials Bioo Scientific Supplied PURPLE CAP - NEXTflex Ligation Mix (remove right before use and store immediately after use at -20 C) WHITE CAP - Nuclease-free Water User Supplied Thermocycler NEXTflex DNA Barcodes for Ion platforms 8 / 16 / 32 / 64 (Cat # , , , ) NEXTflex DNA P1 Adapter and NEXTflex DNA Barcode Adapter (found in DNA Barcodes for Ion platforms kits) 13 µl Purified End-Repaired DNA (from STEP B) 1. For each sample, combine the following reagents on ice in the PCR plate: For 1 μg DNA initial input: 13 µl Purified End-Repaired DNA (from Step B) 5 µl NEXTflex DNA P1 Adapter 5 µl NEXTflex DNA Barcode Adapter 31.5 µl NEXTflex Ligation Mix 54.5 µl TOTAL For ng DNA initial input: 13 µl End-Repaired DNA (from Step B) 6 µl Nuclease-free Water 2 µl NEXTflex DNA P1 Adapter 2 µl NEXTflex DNA Barcode Adapter 31.5 µl NEXTflex Ligation Mix 54.5 µl TOTAL 2. Mix well by pipetting. 3. Apply adhesive PCR plate seal and incubate on a thermocycler for 15 minutes at 22 C. 4. Proceed to Step D: Clean-Up. THE NGS EXPERTS 9

12 STEP C2: Adapter Ligation Non-multiplexed Samples Materials Bioo Scientific Supplied PURPLE CAP - NEXTflex Ligation Mix (remove right before use and store immediately after use at -20 C), NEXTflex DNA Adapter Mix WHITE CAP - Nuclease-free Water User Supplied Thermocycler Purified End-Repaired DNA (from STEP B) 1. For each sample, combine the following reagents on ice in the PCR plate: For 1 μg DNA initial input: 13 µl Purified End-Repaired DNA (from Step B) 5 µl Nuclease-free Water 5 µl NEXTflex DNA Adapter Mix 31.5 µl NEXTflex Ligation Mix 54.5 µl TOTAL For ng DNA initial input: 13 µl End-Repaired DNA (from Step B) 8 µl Nuclease-free Water 2 µl NEXTflex DNA Adapter Mix 31.5 µl NEXTflex Ligation Mix 54.5 µl TOTAL 2. Mix well by pipetting. 3. Apply adhesive PCR plate seal and incubate on a thermocycler for 15 minutes at 22 C. 4. Proceed to Step D: Clean-Up. 10

13 STEP D: Clean-Up Materials Bioo Scientific Supplied WHITE CAP - Resuspension Buffer User Supplied Agencourt AMPure XP Magnetic Beads (room temperature) 80% Ethanol, freshly prepared (room temperature) Magnetic Stand Adapter Ligated DNA (from STEP C) 1. Add 90 µl of AMPure XP Beads to each sample and mix well by pipetting. 2. Incubate at room temperature for 5 minutes. 3. Place the 96 well PCR Plate on the magnetic stand at room temperature for 5 minutes or until the sample appears clear. 4. Remove and discard clear supernatant taking care not to disturb beads. Some liquid may remain in wells. 5. With plate on stand, gently add 200 µl of freshly prepared 80% ethanol to each magnetic bead pellet and incubate plate at room temperature for 30 seconds. Carefully, remove ethanol by pipette. 6. Repeat previous step, for a total of 2 ethanol washes and ensure all ethanol has been removed. 7. Remove the plate from the magnetic stand and let dry at room temperature for 3 minutes. 8. Resuspend dried beads with 53 µl Resuspension Buffer. Mix well by pipetting and ensuring beads are no longer attached to the side of the well. 9. Incubate resuspended beads at room temperature for 2 minutes. 10. Place plate on magnetic stand at room temperature for 5 minutes or until the sample appears clear. 11. Transfer 50 µl of clear sample to new well. 12. Add 90 µl of AMPure XP Beads to each sample and mix well by pipetting. 13. Incubate at room temperature for 5 minutes. 14. Place the 96 well PCR Plate on the magnetic stand at room temperature for 5 minutes or until the sample appears clear. 15. Remove and discard clear supernatant taking care not to disturb beads. Some liquid may remain in wells. 16. With plate on stand, gently add 200 µl of freshly prepared 80% ethanol to each magnetic bead pellet and incubate plate at room temperature for 30 seconds. Carefully, remove ethanol by pipette. THE NGS EXPERTS 11

14 17. Repeat previous step, for a total of 2 ethanol washes and ensure all ethanol has been removed. 18. Remove the plate from the magnetic stand and let dry at room temperature for 3 minutes. 19. Resuspend dried beads with 22 µl Resuspension Buffer. Mix well by pipetting. Ensure beads are no longer attached to the side of the well. 20. Incubate resuspended beads at room temperature for 2 minutes. 21. Place plate on magnetic stand for 5 minutes until the sample appears clear. 22. Transfer 20 µl of clear sample to new well. 23. If you wish to pause your experiment, the procedure may be safely stopped at this step and samples stored at -20 C. To restart, thaw frozen samples on ice before proceeding. 24. Proceed to Step E: Agarose Gel Size Selection. 12

15 NOTE: E-gel systems may be used. Please follow manufacturer's protocols. Final elution should be under 38 μl. STEP E: Agarose Gel Size Selection Materials Bioo Scientific Supplied CLEAR CAP BOTTLE - DNA Binding Buffer, DNA Wash Buffer (Ethanol added, see reagent preparation) YELLOW CAP - Column Elution Buffer ORANGE CAP - 6X Gel Loading Dye, MW Ladder Ready-to-Load 100 bp User Supplied 2% TAE agarose Gel (Certified Low Gelling Temperature Agarose) 1X TAE Buffer SYBR Gold 1.5 ml nuclease-free microcentrifuge tubes Clean razor or scalpel UV transilluminator or gel documentation instrument Gel electrophoresis apparatus Electrophoresis power supply 100% Ethanol (stored at room temperature) Microcentrifuge 1. Add 4 µl of 6X Gel Loading Dye to each sample. 2. Prepare pre-stained SYBR Gold 2% low melt agarose gel by adding 15 µl of SYBR Gold to every 150 ml of cooled 1X TAE and agarose gel buffer. Mix and then pour into gel tray. Load the entire sample into one lane of the gel. If processing more than one sample, it is recommended to run separate gels or leave several empty wells between samples to avoid cross contamination. 3. Load 4 µl of MW Ladder Ready-to-Load 100 bp into one lane, skipping at least two lanes between it and your sample. 4. Run the gel with 1X TAE buffer at V for minutes. 5. Visualize the gel on a UV transilluminator or gel documentation instrument. 6. Use a clean razor or scalpel to cut out a slice of gel from each sample lane. Sizes required are dependent on the downstream kit for Ion platforms used. For 200 bp chemistry, cut a 20 bp slice no larger than 200 bp. For 400 bp chemistry, cut a 20 bp slice no larger than 400 bp. NEXTflex DNA Barcodes for Ion platforms add ~80 bp to each fragment. The user may choose other insert sizes when appropriate. THE NGS EXPERTS 13

16 7. Add 400 µl of DNA Binding Buffer to each gel slice containing sample and mix well. Incubate your sample at room temperature and vortex the sample occasionally until the agarose is completely melted. 8. Add 20 µl of 100% ethanol to each sample and mix well. 9. Transfer the sample to a Clean-Up Spin Column. 10. Centrifuge the Clean-Up Spin Column in a microcentrifuge at 14,000 rpm for 1 minute. 11. Decant the flow through and replace the Clean-Up Spin Column into the same collection tube. 12. Add 700 µl of DNA Wash Buffer to each column. Note: Prior to using the 5X DNA Wash Buffer, 12 ml of 100% ethanol must be added before first use as described in the Reagent Preparation section. 13. Centrifuge the Clean-Up Spin column in a microcentrifuge at 14,000 rpm for 1 minute. 14. Decant the flow through and replace the Clean Up Spin Column into the same collection tube 15. Repeat steps one time. 16. Centrifuge the Clean-Up Spin column in a microcentrifuge at 14,000 rpm for 1 minute to remove any residual ethanol. 17. Place the Clean-Up Spin Column into a clean 1.5 ml nuclease-free microcentrifuge tube. Add 20 µl of Column Elution Buffer to the center of the column. Incubate the column at room temperature for 1 minute. 18. Centrifuge the Clean-Up Spin Column in a microcentrifuge at 14,000 rpm for 1 minute to elute the clean DNA. If you wish to pause your experiment, the procedure may be safely stopped at this step and samples stored at -20 C. To restart, thaw frozen samples on ice before proceeding. 19. Proceed to Step F: PCR Amplification. 14

17 STEP F: PCR Amplification Materials Bioo Scientific Supplied GREEN CAP - NEXTflex Primer Mix, NEXTflex 5X PCR Master Mix WHITE CAP - Nuclease-free Water, Resuspension Buffer User Supplied Thermocycler 96 Well PCR Plate Agencourt AMPure XP Magnetic Beads (room temperature) 80% Ethanol, freshly prepared (room temperature) Magnetic Stand Size-Selected Ligation Product (from STEP E) 1. For each sample, combine the following reagents on ice in the PCR plate. Use the full amount of DNA product recovered from spin column: _ µl Size-Selected Ligation Product (from Step E) _ µl Nuclease-free Water 2 µl NEXTflex Primer Mix 10 µl NEXTflex 5X PCR Master Mix 50 µl TOTAL 2. Mix well by pipetting. 3. Apply adhesive PCR plate seal and place in thermocycler for the following PCR cycles: 20 min 65 C 2 min 95 C 30 sec 95 C 30 sec 58 C 60 sec 72 C 4 min 72 C } 1 µg input: Repeat 4 6 cycles* 100 ng input: Repeat 6 8 cycles* 10 ng input: Repeat cycles* *PCR cycles will vary depending on the amount of starting material and quality of your sample. Further optimization may be necessary. Always use the least number of cycles possible. 4. Remove PCR plate from the thermocycler. Add 70 µl of AMPure XP Beads to each sample and mix well by pipetting. 5. Incubate at room temperature for 5 minutes. 6. Place the 96 well PCR Plate on the magnetic stand at room temperature for 5 minutes until the sample appears clear. THE NGS EXPERTS 15

18 7. Set pipette to 118 µl, gently remove and discard clear sample taking care not to disturb beads. Some liquid may remain in wells. 8. With plate on stand, gently add 200 µl of freshly prepared 80% ethanol to each magnetic bead pellet and incubate plate at room temperature for 30 seconds. Carefully, remove ethanol by pipette. 9. Repeat previous step, for a total of 2 ethanol washes and ensure all ethanol has been removed. 10. Remove the plate from the magnetic stand and let dry at room temperature for 3 minutes. 11. Resuspend dried beads with 22 µl Resuspension Buffer. Mix well by pipetting and ensuring beads are no longer attached to the side of the well. 12. Incubate resuspended beads at room temperature for 2 minutes. 13. Place plate on magnetic stand for 5 minutes until the sample appears clear. 14. Transfer 20 µl of clear sample to a well of a new 96 well PCR Plate. 16

19 STEP G: Library Quantification To determine the Library Dilution factor required for Template Preparation, qpcr and/or Bioanalyzer analysis are necessary. qpcr Quantification: Materials User Supplied qpcr Thermocycler 96-well PCR Plate Ion Library Quantitation Kit (qpcr) /or/ Kapa Library Quant Kit - Ion Torrent 1. Follow the recommended protocol as provided in the user-supplied quantification kit. 2. Proceed to template preparation (emulsion PCR) using the applicable Template Preparation kit for Ion platforms. Bioanalyzer analysis: A Bioanalyzer will allow visualization of the library's size distribution. It can also be used for library quantification but this is not recommended. 1. Analyze the DNA library using an Agilent High Sensitivity DNA chip. Follow manufacturer's instructions for use with Agilent Bioanalyzer. 2. Use Bioanalyzer software to determine library concentration through peak integration. Manual adjustment may be required to integrate across the entire peak. 3. After determining the library concentration via Bioanalyzer, the Dilution Factor can be directly calculated. The Dilution Factor is measured as the library dilution which gives 280 x 106 molecules per 16 μl. Template Dilution Factor = (Library concentration in nm) x [(5 x 10 9 molecules / μl) / 8.3 nm] x [18 μl/(280 x 10 6 molecules)] Example: At 5 nm library concentration, the Dilution factor is: (5 nm) x [(5 x 10 9 molecules / μl) / 8.3 nm] x [18 μl/(280 x 10 6 molecules)] = So, with the subsequent DNA Template prep, the DNA library will be diluted 1:194, or 1 μl of library into 194 μl of Low TE buffer. THE NGS EXPERTS 17

20 LIBRARY VALIDATION Sample Bioanalyzer Trace: A) B) High Sensitivity DNA Chip Output: A) NEXTflex 1 µg, 6 cycle PCR product, 300 bp read (gel image) B) NEXTflex 1 µg, 6 cycle PCR product. 300 bp read (electropherogram) 18

21 APPENDIX A Oligonucleotide Sequences NEXTflex P1 Adapter A Adapter Primer 1 Sequence 5' CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT 5' ATCACCGACTGCCCATAGAGAGGAAAGCGGAGGCGTAGTGGTT 5' CTGAGTCGGAGACACGCAGGGATGAGATGGTT 5' CCATCTCATCCCTGCGTGTCTCCGACTCAG 5' CCACTACGCCTCCGCTTTCCTCTCTATG Primer 2 5' CCATCTCATCCCTGCGTGTC THE NGS EXPERTS 19

22 RELATED PRODUCTS Ion platforms Compatible DNA NGS Kits Catalog # Product NEXTflex DNA-Seq Kit for Ion platforms (8 reactions) NEXTflex DNA-Seq Kit for Ion platforms (48 reactions) NEXTflex DNA-Seq Kit Barcodes for Ion platforms NEXTflex DNA-Seq Kit Barcodes for Ion platforms NEXTflex DNA-Seq Kit Barcodes for Ion platforms NEXTflex DNA-Seq Kit Barcodes for Ion platforms - 64 We also have Illumina-compatible DNA sequencing kits. 20

23 WE WANT TO HEAR FROM YOU! Your feedback is important to us. Tell us what you think of our kits by scanning the QR code or visiting our website at We can t wait to hear from you!

24 THE NGS EXPERTS Bioo Scientific Corporation 7050 Burleson Road, Austin, Texas BiooScientific.com P: F: Bioo Research Products Group Made in the USA