BIOO LIFE SCIENCE PRODUCTS. NEXTflex TM 16S V4 Amplicon-Seq Kit 4 (Illumina Compatible) BIOO Scientific Corp V13.01

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1 BIOO LIFE SCIENCE PRODUCTS NEXTflex TM 16S V4 Amplicon-Seq Kit 4 (Illumina Compatible) Catalog #: (16 reactions) BIOO Scientific Corp V13.01

2 TABLE OF CONTENTS GENERAL INFORMATION... 1 Product Overview... 1 Contents, Storage and Shelf Life... 1 Required Materials not Provided... 2 Warnings and Precautions... 3 NEXTflex 16S V4 AMPLICON PREPARATION PROTOCOL... 4 NEXTflex 16S V4 Amplicon Sample Preparation Flow Chart... 4 Reagent Preparation... 4 STEP A: PCR Amplification... 5 STEP B: Post-PCR Cleanup... 6 LIBRARY VALIDATION... 8 APPENDIX A... 9 Oligonucleotide Sequences... 9 RELATED PRODUCTS Illumina Compatible DNA NGS Kits and Adapters DNA Fragmentation Illumina Compatible RNA NGS Kits and Adapters The NEXTflex 16S V4 Amplicon-Seq Kit is intended for research use only. Scientific Corporation. NEXTflex is a trademark of Bioo

3 GENERAL INFORMATION Product Overview GENERAL INFORMATION The NEXTflex 16S V4 Amplicon-Seq Kit is designed to prepare multiplexed amplicon libraries of the fourth hypervariable (V4) domain of microbial 16S ribosomal RNA (rrna) genes. These libraries are compatible with the Illumina MiSeq platform when used with the provided Custom Sequencing Read Primers and Custom Index Primers. The specialized 16S V4 primers contain the relevant flow cell binding domain, sequencing read primer regions and unique 12 base pair barcode domains. A bead-based PCR cleanup streamlines processing multiple samples at once. There are two main steps involved in 16S V4 amplicon processing: PCR amplification using customized PCR primers and PCR cleanup. The limited number of cleanup steps ensures maximum recovery of amplicons for downstream sequencing. Contents, Storage and Shelf Life The NEXTflex 16S V4 Amplicon-Seq Kit contains enough material to prepare 16 16S V4 samples from bacterial genomic DNA for Illumina compatible sequencing. The shelf life of all reagents is 12 months when stored properly. All components can be safely stored at -20 C. Kit Contents Amount GREEN CAP NEXTflex DNA PCR Master Mix 192 µl ORANGE CAP NEXTflex 16S V4 Forward Primer (5 µm) 32 µl YELLOW CAP NEXTflex 16S V4 Reverse Primer Barcode (5 µm) µl WHITE CAP Resuspension Buffer Nuclease-free Water 1 ml 1.5 ml PURPLE CAP Custom Sequencing Read Primer 1 (100 µm) 55 µl Custom Sequencing Read Primer 2 (100 µm) 55 µl Custom Index Read Primer (100 µm) 55 µl BIOO LIFE SCIENCE PRODUCTS 1

4 Required Materials not Provided 50 ng high-quality bacterial genomic DNA in up to 34 µl nuclease-free water. 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 Thermocycler 2, 10, 20, 200 and 1000 L pipettes / multichannel pipettes Nuclease-free barrier pipette tips 1.5 ml nuclease-free microcentrifuge tubes Vortex 80% Ethanol, freshly prepared (room temperature) BIOO LIFE SCIENCE PRODUCTS 2

5 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. Ensure pipettes are properly calibrated as library preparations are highly sensitive to pipetting error. Try to maintain a laboratory temperature of 20º 25ºC (68º 77ºF). Genomic DNA sample quality may vary between preparations. It is the user s responsibility to utilize high quality Genomic DNA. Genomic 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. It is required that both NEXTflex 16S V4 Forward and Reverse Primers are used during PCR amplification. It is required that the Custom Sequencing Read Primers and the Custom Index Read Primer are spiked in rather than added separately. Spiking in the primers will allow both PhiX and the 16S V4 templates to be read properly. 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 of 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 damage, or expense arising directly or indirectly from the use of this product. BIOO LIFE SCIENCE PRODUCTS 3

6 NEXTflex 16S V4 AMPLICON PREPARATION PROTOCOL NEXTflex 16S V4 AMPLICON PREPARATION PROTOCOL NEXTflex 16S V4 Amplicon Sample Preparation Flow Chart Figure 1: Sample flow chart with approximate times necessary for each step. Starting Material The NEXTflex 16S V4 Amplicon-Seq Kit has been optimized and validated using 50 ng of high-quality bacterial 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. Allow Agencourt AMPure XP Beads to come to room temperature and vortex the beads until liquid appears homogenous before every use. BIOO LIFE SCIENCE PRODUCTS 4

7 STEP A: PCR Amplification Materials Bioo Scientific Supplied GREEN CAP NEXTflex DNA PCR Master Mix ORANGE CAP NEXTflex 16S V4 Forward Primer YELLOW CAP NEXTflex 16S V4 Reverse Primer Barcode 1 4 WHITE CAP Nuclease-Free Water User Supplied Thermocycler 96 Well PCR Plate High-Quality Bacterial Genomic DNA, 50 ng 1. For each sample, combine the following reagents on ice in the PCR plate. _ µl High-Quality Bacterial Genomic DNA (50 ng in at most 34 µl) _ µl Nuclease-free H 2O 12 µl NEXTflex DNA PCR Master Mix 2 µl NEXTflex 16S V4 Forward Primer 2 µl NEXTflex 16S V4 Reverse Primer Barcode 50 µl TOTAL 2. Set pipette to 50 µl, gently pipette the entire volume up and down 10 times. 3. Apply adhesive PCR plate seal and place in thermocycler for the following PCR cycles: 2 min 98 C 45 sec 98 C 60 sec 56 C Repeat 30 cycles* 90 sec 72 C 10 min 72 C *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. BIOO LIFE SCIENCE PRODUCTS 5

8 STEP B: Post-PCR Cleanup 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 1. Add 28 µl of AMPure XP Beads to each clear sample and gently pipette the entire volume up and down 10 times. 2. Incubate at room temperature for 5 minutes. 3. Place the 96 well PCR Plate on the magnetic stand at room temperature until the supernatant appears completely clear. 4. Do not discard the supernatant in this step. Transfer 76 µl of clear supernatant to a new well. Be careful not to disrupt the magnetic bead pellet or transfer any magnetic beads with the sample. The bead pellet binds and removes genomic DNA. 5. Add 19 µl of AMPure XP Beads to each well containing sample and gently pipette the entire volume up and down 10 times. 6. Incubate at room temperature for 5 minutes. 7. Place the 96 well PCR Plate on the magnetic stand at room temperature or until the supernatant appears completely clear. 8. Gently remove and discard 93 µl of the supernatant taking care not to disturb beads. Some liquid may remain in wells. 9. 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. 10. Repeat step 20, for a total of 2 ethanol washes. Ensure all ethanol has been removed. 11. Remove the plate from the magnetic stand and let dry at room temperature for 3 minutes. 12. Resuspend dried beads with 22 µl Resuspension Buffer. Gently, pipette entire volume up and down 10 times mixing thoroughly. Ensure beads are no longer attached to the side of the well. 13. Incubate resuspended beads at room temperature for 2 minutes. 14. Place plate on magnetic stand for 5 minutes until the sample appears clear. 15. Gently transfer 20 µl of clear supernatant to new well. BIOO LIFE SCIENCE PRODUCTS 6

9 16. To ensure cluster generation it is recommended that you quantify your library by gel or Agilent Bioanalyzer. To quantify by gel, load 2 µl of 6X Gel Loading Dye and 10 µl of PCR Product in a SYBR stained 2% low melt agarose gel. 17. Quantitate DNA library templates for optimal cluster density. 18. For multiplexed libraries, transfer 10 µl of each normalized library to be pooled in the well of a new PCR plate. Gently pipette the entire volume up and down 10 times. 19. Once desired sequencing concentration is reached, spike in 20 40% denatured PhiX to balance base bias present in 16S amplicon samples. PhiX must be spiked in rather than run separately. 20. When preparing the MiSeq sequencing cartridge, add Custom Sequencing Read Primer 1, Custom Sequencing Read Primer 2 and Custom Index Read Primer to their respective reservoirs so that the final concentration of each is 0.5 µm. (Add 3.4 µl of 100 µm custom primer to a 680 µl reservoir). For HiSeq runs, the Custom Sequencing Read Primers should each be at a final concentration of 0.5 µm in Illumina s HT1 Buffer. BIOO LIFE SCIENCE PRODUCTS 7

10 LIBRARY VALIDATION LIBRARY VALIDATION Figure 2. Gel validation of the NEXTflex 16S V4 Amplicon PCR product (30 cycles). A B C Desired 16S V4 amplicon PCR product 2% Agarose gel A) E. coli genomic DNA, 250 ng B) MW Ladder Ready-to-Load- 100bp C) 16S V4 amplicon product, 30 PCR cycles, 50 ng E. coli genomic DNA input BIOO LIFE SCIENCE PRODUCTS 8

11 APPENDIX A Oligonucleotide Sequences APPENDIX A NEXTflex 16S V4 Forward Primer 16S V4 Reverse Primer 1 16S V4 Reverse Primer 2 16S V4 Reverse Primer 3 16S V4 Reverse Primer 4 Custom Sequencing Read Primer 1 Custom Sequencing Read Primer 2 Custom Sequencing Index Primer 1 Sequence 5 AATGATACGGCGACCACCGAGATCTACACTATGGTAATTGTGTGCCAGCMGCCGCGGTAA 5 CAAGCAGAAGACGGCATACGAGATTGGTCAACGATAAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 5 CAAGCAGAAGACGGCATACGAGATTGCATACACTGGAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 5 CAAGCAGAAGACGGCATACGAGATACCAGTGACTCAAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 5 CAAGCAGAAGACGGCATACGAGATCATTATGGCGTGAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 5 TATGGTAATTGTGTGCCAGCMGCCGCGGTAA 5 AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 5 ATTAGAWACCCBDGTAGTCCGGCTGACTGACT For an electronic list of the DNA barcodes contact: nextgen@biooscientific.com. Low Level Mulitplexing: Use the following barcode combinations for low level multiplexing in this kit: 2 barcodes: (1, 3) or (2, 4) 3 barcodes: (1, 2, 4) 4 barcodes: any above triplet plus another barcode BIOO LIFE SCIENCE PRODUCTS 9

12 RELATED PRODUCTS RELATED PRODUCTS Illumina Compatible DNA NGS Kits and Adapters Product Catalog Number NEXTflex 16S V4 Amplicon-Seq kit NEXTflex 16S V4 Amplicon-Seq kit NEXTflex 16S V4 Amplicon-Seq kit NEXTflex DNA Barcodes NEXTflex DNA Barcodes NEXTflex DNA Barcodes NEXTflex DNA Barcodes NEXTflex-96 DNA Barcodes NEXTflex DNA Sequencing Kit (8 reactions) NEXTflex DNA Sequencing Kit (48 reactions) NEXTflex Bisulfite-Seq kit (8 reactions) NEXTflex Bisulfite-Seq kit (48 reactions) NEXTflex Bisulfite-Seq Barcodes NEXTflex Bisulfite-Seq Barcodes NEXTflex Msp 1 (8 reactions) NEXTflex Msp 1 (48 reactions) NEXTflex DNA Sequencing Kit (8 reactions) NEXTflex DNA Sequencing Kit (48 reactions) NEXTflex DNA Barcodes NEXTflex DNA Barcodes NEXTflex DNA Barcodes NEXTflex DNA Barcodes NEXTflex-96 DNA Barcodes NEXTflex ChIP-Seq Kit (8 reactions) NEXTflex ChIP-Seq Kit (48 reactions) NEXTflex ChIP-Seq Barcodes NEXTflex ChIP-Seq Barcodes NEXTflex ChIP-Seq Barcodes NEXTflex ChIP-Seq Barcodes NEXTflex-96 ChIP-Seq Barcodes NEXTflex PCR-Free DNA Sequencing Kit (8 reactions) NEXTflex PCR-Free DNA Sequencing Kit (48 reactions) NEXTflex PCR-Free Barcodes NEXTflex PCR-Free Barcodes NEXTflex PCR-Free Barcodes NEXTflex PCR-Free Barcodes NEXTflex Methyl-Seq 1 kit (8 reactions) NEXTflex Methyl-Seq 1 kit (48 reactions) BIOO LIFE SCIENCE PRODUCTS 10

13 DNA Fragmentation Product Catalog Number AIR DNA Fragmentation Kit (10 reactions) AIR DNA Fragmentation Kit (40 reactions) Illumina Compatible RNA NGS Kits and Adapters Product Catalog Number NEXTflex RNA-Seq Kit (8 reactions) NEXTflex RNA-Seq Kit (48 reactions) NEXTflex RNA-Seq Barcodes NEXTflex RNA-Seq Barcodes NEXTflex RNA-Seq Barcodes NEXTflex RNA-Seq Barcodes NEXTflex-96 RNA-Seq Barcodes NEXTflex Directional RNA-Seq Kit (dutp-based) (8 reactions) NEXTflex Directional RNA-Seq Kit (dutp-based) (48 reactions) NEXTflex Directional RNA-Seq Barcodes Set A NEXTflex Directional RNA-Seq Barcodes Set B NEXTflex Directional RNA-Seq Barcodes Set C NEXTflex Directional RNA-Seq Barcodes Set D NEXTflex Small RNA Sequencing Kit (24 reactions) NEXTflex Small RNA Sequencing Kit (48 reactions) NEXTflex Small RNA Barcodes Set A NEXTflex Small RNA Barcodes Set B NEXTflex Small RNA Barcodes Set C NEXTflex Small RNA Barcodes Set D Bioo Scientific also offers library prep kits and barcodes for the Ion Torrent, 5500 SOLiD and SOLiD 4 sequencing platforms. For more information about any of these kits visit our website at Bioo Scientific Corporation 3913 Todd Lane Suite 312 Austin, TX USA Tel: Fax: (512) Made in USA BIOO Research Products Group nextgen@biooscientific.com BIOO LIFE SCIENCE PRODUCTS 11