NEXTFLEX Dual-Indexed DNA Barcodes (Set A) …...NEXTFLEX® Dual-Indexed DNA Barcodes (Set A) (For...
Transcript of NEXTFLEX Dual-Indexed DNA Barcodes (Set A) …...NEXTFLEX® Dual-Indexed DNA Barcodes (Set A) (For...
Bioo Scienti�c Corporation · 7050 Burleson Road, Austin, Texas 78744 · BiooScientific.comP: 1.888.208.2246 · F: 512.707.8122 · Bioo Research Products Group · [email protected]
Made in the USA
THE NGS EXPERTS™
NEXTFLEX® Dual-IndexedDNA Barcodes (Set A)
(For Illumina® Platforms)Catalog #NOVA-514160
©Bioo Scientific Corp. • 2014-2018 V14.01
�is product is for research use only.Not for use in diagnostic procedures.
�is manual is proprietary to Bioo Scienti�c Corp., and intended only for customer use in connection with the product(s) described herein and for no other purpose. �is document and its contents shall not be used or distributed for any other purpose without the prior written consent of Bioo Scienti�c. Follow the protocol included with the kit.
Bioo Scienti�c, NEXT�ex, NextPrep, NextPrep-Mag, AIR, �e NGS Experts, qRNA, Amplicon Studio, and NanoQ are trademarks or registered trademarks of Bioo Scienti�c. All other brands and names contained herein are the prop-erty of their respective owners.
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 ourwebsite at www.biooscienti�c.com/NGSfeedback.
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THE NGS EXPERTS™ 1
NEXTflex™ Dual-Indexed DNA Barcodes (Set A) - 514160
GENERAL INFORMATION 2Product Overview 2Contents, Storage and Shelf Life 2Warnings and Precautions 2
NEXTflex™ RAPID DNA-SEQ SAMPLE PREPARATION PROTOCOL 3NEXTflex™ Rapid DNA-Seq Sample Preparation Flow Chart 3
NEXTflex™ DNA-SEQ SAMPLE PREPARATION PROTOCOL 4NEXTflex™ DNA-Seq Sample Preparation Flow Chart 4
NEXTflex™ DUAL-INDEXED DNA BARCODES PROTOCOL 5NEXTflex™ Dual-Indexed DNA Barcodes Sample Preparation Flow Chart 5
APPENDIX A 6Oligonucleotide Sequences 6Low Level Multiplexing Guidelines 8
APPENDIX B 9Plate Format 9
RELATED PRODUCTS 10
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GENERAL INFORMATION
Product Overview
The NEXTflex™ Dual-Indexed DNA Barcodes are designed to prepare multiplexed single and paired-end genomic DNA libraries for sequencing using Illumina® platforms. The in-dex and flow cell binding sequences are designed within the NEXTflex™ Dual-Indexed DNA Barcodes and attached to the sample insert during adapter ligation. Sample pooling with Dual-Indexed DNA Barcodes allows the user to multiplex up to 192 samples.
Dual-Indexed libraries are libraries prepared with adapters containing two eight base indi-ces: Index 1 (P7 Index) adjacent to the P7 strand, and Index 2 (P5 Index) adjacent to the P5 strand.
During sample preparation, 12 unique P5 index sequences are arranged across the columns of a 96-well plate, and eight unique P7 index sequences are arranged down the rows, which results in 96 uniquely dual-indexed adapters. See Appendix A for index and primer sequences.
Contents, Storage and Shelf Life
The NEXTflex™ Dual-Indexed DNA Barcodes Set A contains 96 dual-indexed barcoded DNA Adapters plated in a 96 well plate. See Appendix B for barcode plate configuration. It is recom-mended that plates are stored at -20°C. The shelf life of each reagent is 12 months when stored properly.
*The Dual-Indexed Adapters are supplied in duplex form. Do not heat the adapters above room temperature.
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 contact Bioo Scientific at [email protected].
• Do not use the kit past the expiration date.
• Ensure pipettes are properly calibrated, as library preparations are highly sensitive to pi-petting error.
• Do not heat the Dual-Indexed DNA Adapters above room temperature.
• Try to maintain a laboratory temperature of 20º– 25ºC (68º– 77ºF).
• The NEXTflex™ Primer Mix must be used during PCR amplification. Inadvertent use of an incorrect primer sequence can potentially result in elimination of the index.
Kit Contents AmountNEXTflex™ Dual-Indexed Adapter 1 - 96* (25 µM) 20 µL
NEXTflex™ Primer Mix (12.5 µM) 1536 µL
THE NGS EXPERTS™ 3
NEXTflex™ Rapid DNA-Seq Sample Preparation Flow Chart
Figure 1: Sample flow chart with approximate times necessary for each step.
NEXTflex™ RAPID DNA SAMPLE PREPARATION PROTOCOL
GENOMIC DNA
FRAGMENT
END-REPAIR & ADENYLATION
ADAPTER LIGATION
OPTIONAL SIZE SELECTION
BRIDGE AMPLIFICATION(CLUSTER GENERATION)
COMPLEXITY REDUCTION(SEQUENCE CAPTURE)
PCR
BEAD CLEANUP
40 Minutes
15 Minutes(Optional Stop Point)
30 Minutes(Optional Stop Point)
30 Minutes(Optional Stop Point)
= N= A= T= Adapters with
Cluster Sequence
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NEXTflex™ DNA-Seq Sample Preparation Flow Chart
Figure 2: Sample flow chart with approximate times necessary for each step.
NEXTflex™ DNA SAMPLE PREPARATION PROTOCOL
FRAGMENTED GENOMIC DNA
END-REPAIR (30 Minutes)(Optional Stop Point)
ADENYLATION (30 Minutes)
LIGATION (15 Minutes)(Optional Stop Point)
BRIDGE AMPLIFICATION(CLUSTER GENERATION)
COMPLEXITY REDUCTION(SEQUENCE CAPTURE)
GEL-FREE SIZE SELECTION (30 Minutes)
PCR (1 Hour)(Optional Stop Point)
BEAD CLEANUP
= N= A= T= Adapters with
Cluster Sequence
THE NGS EXPERTS™ 5
NEXTflex™ Dual-Indexed DNA Barcodes Sample Preparation Flow Chart
NEXTflex™ DUAL-INDEXED DNA BARCODES PROTOCOL
READ 1
Dual Indexed Single Read Sequencing
DNA Insert
Read 1 Primer
INDEX READ 1
P5 Index
P5 Index
Index Seq Primer
INDEX READ 2
P5 Index
P7 Index
P7 Index
P7 Index
Index Seq Primer
READ 1
Dual Indexed Paired End Sequencing
DNA Insert
Read 1 Primer
Index Seq Primer
INDEX READ 1
P5 Index
P5 IndexP7 Index
P5 Index
P7 Index
P7 Index
P7 Index
P5 Index(Chemistry Only Cycles)
Index Seq Primer
INDEX READ 2
READ 2
Read 2 Primer
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Oligonucleotide Sequences
1XXXXXXXX denotes the P5 index region of adapter. The index sequences contained in each adapter are listed below.2XXXXXXXX denotes the P7 index region of the adapter. The index sequences contained in each adapter are listed below.
APPENDIX A
NEXTflex™ Sequence (5’ → 3’)
PCR Primer 1 AATGATACGGCGACCACCGAGATCTACAC
PCR Primer 2 CAAGCAGAAGACGGCATACGAGAT
NEXTflex™Dual-Indexed
Barcode Adapter
AATGATACGGCGACCACCGAGATCTACACXXXXXXXX1ACACTCTTTCCCTACACGACGCTCTTCCGATCTGATCGGAAGAGCACACGTCTGAACTCCAGTCACXXXXXXXX2ATCTCGTATGCCGTCTTCTGCTTG
P5 Index P7 Index
DI 1 GACCTGTA CGTGTAGG
DI 2 ATGTAACT CGTGTAGG
DI 3 GTTTCAGA CGTGTAGG
DI 4 CACAGGAT CGTGTAGG
DI 5 TCAAAAAG CGTGTAGG
DI 6 CGGTGGTA CGTGTAGG
DI 7 TATGCTGC CGTGTAGG
DI 8 AGAAGACT CGTGTAGG
DI 9 TAGCTGCC CGTGTAGG
DI 10 AGCGAATG CGTGTAGG
DI 11 GTTGGACT CGTGTAGG
DI 12 AAACCGTC CGTGTAGG
DI 13 GACCTGTA GACACTAC
DI 14 ATGTAACT GACACTAC
DI 15 GTTTCAGA GACACTAC
DI 16 CACAGGAT GACACTAC
DI 17 TCAAAAAG GACACTAC
DI 18 CGGTGGTA GACACTAC
DI 19 TATGCTGC GACACTAC
DI 20 AGAAGACT GACACTAC
DI 21 TAGCTGCC GACACTAC
DI 22 AGCGAATG GACACTAC
DI 23 GTTGGACT GACACTAC
DI 24 AAACCGTC GACACTAC
P5 Index P7 Index
DI 25 GACCTGTA TGCATACA
DI 26 ATGTAACT TGCATACA
DI 27 GTTTCAGA TGCATACA
DI 28 CACAGGAT TGCATACA
DI 29 TCAAAAAG TGCATACA
DI 30 CGGTGGTA TGCATACA
DI 31 TATGCTGC TGCATACA
DI 32 AGAAGACT TGCATACA
DI 33 TAGCTGCC TGCATACA
DI 34 AGCGAATG TGCATACA
DI 35 GTTGGACT TGCATACA
DI 36 AAACCGTC TGCATACA
DI 37 GACCTGTA CAGTCTGG
DI 38 ATGTAACT CAGTCTGG
DI 39 GTTTCAGA CAGTCTGG
DI 40 CACAGGAT CAGTCTGG
DI 41 TCAAAAAG CAGTCTGG
DI 42 CGGTGGTA CAGTCTGG
DI 43 TATGCTGC CAGTCTGG
DI 44 AGAAGACT CAGTCTGG
DI 45 TAGCTGCC CAGTCTGG
DI 46 AGCGAATG CAGTCTGG
DI 47 GTTGGACT CAGTCTGG
DI 48 AAACCGTC CAGTCTGG
THE NGS EXPERTS™ 7
Note: Dual indexing on paired-end flow cells requires 23 additional cycles of sequencing: eight cycles for the Index 1 (P7 Index) read, eight cycles for the Index 2 (P5 Index) read, and seven, non-imaging, chemistry-only cycles at the beginning of the Index 2 (P5 Index) read. For sin-gle-read flow cells, dual indexing only requires 16 additional cycles of sequencing: eight cycles for the Index 1 (P7 Index) read and eight cycles for the Index 2 (P5 Index) read.
P5 Index P7 Index
DI 49 GACCTGTA TGGCACCT
DI 50 ATGTAACT TGGCACCT
DI 51 GTTTCAGA TGGCACCT
DI 52 CACAGGAT TGGCACCT
DI 53 TCAAAAAG TGGCACCT
DI 54 CGGTGGTA TGGCACCT
DI 55 TATGCTGC TGGCACCT
DI 56 AGAAGACT TGGCACCT
DI 57 TAGCTGCC TGGCACCT
DI 58 AGCGAATG TGGCACCT
DI 59 GTTGGACT TGGCACCT
DI 60 AAACCGTC TGGCACCT
DI 61 GACCTGTA CAAGGTGA
DI 62 ATGTAACT CAAGGTGA
DI 63 GTTTCAGA CAAGGTGA
DI 64 CACAGGAT CAAGGTGA
DI 65 TCAAAAAG CAAGGTGA
DI 66 CGGTGGTA CAAGGTGA
DI 67 TATGCTGC CAAGGTGA
DI 68 AGAAGACT CAAGGTGA
DI 69 TAGCTGCC CAAGGTGA
DI 70 AGCGAATG CAAGGTGA
DI 71 GTTGGACT CAAGGTGA
DI 72 AAACCGTC CAAGGTGA
P5 Index P7 Index
DI 73 GACCTGTA AAAGATAC
DI 74 ATGTAACT AAAGATAC
DI 75 GTTTCAGA AAAGATAC
DI 76 CACAGGAT AAAGATAC
DI 77 TCAAAAAG AAAGATAC
DI 78 CGGTGGTA AAAGATAC
DI 79 TATGCTGC AAAGATAC
DI 80 AGAAGACT AAAGATAC
DI 81 TAGCTGCC AAAGATAC
DI 82 AGCGAATG AAAGATAC
DI 83 GTTGGACT AAAGATAC
DI 84 AAACCGTC AAAGATAC
DI 85 GACCTGTA TGGAGCTG
DI 86 ATGTAACT TGGAGCTG
DI 87 GTTTCAGA TGGAGCTG
DI 88 CACAGGAT TGGAGCTG
DI 89 TCAAAAAG TGGAGCTG
DI 90 CGGTGGTA TGGAGCTG
DI 91 TATGCTGC TGGAGCTG
DI 92 AGAAGACT TGGAGCTG
DI 93 TAGCTGCC TGGAGCTG
DI 94 AGCGAATG TGGAGCTG
DI 95 GTTGGACT TGGAGCTG
DI 96 AAACCGTC TGGAGCTG
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Low Level Multiplexing Guidelines
Each diagonal pair seen below are fully color balanced and are suitable to be used in a pool of two samples. When designing low-plexity index pools, always include two libraries barcoded with a set of two unique and fully color balanced barcodes to avoid laser color complexity is-sues during de-multiplexing. Additional libraries may be safely multiplexed with one set of fully color balanced barcodes in a pool. The following figure illustrates possible pooling strategies.
Each color balanced pool of two barcodes is configured diagonally and shown below in connect-ed light or dark blue wells.
A
B
C
D
E
F
G
H
21 3 4 5 6 7 8 9 10 11 12
THE NGS EXPERTS™ 9
Plate Format
96 Dual-Indexed Barcoded Adapters / Plate; 20 µL (8 reaction) / well Assay Plate: Axygen P-96-450V-C; 500 µL 96 well “V” Bottom, ClearHeat Seal: 4titude Pierce Seal 4ti
APPENDIX B
Directionality of plates is indicated with corner notches
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RELATED PRODUCTS
Illumina Compatible RNA NGS Kits and Adapters
Catalog # Product
5138-01 NEXTflex™ Rapid RNA-Seq Kit (8 reactions)
5138-02 NEXTflex™ Rapid RNA-Seq Kit (48 reactions)
5138-07 NEXTflex™ Rapid Directional RNA-Seq Kit (8 reactions)
5138-08 NEXTflex™ Rapid Directional RNA-Seq Kit (48 reactions)
512911 NEXTflex™ RNA-Seq Barcodes –6
512912 NEXTflex™ RNA-Seq Barcodes – 12
512913 NEXTflex™ RNA-Seq Barcodes – 24
512914 NEXTflex™ RNA-Seq Barcodes – 48
512916 NEXTflex-96™ RNA-Seq Barcodes
5130-01 NEXTflex™ qRNA-Seq™ Kit 4 barcodes (8 reactions)
5130-02 NEXTflex™ qRNA-Seq™ Kit 24 barcodes - Set A (48 reactions)
5130-03 NEXTflex™ qRNA-Seq™ Kit 24 barcodes - Set B (48 reactions)
5130-04 NEXTflex™ qRNA-Seq™ Kit 24 barcodes - Set C (48 reactions)
5130-05 NEXTflex™ qRNA-Seq™ Kit 24 barcodes - Set D (48 reactions)
5130-01D NEXTflex™ Rapid Directional qRNA-Seq™ Kit 4 barcodes (8 reactions)
5130-02D NEXTflex™ Rapid Directional qRNA-Seq™ Kit 24 barcodes - Set A (48 reactions)
5130-03D NEXTflex™ Rapid Directional qRNA-Seq™ Kit 24 barcodes - Set B (48 reactions)
5130-04D NEXTflex™ Rapid Directional qRNA-Seq™ Kit 24 barcodes - Set C (48 reactions)
5130-05D NEXTflex™ Rapid Directional qRNA-Seq™ Kit 24 barcodes - Set D (48 reactions)
5132-01 NEXTflex™ Small RNA Sequencing Kit (24 reactions)
5132-02 NEXTflex™ Small RNA Sequencing Kit (48 reactions)
5132-03 NEXTflex™ Small RNA Sequencing Kit v2 (24 reactions)
5132-04 NEXTflex™ Small RNA Sequencing Kit v2 (48 reactions)
513305 NEXTlfex™ Small RNA Barcode Primers -12 (Set A)
513306 NEXTlfex™ Small RNA Barcode Primers -12 (Set B)
513307 NEXTlfex™ Small RNA Barcode Primers -12 (Set C)
513308 NEXTlfex™ Small RNA Barcode Primers -12 (Set D)
512979 NEXTflex™ Poly(A) Beads (8 reactions)
512980 NEXTflex™ Poly(A) Beads (48 reactions)
512981 NEXTflex™ Poly(A) Beads (100 reactions)
THE NGS EXPERTS™ 11
Illumina Compatible DNA NGS Kits and Adapters
Catalog # Product
4201-01 NEXTflex™ 16S V4 Amplicon-Seq Kit – 4
4201-02 NEXTflex™ 16S V4 Amplicon-Seq kit – 12
4201-03 NEXTflex™ 16S V4 Amplicon-Seq kit – 24
4201-04 NEXTflex™ 16S V4 Amplicon-Seq kit – 48
4201-05 NEXTflex™ 16S V4 Amplicon-Seq kit – 96
4201-06 NEXTflex™ 16S V4 Amplicon-Seq kit – 192
4201-07 NEXTflex™ 16S V4 Amplicon-Seq kit – 288
4202-01 NEXTlfex™ 16S V1-V3 Amplicon-Seq Kit - 4
4202-02 NEXTlfex™ 16S V1-V3 Amplicon-Seq Kit - 12
4202-03 NEXTlfex™ 16S V1-V3 Amplicon-Seq Kit - 48
4202-04 NEXTlfex™ 16S V1-V3 Amplicon-Seq Kit - 1-96
4202-05 NEXTlfex™ 16S V1-V3 Amplicon-Seq Kit - 97-192
4202-06 NEXTlfex™ 16S V1-V3 Amplicon-Seq Kit - 193-288
4202-07 NEXTlfex™ 16S V1-V3 Amplicon-Seq Kit - 289-384
5140-01 NEXTflex™ DNA Sequencing Kit (8 reactions)
5140-02 NEXTflex™ DNA Sequencing Kit (48 reactions)
5144-01 NEXTflex™ Rapid DNA-Seq Kit (8 reactions)
5144-02 NEXTflex™ Rapid DNA-Seq Kit (48 reactions)
5150-01 NEXTflex™ Cell Free DNA-Seq Kit (8 reactions)
5150-02 NEXTflex™ Cell Free DNA-Seq Kit (48 reactions)
514101 NEXTflex™ DNA Barcodes – 6
514102 NEXTflex™ DNA Barcodes – 12
514103 NEXTflex™ DNA Barcodes – 24
514104 NEXTflex™ DNA Barcodes – 48
514105 NEXTflex-96™ DNA Barcodes (Plate Format)
514106 NEXTflex-96™ DNA Barcodes (Tube Format)
514160 NEXTflex™ Dual-Indexed DNA Barcodes (1-96)
514161 NEXTflex™ Dual-Indexed DNA Barcodes (97-192)
5119-01 NEXTflex™ Bisulfite-Seq kit (8 reactions)
5119-02 NEXTflex™ Bisulfite-Seq kit (48 reactions)
511911 NEXTflex™ Bisulfite-Seq Barcodes – 6
511912 NEXTflex™ Bisulfite-Seq Barcodes – 12
511913 NEXTflex™ Bisulfite-Seq Barcodes - 24
5118-01 NEXTflex™ Methyl-Seq 1 Kit (8 reactions)
5118-02 NEXTflex™ Methyl-Seq 1 Kit (48 reactions)
®12
511921 NEXTflex™ Msp 1 (8 reactions)
511922 NEXTflex™ Msp 1 (48 reactions)
5143-01 NEXTflex™ ChIP-Seq Kit (8 reactions)
5143-02 NEXTflex™ ChIP-Seq Kit (48 reactions)
514120 NEXTflex™ ChIP-Seq Barcodes – 6
514121 NEXTflex™ ChIP-Seq Barcodes – 12
514122 NEXTflex™ ChIP-Seq Barcodes – 24
514123 NEXTflex™ ChIP-Seq Barcodes – 48
514124 NEXTflex-96™ ChIP-Seq Barcodes
5140-51 NEXTflex™ Pre-Capture Combo Kit (6 barcodes)
5140-52 NEXTflex™ Pre-Capture Combo Kit (12 barcodes)
5140-53 NEXTflex™ Pre-Capture Combo Kit (24 barcodes)
5140-56 NEXTflex™ Pre-Capture Combo Kit (48 barcodes)
5140-54 NEXTflex™ Pre-Capture Combo Kit (96 barcodes)
514131 NEXTflex™ DNA Barcode Blockers - 6 for SeqCap
514132 NEXTflex™ DNA Barcode Blockers - 12 for SeqCap
514133 NEXTflex™ DNA Barcode Blockers - 24 for SeqCap
514136 NEXTflex™ DNA Barcode Blockers - 48 for SeqCap
514134 NEXTflex™ DNA Barcode Blockers - 96 for SeqCap
5142-01 NEXTflex™ PCR-Free DNA Sequencing Kit (8 reactions)
5142-02 NEXTflex™ PCR-Free DNA Sequencing Kit (48 reactions)
514110 NEXTflex™ PCR-Free Barcodes – 6
514111 NEXTflex™ PCR-Free Barcodes – 12
514112 NEXTflex™ PCR-Free Barcodes – 24
514113 NEXTflex™ PCR-Free Barcodes – 48
DNA Fragmentation
Catalog # Product
5135-01 AIR™ DNA Fragmentation Kit (10 reactions)
5135-02 AIR™ DNA Fragmentation Kit (40 reactions)
�is product is for research use only.Not for use in diagnostic procedures.
�is manual is proprietary to Bioo Scienti�c Corp., and intended only for customer use in connection with the product(s) described herein and for no other purpose. �is document and its contents shall not be used or distributed for any other purpose without the prior written consent of Bioo Scienti�c. Follow the protocol included with the kit.
Bioo Scienti�c, NEXT�ex, NextPrep, NextPrep-Mag, AIR, �e NGS Experts, qRNA, Amplicon Studio, and NanoQ are trademarks or registered trademarks of Bioo Scienti�c. All other brands and names contained herein are the prop-erty of their respective owners.
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 ourwebsite at www.biooscienti�c.com/NGSfeedback.
We can’t wait to hear from you!
Bioo Scienti�c Corporation · 7050 Burleson Road, Austin, Texas 78744 · BiooScientific.comP: 1.888.208.2246 · F: 512.707.8122 · Bioo Research Products Group · [email protected]
Made in the USA
THE NGS EXPERTS™