Bioo Scienti�c Corporation · 7050 Burleson Road, Austin, Texas 78744 · BiooScientific.comP: 1.888.208.2246 · F: 512.707.8122 · Bioo Research Products Group · nextgen@biooscientific.com

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

®2

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 nextgen@biooscientific.com.

• 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

®4

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

®6

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

®8

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

®10

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 · nextgen@biooscientific.com

Made in the USA

THE NGS EXPERTS™