NEXTFLEX® Dual-Indexed DNA Barcodes

  • Enables deep multiplexing of Illumina® DNA libraries for NGS applications
  • 8 nt index contained within adapter sequence
  • Increase your sequencing scale by pooling up to 192 samples on a single flow cell
  • The kits are supplied in a 96-well plate format
  • Compatible with Illumina® next-generation sequencing platforms

  • NOVA-514160


    BARCODES 1-96
    768 rxns
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For research use only. Not for use in diagnostic procedures.

Increase Scale while Reducing Cost

The NEXTFLEX® Dual-Indexed DNA Barcodes are two kits that each contain 96 combinatorial dual indexed barcoded adapters for Illumina® library prep which enable deep multiplexing of NGS sequencing samples compatible with high-throughput automation. They significantly increase scale, while reducing costs by allowing the user to pool up to 192 samples in a single flow cell.

Multiplexing Options

Libraries made using the NEXTFLEX® Dual-Indexed DNA Barcodes can be combined with single index libraries using a dual index sequencing read primer. The NEXTFLEX® Dual-Indexed DNA Barcodes can be used with single, paired-end and multiplex reads, and are compatible with the NEXTFLEX® Rapid DNA-Seq Kit 2.0, the NEXTFLEX® Methyl DNA-Seq Kit, the NEXTFLEX® Cell Free DNA-Seq Kit and other genomic DNA library prep kits.

For larger volume requirements, customized and bulk packaging is available. Please contact [email protected] for further information.


  • NNEXTFLEX® Dual-Indexed Adapter (25 µM)
  • NEXTFLEX® Primer Mix (12.5 µM)


  • Sequences of NEXTflex Dual-Indexed DNA Barcode Indexes – Excel / PDF
The NEXTFLEX® Dual-Indexed DNA Barcodes contain 8 reactions each of 96 unique adapters compatible with Illumina® platforms, enabling the user to multiplex up to 192 samples. This kit is shipped on dry ice.

What Sequencing Primers Do I Use for Dual-Indexed Libraries?

Libraries made with the NEXTFLEX® Dual-Indexed DNA Barcodes differ from libraries made with the NEXTFLEX® DNA Barcodes in that they have two index sequences, as opposed to a single barcode index. Depending on the sequencing platform and flowcell being used, dual-indexed libraries may require the use of special sequencing primers in place of the primers found in the TruSeq® Cluster Kit.

Sequencing of dual-indexed libraries on the MiSeq® does not require the use of the TruSeq® Dual Index Sequencing Primer Box in addition to the MiSeq® Reagent Kit.

Sequencing dual-indexed libraries on the HiSeq® or GAIIx, however, may require the use of the TruSeq® Dual Index Sequencing Box. The TruSeq® Dual Index Sequencing Box, SR (Illumina® cat. # FC-121-1003) is required when sequencing dual-indexed libraries on a single-read flow cell. In this case, the Index 2 (i5) Sequencing Primer Mix, HP9, must be loaded in position 16 on the flowcell. If sequencing dual-indexed libraries on a paired-end flow cell for HiSeq® or GAIIx, the TruSeq® Dual Index Sequencing Box is not required, as the Index 2 primer is grafted on the flow cell.

In summary, sequencing dual-indexed libraries requires additional primers only if sequencing on a single-read flow cell on the HiSeq® or GAIIx. Sequencing on a paired-end flow cell on the HiSeq® or GAIIx or on the MiSeq® does not require additional primers to be purchased in addition to the typical sequencing primer kit for each sequencer.

sequencing primer flow chart

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