Dual Index Sequencing

Accurate Demultiplexing

Unique dual indexes reduce per-sample cost and sequence libraries with the highest accuracy compared to other indexing strategies

Dual Index Sequencing

Understanding Dual Index Sequencing

Unique dual indexes allow you to increase the number of samples sequenced per run and reduce per-sample cost compared to other indexing strategies. With just one unique dual index plate, you can pool 96 samples together.

Dual Index Sequencing Strategies

In addition to unique dual indexes, another strategy for dual index sequencing is to use combinatorial dual indexes. When preparing libraries for multiplexing, we recommend using unique dual indexes. With a dual-indexed library, the sequencing run includes two additional reads. The additional reads allow the post-run analysis software to demultiplex with increased accuracy.

Unique Dual Indexes

To prevent repeated sequences in a well plate, unique dual indexing uses unique identifiers on both ends of the sample.

One unique dual index plate enables 96 samples to be pooled together using 96 unique index 1 (i7) adapters and 96 unique index 2 (i5) adapters.

Combinatorial Dual Indexes

With combinatorial dual indexes, sequences are repeated across the rows and columns of a well plate.

Indexing is limited to index combinations of 8 unique dual pairs, so the index adapters share some sequences. Most libraries share sequences on the i7 or i5 end.

Why Use Unique Dual Indexes?

The benefits of unique dual indexing include:

  • Greater efficiency for multiplexing
  • Reduced per-sample cost by allowing more indexes to be included in a sequencing run compared to combinatorial dual or single indexes
  • Highly purified and manufactured under Good Manufacturing Practice (GMP) conditions
  • Improved design with color balance in mind
  • Mitigates index hopping by filtering hopped reads

Learn more about how unique dual indexes can help ensure high accuracy on Illumina sequencing systems.

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Comparison of UD and CD Index Adapters

Comparison of Unique Dual and Combinatorial Dual Index Adapters

One unique dual index plate enables 96 samples to be pooled together. In contrast, combinatorial dual indexing is limited to index combinations of 8 unique dual pairs.

Illumina Index Adapters Explained

Nextera library prep uses an engineered transposome to tagment genomic DNA, which is a process that fragments DNA and then tags the DNA with adapter sequences in a single step. A limited-cycle PCR step uses the adapters to amplify the insert DNA. The PCR step adds index adapter sequences on both ends of the DNA, which enables dual-indexed sequencing of pooled libraries on Illumina sequencing platforms. These index adapters cannot be used with other library preps.

TruSeq library prep uses adapter-embedded indexes to enable high throughput processing and application flexibility. The universal, methylated adapter design incorporates an index sequence at the initial ligation step. The same TruSeq kit can be used to prepare samples for single-read, paired-end, and multiplexed sequencing on all Illumina sequencing instruments. Both RNA and DNA preparation kits include adapters containing unique index sequences that are ligated to sample fragments at the beginning of the library construction process. This allows samples to be pooled and then individually identified during downstream analysis. These indexes can generally work with other library preps.

Recommended Index Adapters and Library Prep Kits for Key Applications

Application Library Prep Kits Recommended Indexing Solutions
Whole-genome sequencing Illumina DNP Prep IDT for Illumina UD Indexes
RNA sequencing Illumina Stranded mRNA Prep
Illumina Stranded Total RNA Prep
IDT for Illumina – RNA
Amplicon sequencing AmpliSeq for Illumina Panels AmpliSeq UD Indexes for Illumina (for ≤ 24 samples)
AmpliSeq UD Indexes for Illumina (for > 24 samples)
Targeted DNA enrichment Illumina DNA Prep with Enrichment IDT for Illumina Nextera UD Indexes

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Should I Use Unique Dual Indexes or Unique Molecular Identifiers?

Unique dual indexes are useful when multiplexing to mitigate sample misassignment due to index hopping. UDIs can be especially effective when using instruments with patterned flow cells, such as the NovaSeq 6000 system.

Unique molecular identifiers have been shown to reduce the rate of false-positive variant calls and increase sensitivity of variant detection. They are useful for removing PCR duplicates in DNA and cDNA and for detection of low-frequency variants such as highly-multiplexed CNV detection. Learn more about unique molecular identifiers.

Single indexing vs dual indexing

Generally speaking, dual indexing is the preferred route. Dual index sequencing does require extra cycles; however, the reagent kits supply enough to perform the extra cycles. You might still want to perform single indexing if:

  • The instrument does not support dual sequencing (HiSeq 1000/1500 systems)
  • The reads need to be a few bases longer
  • Your schedule does not permit an additional 1-2 hours required to run the second index
NovaSeq 6000

NovaSeq 6000 System

The NovaSeq 6000 System unites the latest high-performance imaging with Illumina patterned flow cell technology. The updated flow cell design reduces spacing between nanowells, significantly increasing cluster density and data output.

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Resources for Dual Index Sequencing

Illumina Adapter Sequences

This document contains the oligonucleotide sequences of Illumina adapters used in several Illumina library prep kits.

Indexed Sequencing Guide

Learn about single-indexed and dual-indexed sequencing on Illumina instruments.

Illumina Adapters Pooling Guide

This document provides recommendations for optimizing color balance across all Illumina systems when pooling indexed libraries.

Illumina Adapters Support

Find helpful resources and support information for IDT for Illumina unique dual indexes.