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Multiplex Sequencing with Index Adapters

Sample Multiplexing Overview

Multiplex sequencing allows large numbers of libraries to be pooled and sequenced simultaneously during a single run on Illumina instruments. Sample multiplexing is useful when targeting specific genomic regions or working with smaller genomes. Pooling samples exponentially increases the number of samples analyzed in a single run, without drastically increasing cost or time.

Individual "barcode" sequences are added to each DNA fragment during next-generation sequencing (NGS) library preparation so that each read can be identified and sorted before the final data analysis. These barcodes, or index adapters, can follow one of two major indexing strategies. The choice of Illumina adapters depends on your library prep kit and application.

Conceptual Overview of Sample Multiplexing

Multiplex Sequencing Highlights

  • Fast High-Throughput Strategy: Large sample numbers can be simultaneously sequenced during a single experiment
  • Cost-Effective Method: Sample pooling improves productivity by reducing time and reagent use
  • Simplified Analysis: Automatic sample identification with "barcodes" using Illumina data analysis software
Multiplexing Facilitates Microbial Studies

Researchers leverage the speed and multiplexing capabilities of Illumina sequencers to identify microbes along the Mississippi River.

Read Interview

Overview of Illumina Index Adapters

Two types of dual indexing options are available for multiplex sequencing: unique dual (UD) indexes and combinatorial dual (CD) indexes.

Unique Dual Indexes

UD indexing uses unique identifiers on both ends of the sample. It involves 96 unique Index 1 (i7) adapters and 96 unique Index 2 (i5) adapters, preventing repeated sequences in a well plate. Along with other best practices, the unique dual indexing strategy will help make sure that your libraries will demultiplex with high accuracy across all Illumina sequencing platforms.

Benefits of UD indexing:
  • Greater efficiency for multiplexing
  • Reduces per-sample cost by allowing more indexes to be included in a sequencing run compared to CD or single indexes
  • Highly purified and manufactured under Good Manufacturing Practice (GMP) conditions
  • Improved design with color balance in mind
  • Index hopping mitigation by filtering hopped reads
Combinatorial Dual Indexes

With CD indexing, combinations of certain i5 indexes with i7 indexes allow for sample identification. CD indexing involves 12 unique Index 1 adapters (i7) and 8 unique Index 2 adapters (i5), so sequences are repeated across the rows and columns of a well plate.

Understanding Unique Dual Indexes

When preparing libraries for multiplexing, we encourage customers to use UD indexing whenever possible.

Read Bulletin

Comparison of UD and CD 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.

Comparison of UD and CD Index Adapters
Recommended Index Adapters and Library Prep Kits for Key Applications
Application Library Prep Kit Recommended Indexing Solution
Whole-genome sequencing Nextera DNA Flex Library Prep Kit IDT for Illumina Nextera UD Indexes
RNA sequencing TruSeq Stranded mRNA
TruSeq Stranded Total RNA
IDT for Illumina – TruSeq RNA UD Indexes
Amplicon sequencing AmpliSeq for Illumina Panels AmpliSeq UD Indexes for Illumina (for ≤ 24 samples)
AmpliSeq CD Indexes for Illumina (for > 24 samples)
Targeted DNA enrichment Nextera Flex for Enrichment IDT for Illumina Nextera UD Indexes

Resources for Using Illumina Adapters

Illumina Adapter Sequences

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

Indexed Sequencing Guide

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

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.

Chan Zuckerberg Biohub and the NovaSeq System

Researchers at the Chan Zuckerberg Biohub are working to conduct innovative experiments and form new collaborations in genomics. The NovaSeq System enables them to bundle many samples together for medical research that may help to improve human health.

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Chan Zuckerberg Biohub and the NovaSeq System

Related Information

  • Minimizing Index Hopping: Find tips and best practices to avoid sequencing read misalignment when using Illumina adapters.
  • High-Throughput Library Prep Automation: Learn about automated liquid handling solutions designed to help labs prepare large quantities of sequencing libraries.
  • Consulting Services: Whether you are newly applying NGS or ramping up high-throughput sequencing, our highly experienced global network is here to help.
Flexible High-Throughput Sequencer

The NovaSeq 6000 System offers scalable throughput and flexibility for virtually any genome, sequencing method, and scale of project.

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