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.
Two types of dual indexing options are available for multiplex sequencing: unique dual (UD) indexes and combinatorial dual (CD) 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.
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.
|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|
This document contains the oligonucleotide sequences of Illumina adapters used in several library prep kits.
Learn about single-indexed and dual-indexed sequencing on Illumina sequencing systems.
This document provides recommendations for optimizing color balance across all Illumina systems when pooling indexed libraries.
Find helpful resources and support information for IDT for Illumina unique dual indexes.