This documentation provides an overview of indexed sequencing for Illumina sequencing systems. Indexed sequencing is a method that allows multiple libraries to be pooled and sequenced together.

Indexing libraries requires the addition of a unique identifier, or index sequence, to DNA samples during library preparation. Analysis Software (AS), Local Run Manager, Instrument Run Setup, or bcl2fastq2 Conversion Software process these tags to identify each uniquely tagged library for downstream analysis.

The number of index sequences added to samples differs for single-indexed and dual-indexed sequencing.

Single-indexed libraries—Adds Index 1 (i7) sequences to generate uniquely tagged libraries.
Dual-indexed libraries—Adds Index 1 (i7) and Index 2 (i5) sequences to generate uniquely tagged libraries.
Unique dual (UD) indexes have distinct, unrelated index adapters for both index reads. Index adapter sequences are eight or 10 bases long.
Combinatorial dual (CD) indexes have eight unique dual pairs of index adapters, so most libraries share sequences on the i7 or i5 end. Index adapter sequences are eight bases long.

During indexed sequencing, the index is sequenced in a separate read called the Index Read, where a new sequencing primer is annealed. When libraries are dual-indexed, the sequencing run includes two additional reads, called the Index 1 Read and Index 2 Read.