Dual-Indexed Sequencing Overview

Dual-indexed sequencing includes two index reads after Read 1: the Index 1 Read and the Index 2 Read.

Sequencing kits include a paired-end flow cell.

The control software performs Read 1, any index reads, and then Read 2 based on the parameters provided for the run in the sample sheet or during run setup.

For all indexing workflows, the Index 1 Read directly follows Read 1. However, for dual-indexing on a paired-end flow cell, the rest of the workflow differs.

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Forward strand—The Index 2 Read occurs before Read 2 resynthesis, so the Index 2 (i5) adapter is sequenced on the forward strand.

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Reverse complement—The Index 2 Read occurs after Read 2 resynthesis, which creates the reverse complement of the Index 2 (i5) index adapter sequence.