Here, we present an in-depth characterization of the index swapping mechanism on Illumina instruments that employ the ExAmp chemistry for cluster generation (HiSeqX, HiSeq4000, and NovaSeq). We discuss best practices for eliminating the effects of index swapping on data integrity by utilizing unique dual indexing for complete filtering of index swapped reads. We calculate mean swap rates across multiple sample preparation methods and sequencer models, demonstrating that different methods can have vastly different swap rates, and show that even non-ExAmp chemistry instruments display trace levels of index swapping. Finally, using computational methods we provide a greater insight into the mechanism of index swapping.

Characterization and remediation of sample index swaps by non-redundant dual indexing on massively parallel sequencing platforms