Abstract
BackgroundHere we present an in-depth characterization of the mechanism of sequencer-induced sample contamination due to the phenomenon of index swapping that impacts Illumina sequencers employing patterned flow cells with Exclusion Amplification (ExAmp) chemistry (HiSeqX, HiSeq4000, and NovaSeq). We also present a remediation method that minimizes the impact of such swaps.ResultsLeveraging data collected over a two-year period, we demonstrate the widespread prevalence of index swapping in patterned flow cell data. We calculate mean swap rates across multiple sample preparation methods and sequencer models, demonstrating that different library methods can have vastly different swapping rates and that even non-ExAmp chemistry instruments display trace levels of index swapping. We provide methods for eliminating sample data cross contamination by utilizing non-redundant dual indexing for complete filtering of index swapped reads, and share the sequences for 96 non-combinatorial dual indexes we have validated across various library preparation methods and sequencer models. Finally, using computational methods we provide a greater insight into the mechanism of index swapping.ConclusionsIndex swapping in pooled libraries is a prevalent phenomenon that we observe at a rate of 0.2 to 6% in all sequencing runs on HiSeqX, HiSeq 4000/3000, and NovaSeq. Utilizing non-redundant dual indexing allows for the removal (flagging/filtering) of these swapped reads and eliminates swapping induced sample contamination, which is critical for sensitive applications such as RNA-seq, single cell, blood biopsy using circulating tumor DNA, or clinical sequencing.
Highlights
We present an in-depth characterization of the mechanism of sequencer-induced sample contamination due to the phenomenon of index swapping that impacts Illumina sequencers employing patterned flow cells with Exclusion Amplification (ExAmp) chemistry (HiSeqX, HiSeq4000, and NovaSeq)
Index swapping of PCR-free genomes on HiSeqX We began multiplexing our PCR-free human whole genome libraries prior to sequencing on HiSeqX in 2015, starting with pools of 8 in February and eventually pools of 24 by November
We ran all aggregated sample BAM (Binary Alignment/Map) files generated during the past year on HiSeqX through “VerifyBamID”, a tool designed to estimate sample % contamination in human sequencing data [9] and confirmed widespread sample contamination in PCR-free libraries at an average of 1.2%
Summary
Preparation of sequencing libraries Library construction was performed using Kapa Biosystems reagents as described by Fisher et al [18] with some slight modifications. Input to Nextera based library prep was 50 ng. DNA input into sheared library prep was 100 ng. Subsequent exome capture for both somatic and germline exomes were performed using the Illumina exome oligo pool with a 38 Mb target design. For stranded RNA-seq, 250 ng of total RNA was used as input into the TruSeq stranded mRNA sequencing kit (Illumina). Dual indexed library oligos were customordered from IDT. For ligation adapters, these were ordered HPLC purified, pre-annealed, and in single use plates each at a concentration of 15 uM. For Nextera PCR primers, these were ordered standard desalted, forward and reverse premixed, and in single use plates at a concentration of 10 uM
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