Characterization and remediation of sample index swaps by non-redundant dual indexing on massively parallel sequencing platforms.
Characterization and remediation of sample index swaps by non-redundant dual indexing on massively parallel sequencing platforms.
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DOI：
10.1186/s12864-018-4703-0
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发表时间：
2018-05-08
期刊：
影响因子：
4.4
通讯作者：
Gabriel S
中科院分区：
文献类型：
--
作者：
Costello M;Fleharty M;Abreu J;Farjoun Y;Ferriera S;Holmes L;Granger B;Green L;Howd T;Mason T;Vicente G;Dasilva M;Brodeur W;DeSmet T;Dodge S;Lennon NJ;Gabriel S
Here 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. Leveraging 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. Index 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. The online version of this article (10.1186/s12864-018-4703-0) contains supplementary material, which is available to authorized users.
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