Unique, dual-indexed sequencing adapters with UMIs effectively eliminate index cross-talk and significantly improve sensitivity of massively parallel sequencing

BMC Genomics
Laura E MacConaillAaron R Thorner

Abstract

Sample index cross-talk can result in false positive calls when massively parallel sequencing (MPS) is used for sensitive applications such as low-frequency somatic variant discovery, ancient DNA investigations, microbial detection in human samples, or circulating cell-free tumor DNA (ctDNA) variant detection. Therefore, the limit-of-detection of an MPS assay is directly related to the degree of index cross-talk. Cross-talk rates up to 0.29% were observed when using standard, combinatorial adapters, resulting in 110,180 (0.1% cross-talk rate) or 1,121,074 (0.29% cross-talk rate) misassigned reads per lane in non-patterned and patterned Illumina flow cells, respectively. Here, we demonstrate that using unique, dual-matched indexed adapters dramatically reduces index cross-talk to ≤1 misassigned reads per flow cell lane. While the current study was performed using dual-matched indices, using unique, dual-unrelated indices would also be an effective alternative. For sensitive downstream analyses, the use of combinatorial indices for multiplexed hybrid capture and sequencing is inappropriate, as it results in an unacceptable number of misassigned reads. Cross-talk can be virtually eliminated using dual-matched indexed adapters. The...Continue Reading

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Citations

Sep 7, 2018·Nature Biotechnology·Ruth E Hanna, John G Doench
May 12, 2019·Journal of Forensic Sciences·Mallory A ClarkDarrell O Ricke
Oct 12, 2019·British Journal of Haematology·Marcus C HansenCharlotte G Nyvold
Dec 18, 2019·Clinical Chemistry·Robert Schlaberg
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Dec 20, 2019·Scientific Reports·Jana BatovskaBrendan C Rodoni
Sep 8, 2018·Nature Protocols·Divya MohanH Benjamin Larman
Jan 30, 2020·BMC Genomics·Sara R SelitskyCharles M Perou
Oct 28, 2020·European Journal of Haematology·Shayu DeshpandeBrian A Walker
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Apr 4, 2021·Viruses·Solomon MainaBrendan C Rodoni
Jul 6, 2021·Molecular Diagnosis & Therapy·Giuseppa De Luca, Mariella Dono

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Methods Mentioned

BETA
PCR
genotyping
xenograft
xenografts

Software Mentioned

ExtractIlluminaBarcodes
IDT
FilterConsensusReads
xGen IDT
Conpair
Picard
IlluminaBasecallsToSam
CallMolecularConsensusReads
VarDict
GroupReadsByUmi

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