Barcode-free next-generation sequencing error validation for ultra-rare variant detection

Huiran Yeom, Yonghee Lee, Taehoon Ryu, Jinsung Noh, Amos Chungwon Lee, Han-Byoel Lee, Eunji Kang, Seo Woo Song and Sunghoon Kwon ()
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Huiran Yeom: Seoul National University
Yonghee Lee: Seoul National University
Taehoon Ryu: Celemics Inc.
Jinsung Noh: Seoul National University
Amos Chungwon Lee: Seoul National University
Han-Byoel Lee: Seoul National University Hospital Biomedical Research Institute
Eunji Kang: Seoul National University
Seo Woo Song: Seoul National University
Sunghoon Kwon: Seoul National University

Nature Communications, 2019, vol. 10, issue 1, 1-8

Abstract: Abstract The advent of next-generation sequencing (NGS) has accelerated biomedical research by enabling the high-throughput analysis of DNA sequences at a very low cost. However, NGS has limitations in detecting rare-frequency variants ( 0.1~1%). NGS errors could be filtered out using molecular barcodes, by comparing read replicates among those with the same barcodes. Accordingly, these barcoding methods require redundant reads of non-target sequences, resulting in high sequencing cost. Here, we present a cost-effective NGS error validation method in a barcode-free manner. By physically extracting and individually amplifying the DNA clones of erroneous reads, we distinguish true variants of frequency > 0.003% from the systematic NGS error and selectively validate NGS error after NGS. We achieve a PCR-induced error rate of 2.5×10−6 per base per doubling event, using 10 times less sequencing reads compared to those from previous studies.

Date: 2019
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DOI: 10.1038/s41467-019-08941-4

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