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

2019; Nature Portfolio; Volume: 10; Issue: 1 Linguagem: Inglês

10.1038/s41467-019-08941-4

2041-1723

Huiran Yeom, Yonghee Lee, Taehoon Ryu, Jinsung Noh, Amos Chungwon Lee, Han‐Byoel Lee, Eunji Kang, Seo Woo Song, Sunghoon Kwon,

Advanced biosensing and bioanalysis techniques

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 (< 1%) because of high sequencing errors (> 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.