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

• Published in: Nature communications Vol. 10; no. 1; p. 977
• Main Authors: Yeom, Huiran, Lee, Yonghee, Ryu, Taehoon, Noh, Jinsung, Lee, Amos Chungwon, Lee, Han-Byoel, Kang, Eunji, Song, Seo Woo, Kwon, Sunghoon
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 28.02.2019; Nature Publishing Group; Nature Portfolio
• Subjects: 45/22; 45/23; 45/56; 45/71; 631/114; 631/61; Bar codes; Bioinformatics; Biomedical research; Cloning; Cloning, Molecular; Cost analysis; Data integrity; Deoxyribonucleic acid; DNA; DNA Barcoding, Taxonomic; DNA, Bacterial - genetics; Error detection; Escherichia coli - genetics; Gene Library; Gene sequencing; Genetic Variation; High-Throughput Nucleotide Sequencing - methods; High-Throughput Nucleotide Sequencing - standards; High-Throughput Nucleotide Sequencing - statistics & numerical data; Humanities and Social Sciences; Lasers; Medical research; Methods; multidisciplinary; Mutation; Next-generation sequencing; Nucleotide sequence; Polymerase Chain Reaction; Quality Control; Science; Science (multidisciplinary); Sequence Analysis, DNA - methods; Sequence Analysis, DNA - standards; Sequence Analysis, DNA - statistics & numerical data
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-019-08941-4

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. Next generation sequencing has difficulty in detecting rare-frequency variants due to high sequencing errors. Here the authors present a barcode-free error validation method that physically extracts erroneous reads to identify true variants.