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

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. 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.