Long-read next generation sequencing approach to close the gaps of short-read sequencing in hereditary breast and/or ovarian cancer

Abstract

Background: Advances in next generation sequencing (NGS) have revolutionized genetic profiling in hereditary breast cancer. The development of molecular barcode NGS enables simultaneous detection of DNA-copy number variation (CNV) and nucleotide variation, therefore replacing the use of multiplex ligation-dependent probe amplification (MLPA). The breakpoints can further be resolved by long-read sequencing with/without coupling of long range PCR (≤10 kb). Methods: Germline DNA from high-risk breast cancer patients were subjected to a 6 genes (BRCA1, BRCA2, TP53, PTEN, PALB2 and CDH1) human BRCA Plus QIAseq DNA panel (Qiagen) for NGS using MiSeq (Illumina). Sequencing data were analyzed by in-house developed bioinformatic pipeline. Cases identified with CNV were further characterized by Min-ION (Oxford Nanopore) long-read sequencing. Result: Eleven retrospective controls with unique BRCA1 or BRCA2 CNV identified by MLPA were tested positive using molecular barcode NGS. Eight new CNV cases, including BRCA1, BRCA2 and PALB2, were found from > 200 prospective samples tested. The breakpoints of 10 cases were characterized using long-range PCR coupled with long-read sequencing and 1 case solely with long-read whole-genome sequencing (N50=19 kb). The rest are under investigation. Conclusions: Incorporating molecular barcodes in NGS enables de-duplication of reads from amplicon sequencing, thus allowing CNV to be accurately identified. The capability of extremely long-read sequencing has proven to be useful in solving genomic breakpoint in the base-pair level. Here we showed the prototype of coupling short- and long-read sequencing, which could be the standard workflow for germline mutation detection. It is potentially applicable in somatic breast and ovarian cancers for targeted therapy.