Nanopore sequencing detects structural variants in cancer

Abstract

Despite advances in sequencing, structural variants (SVs) remain difficult to reliably detect due to the short read length (<300 bp) of 2nd generation sequencing. Not only do the reads (or paired-end reads) need to straddle a breakpoint, but repetitive elements often lead to ambiguities in the alignment of short reads. We propose to use the long-reads (up to 20 kb) possible with 3rd generation sequencing, specifically nanopore sequencing on the MinION. Nanopore sequencing relies on a similar concept to a Coulter counter, reading the DNA sequence from the change in electrical current resulting from a DNA strand being forced through a nanometer-sized pore embedded in a membrane. Though nanopore sequencing currently has a relatively high mismatch rate that precludes base substitution and small frameshift mutation detection, its accuracy is sufficient for SV detection because of its long reads. In fact, long reads in some cases may improve SV detection efficiency.We have tested nanopore sequencing to detect a series of well-characterized SVs, including large deletions, inversions, and translocations that inactivate the CDKN2A/p16 and SMAD4/DPC4 tumor suppressor genes in pancreatic cancer. Using PCR amplicon mixes, we have demonstrated that nanopore sequencing can detect large deletions, translocations and inversions at dilutions as low as 1:100, with as few as 500 reads per sample. Given the speed, small footprint, and low capital cost, nanopore sequencing could become the ideal tool for the low-level detection of cancer-associated SVs needed for molecular relapse, early detection, or therapeutic monitoring.

MinION-based long-read sequencing and assembly extends the Caenorhabditis elegans reference genome

“…The MinION works on the principle of nanopore strand sequencing in real time Jain et al 2016) and results in sequence read lengths from several hundred bases to hundreds of thousands of bases. To date, most studies have used the MinION to sequence small genomes or to partially survey larger genomes to assess chromosomal structure and copy-number variations (Goodwin et al 2015;Loman et al 2015;Norris et al 2016;Wei and Williams 2016). The long read lengths combined with recent improvements in performance and the development of software to assemble genomes from long sequence reads ) make MinION sequencing a viable option for whole-genome sequencing of complex metazoan genomes.…”

Séquençage de l’ADN par nanopores

“…Grâce à des algorithmes fondés sur le maximum de vraisemblance, elle a aussi révélé des isoformes de récepteurs dans des lymphocytes B [28]. Des variants structuraux et des mutations dans le cancer du pancréas et dans la leucémie [29,30] ont pu aussi être détectés dans des études dans lesquelles la méthode s'est montrée complémentaire de celles existantes. Elle a également été testée avec succès pour la détection prénatale d'aneuploïdies dans le sang maternel [31].…”

Structural Variation Detection from Next Generation Sequencing

“…Another study using assembly for SV detection was reported by Pendleton, et al [41], who used 46X PacBio long reads and 80X long molecule Bionano mapping data. Another emerging single molecule sequencing platform is Oxford Nanopore's MinION, a real time nanopore-based DNA sequencing instrument [42][43][44]. With reported features of compact, inexpensive, long read length and fast sequencing data production, the MinION device offers great potential for genome analysis from structural variation perspectives.…”