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.

The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community

“…The problem is acute in cancer, where examples of copy number variants, gene duplications, deletions, insertions, inversions, and translocations are common. For reads that averaged 8 kb in length, Norris et al [45] used the MinION to detect structural variants in a pancreatic cancer cell line. These authors concluded that the MinION allowed for reliable detection of structural variants with only a few hundred reads compared to the millions of reads typically required when using NGS platforms.…”

Oxford Nanopore MinION Sequencing and Genome Assembly

“…ONT has released several versions of flowcells, where data quality and throughput vary significantly [18]. The release of version R7+ flowcells to the MAP community received positive feedback from different users who were using them for different applications.…”

“…The release of version R7+ flowcells to the MAP community received positive feedback from different users who were using them for different applications. Norris et al [18] compared the data quality of different flowcell versions. Using R7 flowcells, the average base accuracy was only 67.4% for the reads produced, with 24.2% mismatched bases, 7.5% insertions, and 8.3% deletions.…”

“…To demonstrate nanopore sequencing’s ability to detect SVs, Norris et al [18] designed an experiment to see how well a set of 10 well-characterized SVs could be detected. These SVs included 2 interstitial deletions, 4 translocations, and 4 inversions.…”

Developments beyond blood group serology in the genomics era

“…Technologies that have emerged earlier this decade provide linked-read sequencing, which uniquely barcodes individual long molecules prior to fragmentation and NGS (Zheng et al, 2016). Long-read sequencing (or third generation MPS) has already been used to sequence the human genome, and has demonstrated its potential to resolve complex structural rearrangements in patients with congenital chromothripsis and cancer, indicating its utility for use in clinical and diagnostic settings (Jain et al, 2018;Norris et al, 2016;Cretu Stancu et al, 2017;Pollard et al, 2018).…”