Winston Timp | Search bioRxiv

bioRxiv2016

Detecting DNA Methylation using the Oxford Nanopore Technologies MinION sequencer

Nanopore sequencing instruments measure the change in electric current caused by DNA transiting through the pore. In experimental and prototype nanopore sequencing devices it has been shown that the electrolytic current signals are sensitive to base modifications, such as 5-methylcytosine. Here we quantify the strength of this effect for the Oxford Nanopore Technologies MinION sequencer. Using synthetically methylated DNA we are able to train a hidden Markov model to distinguish 5-methylcytosine from unmethylated cytosine in DNA. We demonstrate by sequencing natural human DNA, without any special library preparation, that global patterns of methylation can be detected from low-coverage sequencing and that the methylation status of CpG islands can be reliably predicted from single MinION reads. Our trained model and prediction software is open source and freely available to the community under the MIT license.

Genomics↗

bioRxiv2015

Nanopore sequencing detects structural variants in cancer

Despite advances in sequencing, structural variants (SVs) remain difficult to reliably detect due to the short read length (<300bp) 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 20kb) 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.\n\nWe 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.

Genomics↗