Whale watching with BulkVis: A graphical viewer for Oxford Nanopore bulk fast5 files

Abstract

AbstractMotivationThe Oxford Nanopore Technologies (ONT) MinION is used for sequencing a wide variety of sample types with diverse methods of sample extraction. Nanopore sequencers output fast5 files containing signal data subsequently base called to fastq format. Optionally, ONT devices can collect data from all sequencing channels simultaneously in a bulk fast5 file enabling inspection of signal in any channel at any point. We sought to visualise this signal to inspect challenging or difficult to sequence samples, or where flow cell performance is modified by an external agent, such as ‘Read Until’.ResultsThe BulkVis tool can load a bulk fast5 file and overlays MinKNOW classifications on the signal trace. Users can navigate to a channel and time or, given a fastq header from a read, jump to its specific position. BulkVis can export regions as Nanopore base caller compatible reads. Using BulkVis, we find long reads can be incorrectly divided by MinKNOW resulting in single DNA molecules being split into two or more reads. The longest seen to date is 2,272,580 bases in length and reported in eleven consecutive reads. We provide helper scripts that identify and reconstruct split reads given a sequencing summary file and alignment to a reference. We note that incorrect read splitting appears to vary according to input sample type and is more common in ‘ultra long’ read preparations.AvailabilityThe software is available freely under an MIT license athttps://github.com/LooseLab/bulkVis. The software requires python3 to run.