BulkVis: a graphical viewer for Oxford nanopore bulk FAST5 files

Area of Science:

• Genomics
• Bioinformatics
• Molecular Biology

Background:

• Oxford Nanopore Technologies (ONT) MinION is a versatile sequencing platform.
• FAST5 files store raw signal data, which is base called into FASTQ format.
• Bulk FAST5 files allow simultaneous data collection for signal inspection.

Purpose of the Study:

• To develop a tool for visualizing raw signal data from ONT sequencers.
• To inspect challenging or difficult-to-sequence samples by examining signal traces.
• To identify and address potential issues in the base calling or read processing pipeline.

Main Methods:

• Development of the BulkVis tool to load and visualize bulk FAST5 files.
• Overlaying MinKNOW classifications and reference mappings onto signal traces.
• Enabling navigation to specific channels, time points, or reads using FASTQ headers.

Main Results:

• BulkVis identified instances where MinKNOW incorrectly divides long reads into multiple segments.
• The longest observed split read spanned over 2.2 million bases across eleven consecutive reads.
• Incorrect read splitting was found to be dependent on sample type and more prevalent in ultra-long read preparations.

Conclusions:

• The BulkVis tool aids in the inspection of raw sequencing signal data.
• Incorrect read splitting by MinKNOW can lead to fragmented long reads.
• Helper scripts are provided to identify and reconstruct split reads for improved data accuracy.