![]() To navigate the bulk fast5 file a specific region can be input as channel and time coordinates, in the format channel: start time-end time. Basic metadata associated with the bulk fast5 file are displayed to the user. Individual files can be selected through the interface and specific channels plotted to the screen ( Figure 1). In some cases there is no apparent reason for the read to have been split, but in many others we observe examples of reads that exhibit unusual signal patterns prior to the incorrect split.īulkVis runs as a bokeh app and scans a folder containing bulk fast5 files at startup. This incorrect splitting of reads appears to correlate with read lengths such that ultra long reads (colloquially referred to as ‘whales’) are more likely to be affected. In the course of developing BulkVis, we observed examples of reads incorrectly segmented by MinKNOW leading to a reduction in the read lengths reported. These reads can then be called by a Nanopore compatible base caller. ![]() BulkVis also provides a feature to create read fast5 files from a selected region of a bulk fast5 file. BulkVis can annotate signal features based on the metadata within the bulk fast5 file and provides a simple method to relate a base called read back to the channel and time in the data stream from which it originated. ![]() To visualise bulk fast5 files, we developed BulkVis using the bokeh visualisation package 7. The bulk fast5 file includes raw signals for every channel and metadata including the classifications made by MinKNOW on the raw signal stream (see Supplementary Table 1). ONT provide an optional bulk fast5 file format to capture the full complement of data from every channel on the sequencing device 6. To better understand these events and to view the effects of user intervention on sequencing when developing methods for read until or using difficult samples, we wished to visualise the entire data stream from the MinlON device. The real time partitioning of the data stream into reads results in the loss of information about the current state both before and after a read as these events are not recorded in a read fast5 file. At its most real time, the sequence of the DNA can be determined as the molecule is passing through the pore, enabling approaches such as ‘Read Until’ where specific molecules can be dynamically rejected from the sequencer according to user customisable parameters 5. These files are subsequently base called to retrieve the underlying sequence. MinKNOW processes the continuous data stream from the MinION device into individual read fast5 files that contain the raw signal data. To do this, the software controlling sequencing (MinKNOW) monitors the state of each channel in real time to determine if the signal observed represents nucleic acid. This uniquely enables rapid analysis of sequence data ideal for both field and clinical work 3, 4. The real time nature of nanopore sequencing means that reads are written to disk as soon as the DNA has translocated through the pore. As single stranded DNA passes through the aperture of the nanopore it creates characteristic disruptions in current flow dependent on the specific sequence in the pore at that moment 2. ![]() A potential is applied across the membrane and creates a current flow through the nanopore. Oxford Nanopore Technologies (ONT) range of sequencing platforms (MinION, GridION, and PromethION) utilise biological nanopores, embedded in a synthetic membrane, to sequence individual single stranded molecules of DNA 1. ![]()
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