Updates from v0.1.10:
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Influenza B virus is now supported. This necessitated several changes, hence the rationale for incrementing the minor version to 0.2.x. Since the bioinformatic strategy remains the same, this was not deemed to be a major update to version 1.x.x.
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Implementing influenza B support required changes to the formatting of headers in the FluViewer db. Sequences must now be annotated with species in addition to segment and subtype. A new default FluViewer db has been provided that includes some recent influenza B virus reference sequences.
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Implementing influenza B support also changed how consensus sequences are quality-filtered based on their length. Previously, ranges of permitted sequence lengths were hard-coded for each segment. Since influenza A and B viruses have evolved different length distributions for their segments, those ranges led to the rejection of some valid influenza B segment consensus sequences. Now, the range of accepted segment lengths is determined from the FluViewer db provided by the user. Before each analysis, FluViewer scans the contents of the provided to ensure the headers have proper formatting and to gather the length distribution for each species/segment/subtype combination in the db. Consensus sequences generated by FluViewer must not be more than the length tolerance shorter than the shortest example of that species/segment/subtype in the db. Similarly, they must not be more than the length tolerance longer than the longest example of that species/segment/subtype in the db. The length tolerance is a percentage that can be set by the user using the -t option (default is 1%).
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Implementing influenza B support also changed how mixed infections are handled. FluViewer will fail any specimen if more than one influenza species is detected, more than one influenza A HA or NA subtype is detected, or more than one influenza B lineage is detected. There is a new, experimental “allow mixtures” mode that can be activated with the -m flag. If activated and a mixture of species or subtypes/lineages is detected, FluViewer will proceed with its analysis and generate only consensus sequences for species/segment/subtype combinations detected with a subtype other than “none”. For example, in a mixture of influenza A H1N1 and influenza A H3N2, consensus sequences would be generated for H1, H3, N1, and N2 segments, but not for any of the internal segments (ie PB2, PB1, PA, NP, M, and NS). Intrepid users may attempt to create a custom db where internal segments from sufficient divergent lineages have been given their own “subtype” codes to attempt recovery of more segments from mixed infections, but proceeding with caution is advised and this is not currently supported.
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Normalization and downsampling of reads by BBNorm is no longer part of FluViewer. This was included in previous versions to address uneven amplification by commonly used amplicon-based lab protocols. This step was resource intensive, but might be unnecessary for many users. As such, it was removed, and downsampling/normalization is now left at the discretion of the user, much like trimming of amplification primers and sequencing adapters.
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Runtime inputs provided by the user are now parsed for characters reserved by the system (eg |:;/). This is to prevent injection of destructive/malicious code in runtime parameters.