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The consensus of C is updated whenever a read is removed, which may result in a re-comparison between the updated consensus with each of the constituent read.
Abiding by this definition, we validate each contig by iteratively aligning each constituent read to the consensus, discard those that do not satisfy the criteria and update the contig consensus base calls accordingly (see Methods).
We set the following two goals for validating a contig: 1) Put an explicit distance constraint ma x d between the consensus of each contig and each of its constituent read.
Contig validation procedure is presented in Additional file 1: Algorithm 3. First, for each contig C, a consensus is generated and is compared against every constituent read r. r is retained only if the edit distance between r and the consensus is within d.
We developed a rigorous set of rules for this visual examination that took into account the fact that the alignments consisted of sequences of only 101 nt, limiting the ability to identify "in-phase" variants to instances in which these occur on the same constituent read of an alignment.
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It appears that the automated assembly was conservative in trimming reads for low quality ends, and in not extending contigs beyond apparent length differences in the constituent reads.
All subsequent analyses were based on these contigs, hereafter referred as transcripts, and their constituent reads.
Expression patterns were defined by stage specificity and stage bias of the transcript's constituent reads.
We represent each contig C in the form of a multiple sequence alignment of its constituent reads.
For perfect assemblies, we also annotated contigs and singletons based on the transcripts that constituent reads were originally sourced from.
For each read data, VICUNA outputs consensus sequence(s) and for each consensus sequence, the multiple sequence alignment of its constituent reads.
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Since I tried Ludwig back in 2017, I have been constantly using it in both editing and translation. Ever since, I suggest it to my translators at ProSciEditing.

Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com