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In the first step, we sequentially removed sequences with high similarity to any others until all remaining sequences had a pairwise difference larger than or equal to 2.5%.
We removed sequences with lengths not equal to a multiple of three, with premature stop codons, or with a continuous string of >3 ambiguous N symbols.
The MG-RAST pipeline assessed the quality of sequences, removed short sequences (multiplication of standard deviation of length cutoff of 2.0) and removed sequences with ambiguous bp (non-ACGT; maximum allowed number of ambiguous base pair was set to 5).
The total gene diversity within all the detoxification and stress response-related genes expressed in B. huntii was approximately 584 genes (Additional file 1: Table S1), after we removed sequences with high similarity to microorganisms and plants.
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The program also removes sequences with "AAAA", "TTTT", "GGGG", "CCCC", "TGGC" motifs from siRNA efficient candidates.
Quality control included removing sequences with ambiguous base calls, or ones that did not match the primers perfectly [90].
Pyrosequence reads were denoised with the denoising algorithim described by Quince et al [23], [44], including removing sequences with a mean window quality score <25.
To remove sequences with potential sequencing errors, 15 base pairs on either side of the polymorphic position were compared to the consensus; if a second polymorphism was detected, this sequence read was eliminated from the analysis.
Filtering for sequence ambiguity was performed by removing sequences with homopolymers and removing sequences with a high rate of re-sampling.
This could include removing sequences with a low average quality, removing sequences with too many low-quality individual bases, or masking low-quality positions with Ns.
All bZIP domain sequences were aligned using MAFFT 7 [ 39] to manually check and remove sequences with incomplete domains.
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CEO of Professional Science Editing for Scientists @ prosciediting.com