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In addition, we removed sequences of metazoan taxa with currently unreliable phylogenetic position (Mesozoa, Myxozoa, Chaetognatha and Placozoa) from the database.
To increase the quality of the alignments, we visually inspected the results and removed sequences of low quality, corrected the alignment when necessary, and removed sites with more than 25% gaps or sequences with more than 25% gap characters in the motif.
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After removing sequences of poor-quality or insufficient length by Geneious Pro 8.0.2 software (Biomatters Ltd., Auckland, New Zealand), the remaining sequences were aligned using the MAFFT software (Katoh et al. 2002).
Metasecretome pyrosequencing reads were trimmed with SeqClean [ 74] to remove sequences of pDJ01 vector and VCSM13d3 helper phage.
Cre recombinase is a bacteriophage enzyme, which can remove sequences of DNA that lie between engineered loxP sites (referred to as "floxed" sequences).
The codon substitution analysis was carried out using CodAlignView (Jungreis I, Lin M ,Kellis M. CodAlignView: a tool for visualizing protein-coding constraint) and processed with a python script to remove sequences of codons.
After removing sequences of insufficient quality and sequences that could not be adequately assigned, nearly 156,000 sequences remained from the four human habitats, including 30,521 reads from nasopharynx, 28,309 reads from saliva, 43,708 reads from dominant hands, and 54,179 reads from feces respectively (Table 1).
First, we preprocessed the assemblies by 1) removing sequences of ≤ 90 nucleotides length and 2) clustering ≥ 95% identical sequences using CD-HIT-EST (Li and Godzik 2006; Fu et al. 2012) by maintaining only the longest sequence from each cluster (fig. 3).
An initial set of 26,112 3' ESTs derived from FLcDNA libraries was combined with 81,407 3' ESTs from standard EST libraries [ 11] to generate a starting set of 107,519 3'-end ESTs, which resulted in 90,368 high-quality ESTs after filtering to remove sequences of low quality and contaminant sequences from yeast, bacteria and fungi.
Additionally, the following low-scoring sequences were removed: sequences consisting of greater than 66% of poly-A -T -G or -C; and sequences with fewer than 30 high quality bases.
Next, we removed sequences suspected of containing poly-Adenine tails by discarding any read composed of greater than 33% Adenine.
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