Computational protocol: The Role of +4U as an Extended Translation Termination Signal in Bacteria

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Protocol publication

[…] The bacterial genomes were retrieved from GenBank, and coding sequences (CDSs) were extracted by using DAMBE () for computing ITE. An alternative set of HEGs consists of all ribosomal protein genes extracted from DAMBE () based on genomic annotation. We also extracted small subunit rRNA (ssu rRNA) genes from each species for building a phylogenetic tree for computing independent contrasts. For each stop codon, their nc_tRNAs () were compiled. No tRNA had anticodons AUA or ACA in the species studied and, thus, they are not included in . [...] Variables measured from a set of species are typically not independent because of shared ancestry. Phylogeny-based independent contrasts () were computed to alleviate this problem. We aligned ssu rRNA sequences aligned by MAFFT () with the LINSI option, which generates the most accurate alignment (“–localpair” and “–maxiterate = 1000”). PhyML () was used for phylogenetic reconstruction, with general time reversible (GTR) substitution model and six categories of gamma-distributed rates. The resulting tree () was used for computing independent contrasts (), as numerically illustrated in . The same approach was used to reconstruct the tree for the 19 species (indicated in ) in ITE-related analysis.Because the bacterial species involve deep phylogeny with limited resolution close to the root node, we assessed the effect of different trees on the results of independent contrasts by using 100 bootstrapped trees. DAMBE takes a file with the 100 trees and automatically performs independent contrasts for each tree. We have also used a tree built with PhyPA, suitable for deep phylogenetic relationships (). The PhyPA is based on pairwise sequence alignment using default option simultaneously estimated distances based on a TN93 model (). […]

Pipeline specifications

Software tools DAMBE, MAFFT, PhyML
Application Phylogenetics
Organisms Escherichia coli, Saccharomyces cerevisiae
Chemicals Nucleotides