Computational protocol: Clear Genetic Distinctiveness between Human- and Pig-Derived Trichuris Based on Analyses of Mitochondrial Datasets

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Protocol publication

[…] Sequences were assembled manually and aligned against the complete mt genome sequences of other nematodes (available publicly) using the computer program Clustal X 1.83 to infer gene boundaries. The open-reading frames (ORFs) and codon usages of protein-coding genes were predicted using the program MacVector v.4.1.4 (Kodak), and subsequently compared with that of Trichinella spiralis . Translation initiation and translation termination codons were identified based on comparison with those reported previously . Codon usages were examined based on the relationships between the nucleotide composition of codon families and amino acid occurrence, for which codons are partitioned into AT rich codons, GC-rich codons and unbiased codons. The secondary structures of 22 tRNA genes were predicted using tRNAscan-SE and/or manual adjustment . [...] Amino acid sequences inferred from the 12 protein-coding genes (i.e. not atp-8) common among all of the nematodes included here were concatenated into a single alignment, and then aligned with those of 9 other enoplid nematodes (Trichinella spiralis, GenBank accession number NC_002681; Xiphinema americanum, NC_005928; Hexamermis agrotis, NC_008828; Agamermis sp., NC_008231; Romanomermis culicivorax, NC_008640; Romanomermis iyengari, NC_008693; Romanomermis nielseni, NC_008692; Strelkovimermis spiculatus, NC_008047; Thaumamermis cosgrovei, NC_008046), using the chromadorean nematode, Brugia malayi (NC_004298) as the outgroup. Any regions of ambiguous alignment were excluded using Gblocks (; Talavera and Castresana 2007) using stringent selection criteria (do not allow many contiguous nonconserved positions). Phylogenetic analyses were conducted using three methods: Bayesian inference (BI) analysis was conducted with four independent Markov chain runs for 1,000,000 metropolis-coupled MCMC generations, sampling a tree every 100th generation in MrBayes 3.1.1 ; the first 2,500 trees represented burn-in, and the remaining trees were used to calculate Bayesian posterior probabilities (pp). Maximum likelihood (ML) analyses were performed using PhyML 3.0 , and the (mtREV for amino acid sequences and GTR for rrnL nucleotide sequences) models were determined based on the Akaike information criterion (AIC). Bootstrap support was calculated using 100 bootstrap replicates. Maximum parsimony (MP) analysis was conducted using PAUP 4.0 Beta 10 , with indels treated as missing character states; 1,000 random additional searches were performed using TBR. Bootstrap support was calculated using 1,000 bootstrap replicates, and 100 random taxon additions in PAUP. Phylograms were drawn using the program Tree View v.1.65 . […]

Pipeline specifications

Software tools Clustal W, MacVector, tRNAscan-SE, Gblocks, MrBayes, PhyML, PAUP*
Applications Genome annotation, Phylogenetics
Organisms Sus scrofa, Trichuris trichiura, Homo sapiens, Trichuris suis, Canis lupus familiaris
Diseases Infection, Trichuriasis