Computational protocol: Genetics and physiology of cell wall polysaccharides in the model C4 grass, Setaria viridis spp

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Protocol publication

[…] The CesA and Csl sequences of Setaria italica, sorghum (Sorghum bicolor), barley (Hordeum vulgare), rice (Oryza sativa) and Brachypodium (Brachypodium distachyon) were obtained from the public databases Phytozome 9.0 [], NCBI GenBank [], RGAP [] and GRAMENE []. The hmmalign program in the HMMER package [] was used to assign the retrieved full length protein sequences to the cellulose synthase pfam HMM (PF03552). The full length protein alignment was back-translated to codons using pal2nal [] and residue assignments with a posterior probability of <0.6 were manually removed. The Bayesian Markov Chain Monte Carlo (MCMC) package BEAST v1.8.0 [] was used to reconstruct the phylogenetic tree of Setaria, sorghum, barley and rice Csl and CesA genes. The final codon alignment was partitioned into the three separate codon positions, and each partition was unlinked, that is, substitution model parameters, rate heterogeneity model and base frequency were free to vary across partitions. Two analyses, replicated once, were reconstructed; one with a relaxed-clock (log-normal distribution of nucleotide rate variation), and one with a strict clock prior. The GTR + IG substitution model, as selected by jModelTest, and a Yule tree prior were used. Convergence was monitored in TRACER v1.5 [] by assessing the Effective Sample Size (ESS) and likelihood of the estimated parameters. Each analysis was run for at least 75,000,000 states, or until stationarity, logging every 1000 states. CesA, CslD, and CslE clades on the tree were collapsed using nw_utils []. […]

Pipeline specifications

Software tools HMMER, PAL2NAL, BEAST, jModelTest
Databases Pfam Gramene Phytozome RGAP
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Setaria viridis, Homo sapiens, Setaria italica, Sorghum bicolor, Oryza sativa, Brachypodium distachyon, Hordeum vulgare