Computational protocol: Dynamic evolution of bitter taste receptor genes in vertebrates

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[…] We identified T2R genes from each vertebrate species using the following methods. We first collected previously published T2R gene sequences from human, mouse, dog, opossum, chicken, frog and zebrafish genomes as query sequences. Next, we conducted a TBLASTN [] search using the E-value 1e-10 against each genome sequence. There were so many TBLASTN query results that hit the same genomic region that we extracted non-overlapping sequences, each of which showed the lowest E-value among the hits to a given region. Functional, intact T2R genes were identified from these blast-hit sequences using the following approach. First, we collected the blast-hits that were >100 amino acids long. Then, each of the blast-hit sequences was extended in both 3' and 5' directions along the genome sequences. Obtained sequences were confirmed by BLASTP searches against the NCBI databases to ensure that genuine T2R genes were identified. Finally, the coding sequences with proper ATG and the stop codon were extracted (the average functional T2R gene was ~300 amino acids long). Sequences that contained interrupting stop codons or frameshifts were regarded as pseudogenes and the remaining sequences containing either initiation codons or stop codons were considered partial T2R genes. [...] The translated amino-acid sequences were aligned using the program FFT-NS-I nested in Mafft version 5 []. The phylogenetic T2R gene tree (Figure ) was constructed using MEGA3 software [] and the Neighbor-Joining [] method with the protein JTT matrix model, and was evaluated by 500 bootstrap replications. […]

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