Computational protocol: Molecular identification of methane monooxygenase and quantitative analysis of methanotrophic endosymbionts under laboratory maintenance in Bathymodiolus platifrons from the South China Sea

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Protocol publication

[…] Three subunits of methane monooxygenase, abbreviated as pmoA, pmoB and pmoC, were identified from draft genome data of the B. platifrons methanotrophic endosymbiont (L Li, 2017, unpublished data) by TBLASTN, using the homologous pmoA, pmoB and pmoC of Methylococcus capsulatus (Bath) as query sequences with an E-value cutoff of 1e − 8. The methanotrophic endosymbiont draft genome data were obtained from metagenomic sequencing and assembly of gill tissue of B. platifrons from a cold seep in the South China Sea (22°06′57.144″N, 119°17′6.580″E). The methane monooxygenase amino acid sequences were predicted by DNAstar (version 4.05) and further verified by BLASTP against the NCBI non-redundant (nr) protein database (https://blast.ncbi.nlm.nih.gov/Blast.cgi). [...] Homologous protein sequences and their corresponding coding sequences of methane monooxygenase subunits from type I, type II, and type X methanotrophs were obtained through online BLASTP search through database at NCBI using B. platifrons symbiont methane monooxygenase proteins as queries. Alignments of proteins were conducted in MAFFT v7 (). Coding sequences were aligned according to the protein alignments. ModelGenerator () was used to choose an appropriate model of sequence evolution for the alignment. Bayesian method was used to construct protein and coding sequence (CDS) phylogenetic trees as implemented in MrBayes 3.2 (; ). […]

Pipeline specifications

Software tools TBLASTN, BLASTP, MAFFT, ModelGenerator, MrBayes
Applications Phylogenetics, Metagenomic sequencing analysis, Amino acid sequence alignment
Chemicals Amino Acids, Carbon, Methane