High-throughput taxonomy annotation based on cDNA sequences of methanogenic and methanotrophic functional genes recovered from Amazonian wetlands

Abstract

We present here a plan for a research internship abroad with the main objective to address one of main research goals described in the ongoing principal research proposal (FAPESP 2017/06415-9). The main objective of this research internship abroad is to apply computational approaches for merging, processing and annotation of sequences corresponded to functional cDNA from active microbes associated with the production and consumption of CH4 (methanogenic and methanotrophic) recovered from both natural and agricultural wetlands in floodplains of Amazonian rivers of white (Solimões River), black (Negro River) and clearwater (Tocantins River) in dry-season. The Mothur program (v 1.3.3) will assemble the cDNA sequence readings. The assembled contigs will then be sorted based on their length and on the match with the primers (d 2 errors), spacer (d 2 errors) and barcodes (d 1 error). Barcodes, primers and spacer will be removed. USEARCH 6.0 will remove the putative chimeric readings. The nucleotide sequences will be converted into amino acids sequences of the respective functional genes and the frame change readings will be corrected using the FRAMEBOT program. The mcrA/methanogen, pmoA, pmoA2, mmoX, mcrA/methanotroph and 16S rRNA/NC10 bacterial phylum gene sequences will be aligned and grouped into Operational Taxonomic Units (OTU) with 89% identity cut-off values, 93%, 93%, 89%, 89%, and 97%, respectively (Stackebrandt and Goebel, 1994; Hutchens et al., 2004; Steinberg and Regan, 2008; Degelmann et al., 2010; Yuangeng et al., 2016). Three different activities compose the work plan: (i) Pre-processing raw cDNA sequences; (ii) OTU clustering and taxonomic annotation, (iii) Data normalization and ecological metrics, and (iv) Ecological interpretation of the results.