Computational protocol: Metagenomic profiles of free-living archaea, bacteria and small eukaryotes in coastal areas of Sichang island, Thailand

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Protocol publication

[…] Sequences were categorized based on their appended pyrotag-sequences, and sequences of less than 100 nucleotides were discarded. The sequences were annotated, visualized and computed for taxonomic compositions and evolutionary distances using VITCOMIC (Visualization tool for Taxonomic Compositions of Microbial Community) [] with some modifications. In brief, a library for 16S rDNA species were constructed from NCBI non-redundant [], RDP [,] and Greengenes [] databases, and a library for 18S rDNA species were constructed from NCBI non-redundant [], EMBL [,] and SILVA [] databases. Each read was annotated using BLASTN [] or RNAmmer [] with default parameters, unless identified. Percent relative abundance for each phylum was computed from the frequency of reads in the phylum divided by the total number of identified reads. In VITCOMIC, multiple sequences were aligned following MAFFT 6.713 with default parameters [], genetic distances were calculated following Kimura's two-parameter model [], and a neighbor-joining tree was constructed using PHYLIP 3.69 [,]. Similarity indices of taxonomic compositions among different sample groups were determined based on Lennon and Yue & Clayton theta similarity indices [,]. Additionally, the 16S rDNA data were compared with those of the GOS ( [,,,], using Yue & Clayton theta similarity coefficients (Thetayc) and Smith theta similarity coefficient (Thetan) in mothur [,]. The closer the similarity coefficient to 0.000 indicated the more similarity in community structures. PCoA was plotted in three-dimensions using mothur []. The Tha Wang and Tham Phang 16S rDNA data and the 67 GOS data and their Thetayc were used for an unweighted pair group method with arithmetic mean (UPGMA) clustering by mothur [], given that their multiple sequence alignment were performed using NAST []. The results were manually inspected to ensure properly sequence annotation, clustering, and phylogenetic tree relationship. […]

Pipeline specifications

Software tools VITCOMIC, BLASTN, RNAmmer, MAFFT, PHYLIP, mothur
Application Phylogenetics
Organisms Saccharomyces cerevisiae
Chemicals Oxygen