Computational protocol: Stratified active archaeal communities in the sediments of Jiulong River estuary, China

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Protocol publication

[…] The coverage of the library was calculated with the formula C = 1−(n1/N), where n1 is the number of single-occurrence phylotypes within a library and N is the total number of clones analyzed (Mullins et al., ). The Shannon-Wiener index and Evenness (equitability) were calculated using the equations from Krebs (). The richness was estimated by Chao1 estimator (http://www2.biology.ualberta.ca/jbrzusto/rarefact.php).The 16S rRNA genes retrieved in this study were first submitted to the CHIMERA-CHECK program at the Ribosomal Database Project II (Maidak et al., ) to check and remove chimeric sequences. The non-chimeric sequences were submitted to the BLAST search program on the NCBI (National Center for Biotechnology Information) website (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and RDP (Ribosomal Database Project) website (http://rdp.cme.msu.edu/) to identify close relatives. The ARB-software package (Ludwig et al., ) and SILVA rRNA sequence database (http://www.arb-silva.de/) were used for sequence alignment. Sequences with identities of greater than 97% were tentatively assigned to one OTU (Operation taxonomic units) using the DOTUR (Schloss and Handelsman, ). One sequence per OTU was chosen for the construction of phylogenetic trees. The mcrA genes were translated into amino acids at SIB ExPASy (Expert Protein Analysis System) website (http://web.expasy.org/translate/). Sequence alignments with portions of both the 16S rRNA gene and deduced amino acids sequences of McrA were carried out by CLUSTAL X 1.83 software. The phylogenetic trees were constructed by the neighbor-joining and minimum evolution method by Mega 3.1 software (Kumar et al., ) with the bootstrap analysis used to estimate the confidence of tree topologies (Saitou and Nei, ). The phylogenetic trees presented here were constructed by the neighbor-joining method. […]

Pipeline specifications

Software tools ARB, DOTUR, Clustal W, MEGA
Databases ExPASy
Applications Phylogenetics, 16S rRNA-seq analysis
Chemicals Methane