Computational protocol: A comparative study of microbial diversity and community structure in marine sediments using poly(A) tailing and reverse transcription-PCR

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[…] The PCR products obtained using poly(A) tailing and RT-PCR were cloned into the pCR ®2.1-TOPO ® vector and transformed into competent E. coli DH5α (Life Technologies Japan, Tokyo, Japan). For RT-PCR, the cloned inserts were sequenced using an ABI 3130xl genetic analyzer (Life Technologies Japan) with the primers M13M4, M13rev, 926R/F (), and 1390R (). For the poly(A) tailing method, sequencing was first performed using the M13 primers followed by screening of the 16S rRNA sequence using a hidden Markov model implemented in version 3.0 of the HMMER software package (), as described elsewhere (;). The screened 16S rRNA inserts were sequenced using primers 338R/F (), 515R/F (), 926R/F, and 1390R to assemble full-length 16S rRNA sequences. A primer walking approach was employed for inserts that could not be sequenced using the primers described above. The sequences were trimmed and assembled to obtain consensus sequences using Sequencher software (Hitachi Software, Tokyo, Japan). Chimeric sequences were removed using the UCHIME program () implemented in the Mothur Utility package (). [...] Alignment of all 16S rRNA sequences was performed using the ARB software package (). Since some of the 16S rRNA sequences were fragmented after poly(A) tailing, a 600-bp fragment (corresponding to E. coli 16S rRNA positions 287–886) was used for comparisons of microbial diversity and community structure. Taxonomic assignments were made using Silva taxonomy and the Bayesian classifier. clustering sequences, calculation of diversity indices (i.e., Shannon and Simpson indices) and Libshuff test (;) were performed using the Mothur software package (;). Phylogenetic tree was constructed by ARB software () using the neighbor-joining method () with an Olsen correction. The coverage rate of the used primer set (26F-1492R) at the genus level was evaluated using TestPrime 1.0 program () using SILVA database SSU r114 with RefNR. […]

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