16S rRNA Gene Amplicon Sequencing

Amplicons of 16S rRNA gene were sequenced using Y1 primer. DNA sequences were obtained with ABI PRISM Big Dye Terminator cycle sequencing-ready reaction kit (Applied Biosystems) using ABI 3100 (Perkin Elmer) capillary sequencer, following the manufacturer's instructions. The sequences used for the analyses were those with quality Phrep ≥ 20 [18 (link)]. A search for chimeric sequences was performed with Decipher software [19 (link)] and taxonomic identification to the level of phylum, class, order, family, and genus was made with the Classifier software using confidence limits of 80, 85, 91, 92, and 95% [11 (link)], respectively. Sequences that scored 95% confidence in the Classifier were used to search for similar sequences in MegaBlast (16S rRNA gene of the Bacterial and Archaea) [20 (link)] from the National Center for Biotechnology Information (NCBI) or the SeqMatch Ribosomal Database Project (RDP II) [21 (link)]. These searches were conducted in March 2016.