16s-rRNA NGS sequencing: human gut
DNA was isolated from the collected fecal samples using a ZymoBIOMICS DNA Microprep Kit (Zymo research, USA). Bacterial DNA was amplified with the universal direct 785F forward and reverse 1100R primers. PCR products were purified with AMPure XP magnetic beads (Beckman Coulter, USA) and used for constructing sequencing libraries using Ion AmpliSeq Library Kit 2.0 (Thermo Fisher Scientific, USA). Sequencing was performed on the IonTorrent PGM system using Ion PGM Hi-Q View Sequencing Kit, Ion OneTouch 2System, and Ion PGM Hi-Q View OT2 Kit (Thermo Fisher Scientific, USA). The reads were filtered according to the reading quality based on the expected number of errors using the maximum expected error cutoff 1.0. The samples were pooled and unique sequences were identified before searching for the operational taxonomic units (OTUs). We searched for the OTUs using the UNOISE2 algorithm. Filtration of reads, identification of unique sequences, and clusterization in order to search for the OTUs were performed using either USEARCH v.10.0.240 or VSEARCH v.2.8.2 software. Microbial species in the samples were identified using the SILVA database v.123 (https://www.arb-silva.de). In order to compare relative abundances between different experimental groups, we used generalized linear modelling (GLM) method [23] implemented in DeSEQ2 R package