title: 16S seq for IBS patient 42 at visit 3
design: Total DNA was isolated using the AmpliPrimeDNA-sorb-AM kit (NextBio, Russia) for clinical specimens according to the manufacturers protocol. The isolated DNA was stored at -20C. For qualitative and quantitative assessment of the isolated DNA, we used a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, USA). Next, 16S library was prepared according to the protocol for 16S Metagenomic Sequencing Library Preparation (Illumina, USA) recommended for Illumina MiSeq sample preparation. The first round of amplification of V3-V4 16S rDNA variable regions was performed using the following primers: forward (5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-CCTACGGGNGGCWGCAG-3) and reverse (5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-GACTACHVGGGTATCTAATCC-3). These primers are aimed at the amplification of bacterial (more than 90% taxonomic coverage) but not archaeal (less than 5%) rRNA genes. The amplification program (Applied Biosystems 2720 Thermal Cycler, USA) was the following: 1) 95C  3 min; 2) 30 cycles: 95C  30 s, 55C  30 s, and 72C  30 s; 3) 72C 5 min; and 4) 4C. The derived amplicons were purified using Agencourt AMPure XP (Beckman Coulter, USA) beads according to the manufacturers protocol. The second amplification round was utilized to double-index the samples with a combination of specific primers. The amplification program was the following: 1) 95C  3 min; 2) 8 cycles: 95C  30 s, 55C  30 s, and 72C  30 s; 3) 72C  5 min; and 4) 4C. The products of the second PCR round were purified using Agencourt AMPure XP. The concentrations of the 16S rDNA libraries were measured with a Qubit 2.0 fluorimeter (Invitrogen, USA) utilizing a Quant-iT dsDNA High-Sensitivity Assay Kit. The purified amplicons were mixed equimolarly according to the concentration values. The quality of the libraries was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) and an Agilent DNA 1000 Kit. Sequencing was carried out on a MiSeq machine (Illumina) with a MiSeq Reagent Kit v2 (paired-end reads, 2x250 nt). The obtained sequences of forward and reverse reads were pre-merged using MeFiT and CASPER. For most samples, more than 99.5% of the reads were successfully merged.