16S rRNA sequencing of control sample: tissue-free area of FFPE block
Amplicon-based 16S rRNA libraries were prepared using primers directed to the V3-V4 variable region of the 16S rRNA gene: forward-CCTACGGGNGGCWGCAG and reverse-GACTACHVGGGTATCTAATCC. Two-step PCR was used to amplify the target region with the addition of Illumina sequencing adapters and sample-specific 8-bp dual-index barcodes. All PCR reactions were performed using a Tersus Plus PCR kit (Evrogen, Russia) on a T100 thermal cycler (Bio-Rad, USA). The first stage PCR was performed with the following program: initial denaturation at 95C for 3 min, followed by 30-50 cycles (depending on sample type) of denaturation at 95C for 30 s, annealing at 55C for 30 s, and extension at 72C for 30 s, with final extension at 72C for 5 min, and hold at 4C. PCR products were size verified by 2% agarose gel electrophoresis (~550 bp) and purified using MagPure A4 XP magnetic beads (Magen, China). The second-stage PCR amplification program was as follows: 95C for 3 min followed by 8 cycles of 95C for 30 s, 55C for 30 s, and 72C for 30 s with a final extension at 72C for 5 min, and hold at 4C. PCR amplicons were purified using magnetic beads. The size of the final libraries was examined on an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) and was ~630 bp. The libraries were then mixed equimolarly and diluted to 10pM loading concentration with 40% of the control library PhiX (Illumina, USA). Sequencing was performed on an Illumina MiSeq system using a 300x2 bp paired-end run.