Illumina sequencing of targeted amplicon 16SrDNA V3-V4 region for treated wastewater sample.
DNA was extracted from activated sludge samples in 3 technical replicates using the GeneMatrix Environmental DNA/RNA Extraction kit (Eurx, Gdask, Poland) according to the manufacturer's instructions. The quality of the obtained material was checked by electrophoresis in 1% agarose gel. In order to assess the amount of matrix supplied, measurements were also performed using a Qubit 3.0 fluorimeter (Thermo Scientific) and a dedicated Qubit High Sensitivity DNA kit reagent. Amplification of conserved bacterial 16SrRNA gene fragment covering V3 and V4 regions was done for DNA samples in triplicate with use of the gene specific primers extended on 5 end with Nextera adaptors (Klindworth et al. 2012)(Illumina, Sand Diego, USA). Obtained amplicons of the size c.a. 450bp  were checked on 1% agarose gel and purified by Ampure XP magnetic beads (Beckman Coulter, Brea CA, USA). Amplicon libraries were pooled in equimolar ratio and indexed according to Nextera indexing strategy by PCR (Illumina, Sand Diego, USA). Sample indexing allowed pooling of amplicons for sequencing run and further extraction of the sample sequence reads from large batch of sequencing results data. 16S amplicons were sequenced on MiSeq sequencer in the DNA Sequencing and Oligonuleotide Synthesis Laboratory IBB PAS in paired end mode using 600 cycle v3 chemistry kit (Illumina, San Diego, USA).