16S rRNA amplicon sequencing of soil bacterial community: bulk soil community under Lindane contamination
Total DNA was extracted from 0.5 g of fresh soil, using the NucleoSpin Soil Kit (Macherey-Nagel, Dren, Germany), according to the manufacturers protocol. Three of the five biological replicates were selected for 16S rRNA gene library preparation. Empty tubes were also prepared for each extraction batch, to serve as negative extraction controls. The DNA extracts were quantified with Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific, Waltham, US-MA). 10 ng of DNA extract were used for PCR amplification of the V3-V4 region of the 16S rRNA gene, using the primer pair 335F (5- CADACTCCTACGGGAGGC- 3) and 769R (5- ATCCTGTTTGMTMCCCVCRC -3) with Illumina adapter sequences [Dorn-In,Bassitta,Schwaiger,Bauer & Hlzel 2015]. The PCR reactions were carried out in three technical replicates using the NEBNext High-Fidelity 2X PCR Master Mix (New England Biolabs, Ipswich, US-MA) with an initial denaturation step of 5 min at 98 C, followed by 25 cycles (10 s denaturation at 98 C; 30 s annealing at 60 C; and 30 s elongation at 72 C) and a final elongation step at 72 C for 5 min. A non target control (NTC) and a positive control containing the target gene were also performed in triplicate at the same PCR conditions.   The PCR products were separately checked on 1% agarose gels before the technical replicates were pooled together and purified using the NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel), according to the manufacturers protocol. The amplicon purification was followed by a DNA quality control on a 2100 Bioanalyzer Instrument (Agilent Technologies, Santa Clara, US- CA), using the DNA 7500 kit (Agilent Technologies, United States). After DNA quantification with Quant-iT PicoGreen kit (Life Technologies, United States), 10 ng of the purified 16S rRNA gene amplicon were indexed using the Nextera XT Index Kit v2 (Illumina Inc., San Diego, US-CA) for multiplexed short-read sequencing. The PCR program proceeded with a 30 s initial denaturation step, 8 amplification cycles (10 s denaturation at 98 C; 30 s annealing at 55 C and 30 s elongation at 72 C) and a final elongation time of 5 min at 72 C. The indexed amplicons were purified, checked qualitatively and quantitatively as previously described, diluted to 4 nM and pooled equimolarly. A final amount of 10 pmol DNA was sequenced on a MiSeq System (Illumina, San Diego, US-CA) using the MiSeq Reagent Kit v3 (600 cycle) for paired end sequencing with PhiX (Illumina, San Diego, US-CA) as a spike-in positive control. The raw data were demultiplexed on the MiSeq system, after which the primer and adapter sequences were removed using the software AdapterRemoval V. 2.1.7 [Lindgreen 2012].