16S rRNA amplicon sequencing of soil bacterial community: rhizosphere soil community under HCH contamination
Total DNA was extracted from 0.5g of soil and 0.3g of ground root material (both fresh weight), using the NucleoSpin Soil Kit (Macherey-Nagel, Dren, Germany), according to the manufacturers protocol. Empty sterile extraction tubes were prepared for each soil compartment and root DNA extraction, to serve as negative extraction controls. The concentration of DNA extracts was quantified with a 5200 Fragment Analyzer System (Agilent, US-CA), using the Genomic DNA 50Kb Analysis Kit. Ten ng of the DNA extracts were used to amplify the V3-V4 region of the 16S rRNA gene, using the primer pair 338F (5- GCTGCCTCCCGTAGGAGT- 3) /789R (5 -GGAATCCTCTCTCAC CACATTGCCCAGGCAGACC- 3) with Illumina adapter sequences, which was reported to exclude chloroplast amplification(Dorn-In et al., 2015). The PCR reactions were carried out in three technical replicates using the NEBNext High-Fidelity 2 PCR Master Mix (New England Biolabs, Ipswich, US-MA) with the addition of tetramethyl ammonium chloride at 36mM final concentration in the PCR reaction. Non target control (NTC) and positive controls containing the target gene were also performed in triplicates.  The PCR conditions were the following: initial denaturation step, 30 s at 98 C; 25 cycles (10 s denaturation at 98 C; 30 s annealing at 60 C; and 30 s elongation at 72 C); and the final elongation step at 72 C, 5 min. The technical replicates of the PCR products were checked on 1% agarose gel, and afterwards pooled together for purification using the NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel), according to the manufacturers protocol. The purified product underwent quality check and quantification with the 5200 Fragment Analyzer System (Agilent, US-CA), using the NGS Fragment Kit (1-6000bp). 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 conditions were the following: initial denaturation step, 30 s at 98 C; 8 cycles (10 s denaturation at 98 C; 30 s annealing at 55 C; and 30 s elongation at 72 C); and the final elongation step at 72 C, 5 min. The indexed product was checked on 1% agarose gel and purified with NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel). The quality and concentration of the indexing product were assessed using the above mentioned kit from Agilent. The samples were diluted to 4nM and pooled equimolarly. 10 pmol DNA were sequenced with 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.