Faecal soil total dna extraction kit

Faecal/Soil Total DNA™ extraction kit (Zymo Research Corporation, CA, USA) was used to extract environmental DNA from ground root endosphere and rhizospheric soils according to manufacturer’s instructions and stored at − 20 °C prior to further analysis. For each sample, DNA was extracted in triplicates and pooled together. 16S rRNA gene fragments libraries preparation using 27F and 518R primer pairs, fused with MiSeq adapters and heterogeneity spacers was done according to protocol described by Ogola et al. [18 (link)]. Resultant libraries were sequenced by paired end (300 bp reads) sequencing v.3 chemistry along with its multiplex sample identifiers on the Illumina MiSeq Platform according to standard protocol.
Raw Fastq files from Illumina sequencing have been deposited at the NCBI sequence read archive (SRA) as BioProject ID PRJNA742387. The sequences were curated using the mothur v1.40.5 pipeline implemented in Nephele (v2.2.8) [19 (link)]. Sequences were assigned to operational taxonomic units (OTUs) using a dissimilarity cutoff = 0.03 and classified to representative microbial taxa against the nonredundant SILVA v132 ribosomal RNA database.

Total DNA was extracted from 5 mL of each collected sample using a Faecal/Soil Total DNA™ extraction kit (Zymo Research Corporation, Irvine, CA, USA) according to the manufacturer’s instructions. The resultant DNA concentration and quality were checked at 260 nm wavelength and absorbance ratios of 260/280 nm on a NanoDrop Spectrophotometer (NanoDrop Technology, Wilmington, DE, USA) following which the DNA was preserved at −20 °C until further processing. The extracted DNA was first amplified using the universal bacterial 16S rRNA primers (27F and 1492R) to cover the whole variable region under the following PCR conditions: initial denaturation at 95 °C for 5 min, followed by 32 cycles of melting at 95 °C for 1 min, annealing at 55 °C for 1 min, and elongation at 72 °C for 1 min. This was then superseded by a final elongation step at 72 °C for 10 min. Subsequently, a second PCR run was carried out using the 27F and 518R primer sets, with overhanging adapter sequences that are compatible with Illumina index as described by Ramganesh et al. [2 ]. Cleaning of the resultant PCR products, index library preparation, pooling and sequencing on Illumina Miseq 250^® to generate paired 300-bp high-quality reads of the V1–V3 region were performed according to standard protocol (Illumina Inc., San Diego, CA, USA).