Extraction of metagenomic DNA and next-generation sequencing
Metagenomic DNA extraction from duplicate 1 gram soil samples viz. BS,
SARS, and LARS of each crop were carried out using the Soil DNA
isolation kit, MO, USA, following manufacturer’s protocol, while the
total root + endophyte DNA was obtained using a modified CTAB method
(26). For next-generation sequencing, the V3-V4 hypervariable region of
the 16s rRNA gene was targeted. The library was constructed by random
fragmentation of DNA, and unique 16 bp index sequences were ligated to
each sample. The library was generated for producing 2x300 paired-end
reads in the Illumina MiSeq platform. Sequencing was done at Macrogen
Inc., Korea.