Modification of the soil bacterial metagenome during vermicomposting by Eisenia fetida
DNA was isolated from each sample and16S rDNA PCR was done using specific primers and amplicons were used to generate libraries for Ion Torrent PGM sequencing. The V3 and V6 regions of the Bacterial 16S rRNA gene were amplified by the fusion primer method. The fusion primer is designed to contain the forward adaptor sequence (A adaptor), index, and V3/V6 region-specific forward primer sequence while the reverse primer has a P1 adaptor sequence followed by V3/V6 specific reverse primer sequence (the primer sequences are provided in the supplementary table). Fusion PCR allows for direct template preparation after PCR cleanup. The amplification was performed using the following steps: initial denaturation at 98C for 5 min; 25 cycles of 98C for 10 sec, 58C for 20 sec, 72C for 20 sec; the final extension of 72C for 5 min; hold at 4C. The PCR cleanup was performed using AMPure XP beads and the manual recommended for Ion Torrent fusion amplicon library preparation method. Following the QC of the final PCR on 2% gel electrophoresis and DNA was quantified using a Qubit HS DNA quantitation kit. The equal molar ratio (200 nM) of each library (uniquely indexed) was pooled together and used for downstream steps. The library was serially diluted from 200 nM to 10nM and finally up to100 pM. Next, the 26 pM of the final library was used for template preparation using the V2 DL kit and OT2 template preparation instrument.  The positive ion sphere particles (ISP) enrichment was performed on the OneTouch ES instrument. The enriched library was sequenced using an Ion 200 bp sequencing kit on an Ion-torrent instrument. The demultiplexing and fastq files were generated using the inbuild Ion browser.
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