16S rRNA gene amplicon library preparation and sequencing
DNA was extracted utilizing the FastDNA SPIN Package for Soil (MP Biomedicals) following the producer’s directions. The samples had been transferred to DNA low-binding 96-well plates (Body Star 96, semiskirted), the DNA focus was quantified utilizing double-stranded DNA QuantiFluor (Promega) and normalized to 1 ng µl−1. The 16S rRNA gene amplicon library was generated as follows. PCR amplification, clean-up, and barcoding PCR had been carried out as in [57]. DNA focus was decided as above, and every effectively was normalized to 1 ng µl−1. Equal quantity from every effectively was then mixed right into a pooled 16S rRNA gene amplicon library, and the library was cleaned twice with AMPure magnetic beads utilizing a bead-to-DNA ratio of 0.9 to take away small DNA fragments. The amplicon size distribution of the library was assessed on a 2200 TapeStation utilizing HS D1000 (Agilent), leading to an efficient library measurement of 554–643 bp. Sequencing was carried out for 12 pM samples on a MiSeq desktop sequencer (Illumina) on the Genetic Range Centre Zurich utilizing the MiSeq reagent package v.3 (paired finish, 2 × 300 bp, 600 cyc PE). Denaturation, dilution, and addition of 15% PhiX to the DNA library had been carried out in keeping with the producer’s directions. Customized sequencing primers had been used as described beforehand [30].
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