Boosting biomethanation using magnetite nanoparticles
Amplicon libraries for the archaea 16S rRNA gene variable regions 4-9 (aV49-A) were prepared using a custom protocol. Up to 25 ng of extracted DNA was used as template for PCR amplification, and each PCR reaction (50L) contained 0.2 mM dNTP mix, 0.01 units of Platinum SuperFi DNA Polymerase (Thermo Fisher Scientific, USA), and 500 nM of each forward and reverse primer in the supplied SuperFI Buffer. PCR was done with the following program: Initial denaturation at 98 C for 3 min, 25 cycles of amplification (98 C for 30 s, 62 C for 20 s, 72 C for 2 min) and a final elongation at 72 C for 5 min. The forward and reverse primers used include custom 24 nt barcode sequences followed by the sequences targeting aV49-A: [515FB] GTGYCAGCMGCCGCGGTAA and [SSU1000ArR] GGCCATGCAMYWCCTCTC. Sequencing libraries were prepared from the purified amplicon libraries using the SQK-LSK114 kit (Oxford Nanopore Technologies, UK) according to manufacturer protocol with the following modifications: 500 ng total DNA was used as input, and CleanNGS SPRI beads for library clean-up steps. DNA concentration was measured using Qubit dsDNA HS Assay kit (Thermo Fisher Scientific, USA). Gel electrophoresis using Tapestation 2200 and D1000/High sensitivity D1000 screentapes (Agilent, USA) was used to validate product size and purity of a subset of amplicon libraries.
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