16 S and ITS metabarcoding: microbes from tropical soil samples
eDNA was extracted from Upland, Lowland, and Riparian primary tropical forest soil samples using the MoBio Power Soil DNA extraction kits, according to manufacturer's protocol with a final elution in 50ul molecular grade water. The different eDNA extracts from the soil samples were used for a 2-step PCR amplification of eDNA targeting the nuclear internal transcribed spacer (ITS) ribosomal RNA gene region for fungi, and also targeting the v3 and v4 of 16S ribosomal RNA gene region for bacteria and archaea.  Second PCR used standard primers with Illumnina adapters.  PCR products were checked on a gel, purified using MinElute DNA purification system, and quantified using a Quant-iT PicoGreen dsDNA assay.  For each sample, equimolar amounds of each amplicon were pooled and tagged using Nextera XT Index Kit.  Indexed samples were pooled, purified using magnetic bead purification, quantified using PicoGreen dsDNA assay, average length determined on Agilent Bioanalyzer 2100 before sequencing on an Illumina MiSeq using the V3 sequencing chemistry kit (2 x 300).  10% spike in of PhiX used as a control.
SUB7721007
SAMN15405774_Riparian 2016