Illumina MiSeq Sequencing of Canis lupis familiaris: stool
Genomic DNA was extracted by a mechanical cellular disruption method and treatment with phenol: chloroform. The V4 region of the 16S rRNA gene was amplified using universal bacterial primers with adapters for Illumina-based sequencing and unique barcodes in a 25uL PCR reaction using 2x KAPA HiFi HotStart ReadyMix, 1-15ng of DNA, 0.2umol/L each primer, and ultra pure water under the following program in a thermocycler: Initial denaturation at 95 C for 3 min, followed by 25 cycles of 95 C for 30 s, 55 C for 30 s, and 72 C for 30 s. Product was run on a 1% low melt agarose gel with SYBRSafe, and the 380 basepair region was excised. DNA was extracted from the gel pieces, quantified using qubit reagents, and an equimolar pool was created. Sequencing took place using a 11pmol/L pool with 10% PhiX control on a v3 2x300 kit on the Illumina MiSeq.