16S rDNA amplicon sequencing of African fermented dairy foods
The variable regions of 16S rRNA are amplified to build the sequencing library using Illumina's protocol. 50 ng DNA is used and the variable regions of 16S rRNA are amplified using Phusion polymerase for 25-35 PCR cycles. The primer sequences are:319F-ACTCCTACGGGAGGCAGCAG;806R-GGACTACHVGGGTWTCTAAT. The primer (319F/806R) is designed to target the V3 and V4 regions of 16S rRNA about 469 bp. The length may vary among different species. After one cycle of PCR, sequencing adapters and barcodes are added for further amplification. The amplified library is used for sequencing. PCR AxyPrepTM Mag PCR Normalizer. Illumina cBot system is used to generate clusters for sequencing on MiSeq.