Gut Microbiome Profiling via 16S rDNA Amplicon

Amplification of the variable region V1–V2 of the bacterial 16S rDNA gene was utilized to assess gut microbial diversity. Primers used were 8F-338R (5′-AGAGTTTGATCCTGGCTCAG-3′ and 5′-TGCTGCCTCCCGTAGGAGT-3′) for multiplex Roche 454 GS FLX pyrosequencing. Primer design was carried out according to the manufacturer’s instructions. Initial PCR from each DNA was performed four times [20 (link),21 (link)]. After PCR, the resulting product was checked for size and purity on an agarose-Sybr safe DNA gel stain (Invitrogen, San Diego, CA, USA). The amplicons were purified using a Pure Link kit (Invitrogen, San Diego, CA, USA) and quantified using Qubit and Bioanalyzer. The pool of amplicons were mixed equimolar (four amplicons for cockroach specie) and then prepared for 454-pyrosequencing according to the manufacturer. Cycling conditions were 94°C for 3 min, followed by 30 cycles of 94°C for 30 s, 56°C for 40 s, 68°C for 40 s, and a final extension step at 68°C for 6 min.