16S rRNA amplicon sequencing of mouse fecal samples
The 16S rRNA gene sequencing was performed using a two-step PCR. In the first step, primers targeting the 16S rRNA gene carrying 5’M13/rM13 adapters were used to amplify the V3-V6 region. Duplicate reactions were pooled and purified, and concentration was determined spectrophotometrically. PCR products were used for a second PCR reaction to add a specific multiplex identifier (MID) sequence and the 454-specific Lib-L tag. Amplicons were pooled, concentrated and purified, and finally resuspended in ddH2O. Amplicon-sequencing was performed on a 454 GS FLX Titanium platform from one side (Lib-L-A) according to the recommended procedures.