16S-rRNA gene (V1-V2 region) sequencing of mouse cecum content
Amplicons of the V1-V2 region of the bacterial 16S rRNA gene were prepared using the primer design described by Camarinha-Silva et al. (2014, Env Microbiol), which achieves multiplexing by employing a combination of custom 6-mer barcode sequences directly adjacent to the forward 16S sequence and the adapter integrated index sequences commonly used by Illumina multiplexing kits. Briefly, Amplification and integration of multiplexed adapters was performed in three PCR steps: 1) Enrichment PCR, using 16S-rRNA specific primers amplifying the V1-V2 region, 2) Custom barcode integration PCR, using primers to integrate partial Illumina adapter sequences and a 6-mer barcode sequence at the 5'-end of the insert, 3) Illumina multiplexing PCR: using primers to integrate the remainder of Illumina adapter sequences including an adapter-integrated index sequence, which is read during the index sequencing step. The library was merged at equal molarity with other samples prior to sequencing.