16S rRNA sequencing of mouse gut metagenome in Mgat5 KO and WT mice on an Apc+/min background
Bacterial amplicon sequence analysis targeted a 16S DNA gene fragment comprising the V3/V4 hypervariable regions (16S sense 5-TACGGRAGGCAGCAG-3 and anti-sense 5-CTACCNGGGTATCTAAT-3). 16S amplicon library preparation was performed according to an optimized and standardized protocol (Metabiote, GenoScreen, Lille, France). In brief, PCR of the 16S DNA was performed using 5 ng of genomic DNA according to the manufacturers protocol (Metabiote), utilizing bar-coded primers (Metabiote MiSeq Primers) at 0.2 mol/L final concentration. An annealing temperature of 50C for 30 cycles was used. The Agencourt AMPure XP-PCR purification system (Beckman Coulter, Brea, CA, USA) was used to purify PCR products and following quantification according to the manufacturers instructions, they were multiplexed at equal concentrations. 250bp paired-end sequencing was performed and forward and reverse files were merged by the sequencing company prior to analysis.