BioProject

We propose the first mono-compartmental static culture system specifically designed towards the fermentation of fecal microbiota from exclusively milk-fed infants. More...

We propose the first mono-compartmental static culture system specifically designed towards the fermentation of fecal microbiota from exclusively milk-fed infants. The parallel use of ETH and mGAM culture media, each adapting to different initial microbiota structures, and 16S rRNA gene capture by hybridization allowed a species-level stabilization and characterization of the fecal microbiota of three 2-3 months old infants receiving different milk-based diets. Fresh feces were diluted in culture media to the 1/100th and incubated at 37°C for 48h or 72h. Samples were collected at t0 and at the end of fermentation. DNA was extracted by applying a method adapted from Godon et al., 1997, i.e. International Human Microbiome Standards Standard Operating Protocol for Fecal Samples (IHMS SOP) 07 V1. Probes were previously designed to target 16S rRNA gene. Biotinylated RNA capture probes were obtained by in vitro transcription following the protocol described by Ribière et al., 2016. Gene capture by hybridization was conducted as follow : 500 ng of Nextera Illumina sequencing library were constructed individually for each sample, captured DNA was then PCR-amplified with 25 cycles using primers fully complementary to Illumina adapters. To increase the enrichment efficiency, a second round of capture was performed from the first-round capture products. Captured DNA was finaly sequenced with two Illumina MiSeq 2 × 300 bp runs. Less...