Computational protocol: Lactobacillus fermentum CRL1446 Ameliorates Oxidative and Metabolic Parameters by Increasing Intestinal Feruloyl Esterase Activity and Modulating Microbiota in Caloric-Restricted Mice

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Protocol publication

[…] The bacterial V3-V4 16S rRNA regions were amplified by PCR using primer pairs 343F (5′-TACGGRAGGCAGCAG-3′) and 802R (5′-TACNVGGGTWTCTAATCC-3′) according to Polka et al. []. The PCR product pool was sent to Fasteris SA (Geneva, Switzerland) to be sequenced using the TruSeq™ DNA sample preparation kit (Illumina Inc., San Diego, CA, USA). High-throughput sequencing was carried out in one lane MiSeq Illumina instrument (Illumina Inc.) using the V2 chemistry in a 2 × 250 configuration sufficient to cover the entire amplicon length, estimated to be ~450 bp. Paired-end assembly and quality filtering was performed by using Flash software []. Sample de-multiplexing was carried out using DNA barcoding information per sample and Mothur platform. The assembled and barcode/primer-free sequences were processed for chimera removal using Uchime algorithm [] and SILVA reference set of 16S sequences [].Diversity indexes were calculated with Mothur platform using default parameters and average method in the clustering step []. Different alpha diversity parameters such as the Observed (Sobs) and Chao’s richness, as well as the Shannon’s diversity and evenness indexes, which were computed from OTUs (operational taxonomic units) clustered at 97% and using a normalized subset of 23,000 sequences randomly selected after multiple shuffling (10,000×) of the original dataset. Taxonomy assignation at Phylum and Genus levels of the subset of high-quality reads was performed with Bayesian RDP Classifier [], using an assignment confidence cutoff 0.8. Beta diversity was analyzed by performing Unifrac-weighted analysis []. […]

Pipeline specifications

Software tools mothur, UCHIME, RDP Classifier, UniFrac
Application 16S rRNA-seq analysis
Organisms Lactobacillus fermentum, Mus musculus
Chemicals Cholesterol, Glucose, Glutathione