Computational protocol: Effect of a Protein Supplement on the Gut Microbiota of Endurance Athletes: A Randomized, Controlled, Double-Blind Pilot Study

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Protocol publication

[…] For sequencing, a DNA fragment comprising the bacterial hypervariable regions V3 and V4 of 16S rRNA gene was amplified using the primer pair 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′ and 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′ []. The amplicon of 459 bp was visualized in a 0.8% agarose gel stained with ethidium bromide, and bands were cut and cleaned using the MinElute Gel Extraction Kit (Qiagen, Hilden, Germany). DNA amplicons were sequenced on a MiSeq Illumina platform (Illumina, San Diego, CA, USA). Sequence outputs were analyzed using the Quantitative Insights into Microbial Ecology (QIIME) program, version 1.9.1 [], using QIIME default parameters except for split library demultiplexing (phred quality threshold of 20 and better). The 16S pair-end reads were assembled using the script, which joins forward and reverse demultiplexed reads. The output file was processed for quality filtering by High-quality sequences were grouped into Operational Taxonomic Units (OTUs) with a sequence identity threshold of 97%, and taxonomy was assigned by interrogating the high-quality sequences with the Greengenes database (13_8). Beta-diversity was evaluated by calculating weighted and unweighted Unifrac distances []. To study alpha-diversity, Shannon and Simpson diversity indices were calculated. Linear discriminant analysis (LDA) coupled with effect size (LEfSe) was performed to identify bacterial taxa differentially represented between groups [] […]

Pipeline specifications

Software tools QIIME, UniFrac, LEfSe
Databases Greengenes
Applications Metagenomic sequencing analysis, 16S rRNA-seq analysis
Organisms Bacteroidetes, Bifidobacterium longum
Chemicals Ammonia, Fatty Acids, Volatile, Malondialdehyde