Computational protocol: Bacterial communities and metabolic activity of faecal cultures from equol producer and non-producer menopausal women under treatment with soy isoflavones

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Protocol publication

[…] DNA samples obtained from the primary and secondary cultures were subjected to PCR amplification of the variable region V4-V5 of the 16S rRNA gene using the universal prokaryotic primers 515F and 909R []; these were designed to include Illumina adapters. Double indexed amplicons were generated by the protocol of Caporaso et al. [] with minor modifications. In brief, DNA from the samples was extracted in duplicate and amplified in triplicate; in total, 72 amplification reactions were obtained. These were subsequently paired-end sequenced in an Illumina Miseq System (Illumina, San Diego, CA, USA) by the StarSeq Company (Mainz, Germany), and treated as independent replicates.Bioinformatic analysis was performed using the Mothur software package (v.1.34.1) following the MiSeq Standard Operating Procedure (SOP) []. Briefly, sequences longer than 380 bp in length, or shorter than 370 bp, and those containing ambiguous base pairs or homopolymers regions of >8 bp were removed. All other sequences were aligned using the SINA alignment service of the SILVA 16S rRNA sequence database. Chimera removal was performed using the UCHIME algorithm []. A random subset of 35,000 sequences per sample was used to balance numbers of reads among samples. Sequences were then clustered into operational taxonomic units (OTUs) using a 0.03 dissimilarity cut-off. Sequences were taxonomically classified using the Ribosomal Database Project (RDP) database. The Bayesian classifier with an 80% confidence threshold was used in the taxonomic assignment with the genus level as the lowest taxonomic unit considered. The MOTHUR program was also used to perform weighted UniFrac analysis, which was employed to assess the similarity of the microbial communities between samples. Construction of a heatmap was performed using the R statistical software. Clustering was accomplished using the complete linkage method with Euclidean distance measure. Multivariable statistical analysis was performed by principal coordinates analysis (PCoA) and non-metric multidimensional scaling (NMDS) with two dimensions. Differences in the microbial composition of faecal cultures with and without isoflavones were sought by analysis of molecular variance (AMOVA) and analysis of similarities (ANOSIM). The identification of differentially abundant taxa was assessed using the Metastats software []. Multiple hypothesis tests were adjusted using the false discovery rate (FDR) correction []; an FDR q-value threshold of 0.25 was used to identify significant differences. In an attempt to assign the differential OTUs at the species level, manual sequence comparisons were performed against the Greengenes 16S rRNA gene database. […]

Pipeline specifications

Software tools mothur, UCHIME, UniFrac, Metastats
Databases Greengenes
Applications Metagenomic sequencing analysis, 16S rRNA-seq analysis
Chemicals Fatty Acids, Volatile, Isoflavones, Polyphenols, Equol