Computational protocol: The Salivary Microbiome in Polycystic Ovary Syndrome (PCOS) and Its Association with Disease-Related Parameters: A Pilot Study

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Protocol publication

[…] Raw reads were processed using the open-source software mothur V1.35.0 according to the protocol by Kozich et al. (April 2015), with the following adaptations: no maxlength was defined during the screening step, start (1046) and end (6426) positions were adapted to the V1-V2 region, and a difference of 3 bases was permitted during the precluster step (based on the recommendation by the authors to allow one mismatch per 100 bp; Kozich et al., ). Chimeric sequences were removed by UCHIME (Edgar et al., ). After removal of non-bacterial sequences, classified using the SILVA119 database (www.arb-silva.de), the remaining sequences were degapped, deuniqued, split into individual samples, and formatted for use with the open-source software QIIME 1.8.0 (Caporaso et al., ). Open reference operational taxonomic unit (OTU) picking was performed in QIIME using UCLUST against the Greengenes 13.8 database (DeSantis et al., ). An OTU was defined as a group of sequences with a similarity of 97% or more. Based on the mock community sequencing results, a relative abundance cutoff of 0.1% was applied for subsequent analyses. Faith's phylogenetic diversity and the number of observed OTUs were used as metrics for alpha rarefaction, which was performed in QIIME. Principal coordinate analyses (PCoA) were based on unweighted and weighted UniFrac distances and calculated in QIIME (Lozupone and Knight, ). Taxa summaries were performed in QIIME. All samples were normalized to the sample with the lowest read count for alpha and beta diversity comparisons. For taxa comparisons, relative abundances based on all obtained reads were used.Raw sequencing data are available in NCBI's short read archive (SRA) under the accession number SRP077213. [...] Nonparametric student's t-tests using Monte Carlo permutations were used for alpha diversity comparisons, Mann–Whitney U-tests for taxa comparisons, and Adonis for category comparisons of distance matrices, all calculated in QIIME. Benjamini–Hochberg false discovery rate (FDR) correction was used to correct for multiple hypothesis testing where applicable.All remaining statistical calculations were performed in IBM SPSS Statistics Version 22. Depending on the statistical distribution of the variable, unpaired t-tests or Mann–Whitney U-tests were used to compare groups. Fisher's Exact tests were used to compare categorical parameters. All data are expressed as median and interquartile range (IQR). […]

Pipeline specifications

Software tools mothur, UCHIME, QIIME, UCLUST, UniFrac, SPSS
Databases Greengenes
Applications Miscellaneous, Phylogenetics, 16S rRNA-seq analysis
Organisms Homo sapiens
Diseases Infertility, Polycystic Ovary Syndrome, Mastocytosis, Systemic
Chemicals Testosterone