Computational protocol: Comparison of placenta samples with contamination controls does not provide evidence for a distinct placenta microbiota

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Protocol publication

[…] The 16S rRNA gene reads were analyzed using the QIIME software package [] with default parameters augmented by the package qiimer (github.com/kylebittinger/qiimer) and the R programming language [, ]. Reads were removed from the analysis if they did not match a 12-base Golay barcode with 1 or fewer errors, if the paired reads failed to overlap by 35 bases, if the overlapped region differed by more than 15 %, or if they had more than 3 base calls below Q20. Operational taxonomic units (OTUs) were created by clustering the reads at 97 % identity using UCLUST []. Representative sequences from each OTU were aligned using PyNAST [], and a phylogenetic tree was inferred using FastTree v. 2.1.3 [, ] after applying the standard Lane mask for 16S rRNA gene sequences []. Pairwise UniFrac distances were computed using QIIME [], and permutational tests of distance were performed using the vegan library for the R programming language []. Principal coordinates analyses were performed with the APE library for R []. Taxonomic assignments were generated by the UCLUST consensus method of QIIME 1.8 [], using the GreenGenes 16S rRNA gene database v. 13_8 []. Bayesian analysis of qPCR data was performed using the R library BEST []. […]

Pipeline specifications

Software tools QIIME, UCLUST, PyNAST, FastTree, UniFrac, APE
Applications Phylogenetics, 16S rRNA-seq analysis
Organisms Homo sapiens