Computational protocol: Skin microbiome before development of atopic dermatitis: Early colonization with commensal staphylococci at 2 months is associated with a lower risk of atopic dermatitis at 1 year

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Protocol publication

[…] Sequences were preprocessed with mothur version 1.35.1. Briefly, 454 flowgram data were trimmed and denoised, and chimera checking was completed with the mothur implementation of UCHIME. Sequences were classified by using the Ribosomal Database Project naive Bayesian classifier. Sequences classified as chloroplast or mitochondria were discarded. Site-specific definition of operational taxonomic units (OTUs; groups of sequences that share a specific level of similarity) and downstream analyses was performed in mothur. Within the samples from each time point or site, pairwise distances were calculated, and OTUs were defined at 97% nucleotide similarity. Within-sample (Shannon diversity) and between-sample (theta index) measurements were performed based on these OTU definitions, with subsampling to 1000 sequences per sample. Rarefaction curves level off by this value, suggesting adequate sequencing coverage; any samples with fewer than 1000 sequences after preprocessing were removed from analysis (see in this article's Online Repository at www.jacionline.org). Differentially abundant OTUs were detected by using the metastats command in mothur.The sequences classified to the Staphylococcus genus by using the RDP naive Bayesian classifier were then placed on a phylogenetic reference tree using “-keep-at-most 1000 max-pitches 1000.” Taxonomy was assigned by using the guppy program in pplacer, with a likelihood cutoff set to 0.65, as previously described. […]

Pipeline specifications

Software tools mothur, UCHIME, Pplacer
Applications Phylogenetics, 16S rRNA-seq analysis
Organisms Staphylococcus aureus, Homo sapiens, Epipremnum aureum