Computational protocol: Microbiota in Breast Milk of Chinese Lactating Mothers

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Protocol publication

[…] Data processing was done with mothur software v.1.32.1 []. Paired-end sequences were joined as described []. The sequences were aligned to Silva reference alignment and trimmed so that complete overlap was achieved. A preclustering step with 2%-linkage algorithm was performed []. Chimeras were removed using uchime algorithm using de novo option []. The sequences were classified with RDP classifier (version 9) using 0.8 confidence level. The sequences were clustered into OTUs using 0.03 similarity cut-off value. The distances among sequences were calculated as pairwise uncorrected distance, where gaps were considered a single position, and clustering using average neighbor method. Before calculating beta diversity measures and statistical analyses, the number of sequences per sample was made uniform by random subsampling of 2226 reads from each sample. This eliminated samples that had fewer than 2226 sequencing reads: 8 out of 30 samples from the first data set, and 2 out of 60 samples from the second data set. […]

Pipeline specifications

Software tools mothur, UCHIME, RDP Classifier
Application 16S rRNA-seq analysis
Organisms Homo sapiens