Computational protocol: Intestinal Microbial Ecology and Environmental Factors Affecting Necrotizing Enterocolitis

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Protocol publication

[…] DNA was amplified using a primer set based on universal primers 27F (AGAGTTTGATCCTGGCTCA) and 533R (TTACCGCGGCTGCTGGCAC) to which titanium adaptor sequences and barcodes were added. Cleansed PCR products were pooled in equimolar amounts and submitted for sequencing using 454-Titanium chemistry. From the resulting raw data set, low quality sequences or sequences with a length less than 150 nucleotides were removed. We used the ESPRIT-tree algorithm, which maintains the binning accuracy of ESPRIT while improving computational efficiency to bin sequences into Operational Taxonomic Units (OTUs) using similarity levels from 99% (species/strain level) to 80% (phylum level). We used QIIME to calculate Chao rarefaction diversity and UniFrac distances for comparing α and β diversity respectively.For comparison between the current and the earlier sequence dataset we pooled sequences from both reads and reassigned OTUs using ESPRIT-tree. We then identified OTUs that contained the sequences that were found to differ between cases and controls in either dataset to determine sequence distribution in them. […]

Pipeline specifications

Software tools ESPRIT-Tree, QIIME, UniFrac
Application 16S rRNA-seq analysis
Organisms Homo sapiens
Diseases Intestinal Diseases, Pneumonia, Enterocolitis, Necrotizing