Computational protocol: Microbiota That Affect Risk for Shigellosis in Children in Low-Income Countries

Similar protocols

Protocol publication

[…] DNA was amplified by using universal primers specific for the V1–V3 region of the 16S rRNA gene in bacteria (518R [5′-CAATTACCGCGGCTGCTGG-3′] and 27F [5′-AGAGTTTGATCCTGGCTCAG-3′]). Individual reads were filtered for quality by using custom in-house scripts that removed low-quality sequences as described (). Remaining high-quality sequences were separated into sample-specific sets according to barcodes. Conservative operational taxonomic units (OTUs) were clustered by using DNACLUST with parameters (–r 1 and 99% identity clusters) to ensure that the definition of an OTU was consistent across all samples (). For taxonomic identification, a representative sequence from each OTU was aligned to the Ribosomal Database (RDP) (rdp.cme.msu.edu, release 10.4) by using BLASTn (http://blast.ncbi.nlm.nih.gov/Blast.cgi) with long word length (–W 100) to detect only nearly identical sequences (). Sequences without a nearly identical match to the RDP (>100-bp perfect match and >97% identity, as defined by BLAST) were marked as being unassigned and assigned a unique OTU identifier. If a sample contained taxa classified as C. jejuni, we identified this sample as positive for C. jejuni. […]

Pipeline specifications

Software tools DNACLUST, BLASTN
Application 16S rRNA-seq analysis
Organisms Homo sapiens, Bacteria
Diseases Dysentery, Bacillary, Escherichia coli Infections