Computational protocol: Patent Human Infections with the Whipworm, Trichuris trichiura, Are Not Associated with Alterations in the Faecal Microbiota

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Protocol publication

[…] The DNA amplicons were pyrosequenced using a GS FLX Titanium 454 (Roche Diagnostics, Oakland) machine following the manufacturer's Lib-L kit protocols and were initially processed using the “Amplicon” configuration of the 454 Sequencing System software. This sequence data is available in the European Nucleotide Archive (ENA) short-read archive under the sample accession number ERS236495 and/or the study accession number ERP002465. Downstream data analysis was performed using the mothur software package . Briefly, reads were filtered for quality by truncating them once average quality scores dropped below 35 across a rolling window of 50 bases. Following this step all reads less than 350 bases in length, those with any mismatches to the barcode or 16 S rRNA gene primer sequence, and those with any ambiguous bases or with homopolymeric stretches of longer than 8 bases were discarded. Chimeras were removed using Perseus software , as implemented in mothur. Following these data cleaning steps a median of 2286 sequences remained per sample. Sequences were aligned to the reference SILVA database provided in mothur, then clustered into Operational Taxonomic Units (OTUs) at 97% sequence identity using the default average neighbour setting. Phylogenetic classifications were assigned to each OTU at all taxonomic levels from Phylum to Genus using the reference Ribosomal Database Project database (RDP) provided in mothur. Classifications for selected OTUs, typically the most abundant ones, were further verified by checking similarities using MegaBLAST against the NCBI nucleotide archive . Shannon diversity index scores were calculated using mothur , after samples were randomly subsampled to a depth of 500 sequence reads per sample to ensure that final diversity scores were not influenced by differential sequencing depths. […]

Pipeline specifications

Software tools mothur, Perseus, BLASTN
Databases ENA
Application Phylogenetics
Organisms Homo sapiens, Trichuris trichiura, Ascaris lumbricoides
Diseases Infection, Coinfection