Computational protocol: Impact of Experimental Hookworm Infection on the Human Gut Microbiota

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Protocol publication

[…] This study was approved and carried out in strict accordance and compliance with the National Statement on Ethical Conduct in Research Involving Humans guidelines of the National Health and Medical Research Council of Australia (www.nhrmc.gov.au/publication/humans/contents.htm; www.nhmrc.gov.au/funding/policy/researchprac.htm). The Prince Charles Hospital (Brisbane, Australia) and James Cook University Human Research Ethics Committees approved the study. Written informed consent was obtained from all subjects enrolled in the study. None of the subjects had received any antibiotic treatment prior to sample collection. This study was registered as a clinical trial at ClinicalTrials.gov (NCT00671138). Briefly, 8 otherwise healthy volunteers with HLA-DQ2+ CeD on a long-term gluten-free diet (since >6 months) were recruited and infected percutaneously with 20 infective third stage larvae (iL3) of N. americanus []. Prior to experimental infection (T0), as well as at 8 weeks post-infection (T8), individual fecal samples (approximately 10 g each) were collected from each subject and stored at −20°C until subsequent DNA extraction. Genomic DNA was extracted directly from each sample using the PowerSoil DNA isolation kit (MoBio, USA), according to the manufacturer's instructions. Parallel sequence data sets were generated for individual DNA samples by sequencing of 2 separate fragments of the 16S rRNA gene that encode the V1-V3 and the V3-V5 hypervariable regions on a 454 GS-FLX Titanium System (Roche) using universal primers described elsewhere [, ]. Forward primers incorporated GS Titanium adapters as well as a sample-specific barcode sequence. Raw sequence data have been deposited in the NCBI Sequence Read Archive under study accession number SRP041283.Sequence data were processed using the Quantitative Insights Into Microbial Ecology (QIIME) software suite []. Briefly, after filtering of low-quality reads, all remaining sequences were de-multiplexed according to barcode, including error-correction to reduce the possibility of sample misassignment. Chimeric sequences were removed using UCHIME v 3.0.617. Sequences were subsequently clustered into operational taxonomic units (OTUs) on the basis of similarity to known bacterial sequences in the Greengenes database (http://greengenes.secondgenome.com/; cut-off: 97% sequence similarity) using the UCLUST software; then, sequences were assigned to taxonomy using the Ribosomal Database Project (RDP) Classifier with the confidence level set at 0.8. Statistical analyses and data mining were conducted using the Calypso software (http://bioinfo.qimr.edu.au/calypso/). All analyses were conducted separately for each data set (V1–V3 and V3–V5). Shannon diversity and richness were compared by paired t-test and rarefaction curves. Differences in abundance of individual OTUs were assessed by paired t-test on relative abundance values, and P values were corrected for multiple testing by false discovery rate (FDR). Anosim (Jaccard distance), redundancy analysis (RDA; including human subject and week as explanatory variables), principal coordinates analysis (PCoA; Jaccard distance), and rarefaction analyses were run in Calypso with default parameters. Anosim was run twice for each variable region, with samples labeled by infection status in the first run and by subject ID in the second run, respectively. […]

Pipeline specifications

Software tools QIIME, UCHIME, UCLUST, RDP Classifier
Databases SRA ClinicalTrials.gov Greengenes
Application 16S rRNA-seq analysis
Organisms Necator americanus, Homo sapiens
Diseases Hookworm Infections, Infection