Computational protocol: Effects of Organic Amendments on Microbiota Associated with the Culex nigripalpus Mosquito Vector of the Saint Louis Encephalitis and West Nile Viruses

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Protocol publication

[…] Using AXIOME to manage sequences analysis (), 16S rRNA gene reads were assembled by PANDAseq version 2.10 (), with a quality threshold of 0.9 (which rejects sequences with low-quality scores), a minimum overlap of 10 bases, and a minimum assembled length of 100 bases, and sequences with ambiguous nucleotides were rejected. Operational taxonomic units (OTUs) were picked at 97% identity using the UPARSE algorithm USEARCH version 7.0.1090 () with de novo chimera checking. Taxonomic classification was performed on the representative sequence of each OTU using RDP version 2.2 () via QIIME (), trained against the Greengenes (August 2013 revision) () reference set with a minimum posterior probability of 80%. Sequences were rarefied to the lowest number of sequences per sample (i.e., 43,611) for alpha and beta diversity analyses. To determine microbial community differences among mosquito samples originating from high- and low-nutrient treatments, principal coordinate analysis (PCoA) and nonmetric multidimensional scaling (NMDS) ordinations, based on the Bray-Curtis dissimilarity measures, were conducted using the vegan R package version 2.2-0 (). In addition, a PCoA ordination based on UniFrac distance measures was carried out with QIIME to determine bacterial community differences among samples of multiple treatments and life stages. Multiresponse permutation procedures (MRPP) were used to test differences among sample groups based on distance measures. Core bacterial taxa were determined based on OTUs represented by at least one sequence per sample in all samples ().Repeated-measures analysis of variance (ANOVA) using JMP () was conducted to assess differences in environmental variables (e.g., nutrients, pH, dissolved oxygen, and microeukaryotes) and larval mosquito abundance in the water column between the two treatments. One-way ANOVA was performed to assess differences in mean abundance of total counts of small and large sestonic particles. Means were separated by Tukey’s test at P < 0.05, after performing Bonferroni correction on calculated P values. […]

Pipeline specifications

Databases Greengenes
Application 16S rRNA-seq analysis
Diseases Encephalitis
Chemicals Oxygen