Bioinformatic processing and statistical analysis of microbiome data

The obtained sequence libraries were subjected to quality control using trimmomatic (v0.32) [53]. Reads were merged using FLASH (v1.2.7) [54]. Reads were formatted for use with the UPARSE workflow [55], prior to chimeric read removal, de-replication and clustering into Operational Taxonomic Units (OTUs) at 97% sequence similarity using USEARCH7. Taxonomy was assigned using RDP classifier [56] as implemented in QIIME [57], using Silva release 132 as the reference database [58].

The statistical analysis and visualization of microbial community data was performed in R version 3.5.1 [59] via RStudio version 1.1.463 (http://www.rstudio.com), using the R packages ampvis2, vegan and ggplot2 [60–62]. Beta diversity was calculated for microbiome comparison between housefly from different locations using Bray-Curtis dissimilarity [63], and visualized using non-metric multi-dimensional scaling (NMDS). The microbial community structure was visualized using heatmaps. Relationships between prevalence of potential pathogenic organisms based on literature and the sampled populations were explored using hierarchical clustering using Bray-Curtis distances.