In a comment in this issue, Holmes (2021) uses an earlier study by Kwan et al.,(2017) as a case study to suggest improvements on sampling and statistical analysis of host-associated microbiome analyses. This is an interesting suggestion that is not commonly used in bacterial microbiome studies or ecological studies in general but may be a useful approach to assess differences in microbiome species richness and may be advantageous when the mean pattern does not accurately represent the community as a whole such as when extreme ends of the distribution may have different relationship with a predictive variable (Cade & Noon, 2003; Koenker & Bassett, 1978). GEOGRAPHIC SAMPLING AND SPATIAL CONSIDERATIONS The research objective of Kwan et al.,(2017) was to examine how microbiome species diversity and composition varies between key groups such as tick life stages or between pathogen infected or uninfected ticks in a particular site. Due to low rates of infection prevalence of~ 6%(Swei et al., 2015), nymphal tick collections necessarily had to exceed the final number of ticks analysed by 16S amplicon sequencing and the selection of infected ticks was more restricted than the selection of uninfected ticks because the available samples were more limited.[Extracted from the article]

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