Guideline for the analysis of the microbial communities of the human upper airways

Abstract

The recent COVID-19 pandemic prompted a rapid-growing interest in the investigation of the human microbiota of the upper airways. In fact, the resident microbial community of this body district may have an influence on the onset of SARS-CoV-2 infection and its clinical course in terms of presence, symptom severity, and outcomes. However, several microbiological methodologies are available to study the human microbiota, reflecting the extensive fragmentation of methodological approaches. We investigate the impact of two critical steps that can induce biases in the downstream analyses, i.e. sampling method and microbial DNA extraction kit employed. We observed major discrepancies regarding the total amount of prokaryotic DNA that could be retrieved from a biological sample and the proportion between bacterial DNA and human host DNA. Moreover, shotgun DNA sequencing and taxonomic profile reconstruction also revealed correlations between sampling methods and the procedures applied for microbial DNA extraction. Based on all the data collected in this study, we formulate indications regarding the most efficient and reliable methodological procedures for the metagenomic analyses of the upper airways' microbiota to maximize accuracy and reproducibility.

Keywords: Metagenomics; microbiome; microbiota; shallow shotgun.

Figure 1.

Evaluation of the performances achieved by different commercial available DNA extraction kits. Panel a shows the Whiskers plot representing the average amount of extracted DNA from the samples included in the study and treated with the different DNA extraction kits. The y‐axis reports the average amount of extracted DNA, while the x‐axis indicates the different DNA extraction kits and different sampling methods. The boxes are determined by the 25th and 75th percentiles. The whiskers are determined by 1.5 IQR (Interquartile range). The line in the boxes represented the median, while the square represents the average. Panel b reports a bar blot indicating the percentage of reads associated with eukaryotic sequences based on the DNA extraction kits and sampling methods. Panel c indicates bar plots regarding the species richness of each sample sequenced and the average, basing on the DNA extraction kits and sampling methods.

Figure 2.

Comparison of the taxonomic profiles of each extracted sample with the different extract kits. Panel a shows the principal coordinate analysis (PCoA) of the samples obtained through oropharyngeal swabs. Each group includes a specific sample of an individual extracted with a specific DNA extraction kit. Panel b displays the PCoA of the saliva samples. Each group includes a specific sample of an individual extracted with a specific DNA extraction kit. Panel c reveals the PCoA of the samples obtained through nasopharyngeal swabs. Each group includes a specific sample of an individual extracted with a specific DNA extraction kit.