A preliminary evaluation of a fast, low-cost, and high-throughput nucleic acid extraction method for bacterial microbiota profiling in low-microbial biomass samples

Abstract

The respiratory tract is colonized with low-density microbial communities, which have been shown to impact human respiratory health through microbiota-host interactions. However, a lack of fast and cost-effective nucleic acid extraction method for low-microbial biomass samples hinders investigation of respiratory microbiota. Here, we performed a pilot study to assess the suitability of the NAxtra nucleic acid extraction protocol for profiling bacterial microbiota in respiratory samples. A small number of nasopharyngeal aspirate (n = 8), nasal swab (n = 8), and saliva samples (n = 8) were collected, nucleic acids were isolated using the NAxtra protocol, and 16 S rRNA gene sequencing was performed to characterize bacterial microbiota, which were compared to the same sample types from previous studies using other protocols. The bacterial composition in nasal and saliva samples were consistent with previous reports. Saliva microbiota was significantly richer than nasal microbiota and varied less among individual samples than nasal microbiota. Bacterial composition in nasal samples was distinct from nasopharyngeal aspirates, but closer to saliva samples. A sequencing depth of 50,000 reads/sample was sufficient for microbiota profiling in low biomass respiratory samples. Our pilot study indicates the potential of the NAxtra protocol for bacterial microbiota characterization of low-microbial biomass samples and supports a more comprehensive study to fully evaluate the value of the NAxtra protocol in microbiota research and clinical diagnostics of respiratory pathogens.

Keywords: 16S rRNA gene sequencing; Bacteria; Low-microbial biomass; Microbiota; Nucleic acid extraction; Respiratory samples.

Fig. 1

Bacterial microbiota diversity and composition in different samples. Box plots showing the alpha diversity indices represented by (A) observed amplicon sequence variants (ASVs), and (B) Shannon diversity. Boxes represent the interquartile range between the 25th and 75th percentiles, and the horizontal line inside the box denotes the median value, and the diamond represents the mean value. Asterisks refer to the Benjamini–Hochberg corrected p value, where *p < 0.05, **p < 0.001. (C) Beta diversity illustrated by principal coordinates analysis (PCoA) based on Bray–Curtis distance. (D) Venn diagram showing the numbers of common and distinct ASVs among different sample types.

Fig. 2

Abundance of bacterial taxa in different samples. (A) Genus level. (B) Family level. The 20 most abundant genera and families are indicated in different colors, the remaining taxa are binned into the ‘Others’ category.

Fig. 3

Rarefaction curves. The curve is created for each sample to assess the saturation at different sequencing depths for recovery of bacterial taxa presented by amplicon sequence variants (ASVs). Nasopharyngeal aspirate and nasal swab samples reach a plateau more rapidly (~ 10,000 reads) compared to saliva samples (~ 50,000 reads).