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. 2025 Jun 20;6(2):103740.
doi: 10.1016/j.xpro.2025.103740. Epub 2025 Apr 8.

Protocol for microbial profiling of low-biomass upper respiratory tract samples

Mari-Lee Odendaal  1 , Mei Ling J N Chu  2 , Kayleigh Arp  3 , Gina J van Beveren  4 , Giske Biesbroek  5 , Justyna Binkowska  6 , Thijs Bosch  7 , James A Groot  7 , Raiza Hasrat  3 , Sjoerd Kuiling  7 , Elske M van Logchem  7 , Paula Lusarreta Parga  6 , Wouter A A de Steenhuijsen Piters  3 , Debby Bogaert  8
Affiliations

Protocol for microbial profiling of low-biomass upper respiratory tract samples

Mari-Lee Odendaal et al. STAR Protoc. .

Abstract

The upper respiratory tract (URT) microbiota plays a role in both acute and chronic respiratory health outcomes and consists of multiple ecologically distinct niches, all of which have low bacterial biomass. Here, we present a protocol for microbial profiling of low-biomass URT samples. We describe steps for collecting, storing, and extracting DNA. We then detail procedures for performing 16S rRNA sequencing, using an Illumina MiSeq platform, to characterize microbial communities. For complete details on the use and execution of this protocol, please refer to Odendaal et al.1.

Keywords: Bioinformatics; Health Sciences; Microbiology; Molecular Biology; Sequence analysis; Sequencing.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Schematic layout of work-flow through associated laboratories
Figure 2
Figure 2
Schematic overview of the DNA extraction process
Figure 3
Figure 3
Schematic overview of the library purification and sequencing process
Figure 4
Figure 4
Schematic overview of microbiome analyses (A) Shannon diversity and bacterial density visualized as boxplots. Boxplots display the 25th and 75th percentiles, the median, and values within 1.5 times the interquartile range. Linear models can be used to calculate the p-values. (B) Principal coordinate analysis (PCoA). (C) Heatmap of the differential abundance analysis results. The color corresponds to the direction of the association.
See this image and copyright information in PMC

References

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