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Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing

Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing

Published on: August 25, 2018

Reducing bias in microbiome research: Comparing methods from sample collection to sequencing.

Jolanda Kool¹, Liza Tymchenko¹, Sudarshan A Shetty^1,2

¹Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, Netherlands.

Frontiers in Microbiology

|April 17, 2023

Summary

This summary is machine-generated.

Technical variations significantly impact microbiome study results. Immediate frozen storage and specific DNA input/PCR cycle counts are crucial for accurate fecal microbiota profiling, offering a compromise for challenging logistics.

Keywords:

16S rRNA gene sequencing gut human studies microbiome microbiota reproducible analysis

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Area of Science:

Microbiology
Genomics
Bioinformatics

Background:

Microbiota profiles are highly susceptible to technical variations, hindering result comparability.
Standardizing workflows is essential for reliable microbiome research.

Purpose of the Study:

To identify and assess biases introduced by technical variations in microbiome studies.
To compare different sample collection, preservation, and processing methods.

Main Methods:

Evaluated sample stabilization solutions (OMNIgene·GUT, Zymo Research) versus immediate freezing.
Analyzed DNA extraction, DNA input, and PCR cycle variations.
Utilized mock communities and human fecal samples with positive controls to assess batch effects.

Main Results:

Stabilization buffers limited Enterobacteriaceae overgrowth at room temperature (RT).
RT-stabilized samples differed from frozen samples in bacterial phyla abundance.
Mechanical cell disruption and PCR cycle number significantly impacted microbiota composition and contaminant levels.

Conclusions:

Mechanical cell disruption and immediate frozen storage are critical for fecal microbiota studies.
RT-stabilized samples offer a viable alternative for challenging logistics.
Optimal parameters for library preparation include ~125 pg input DNA and 25 PCR cycles to minimize contaminants.