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RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing

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A quantitative SMRT cell sequencing method for ribosomal amplicons.

Bethan M Jones¹, Adam B Kustka¹

¹Department of Earth and Environmental Sciences, Rutgers University-Newark, Newark, NJ 07102, United States.

Journal of Microbiological Methods

|February 5, 2017

Summary

We developed quantitative SMRT (qSMRT) sequencing to simultaneously classify and quantify microbial communities. This method accurately measures ribosomal amplicons from environmental samples, revealing ecosystem diversity and function.

Keywords:

18S Quantitative Ribosomal Ross Sea SMRT Sequencing Silicoflagellate qSMRT

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Area of Science:

Microbial ecology
Molecular biology
Environmental science

Background:

Advancements in sequencing technologies offer new ways to study microbial communities.
Pacific Biosciences Single Molecule Real-Time (SMRT) sequencing provides long reads at low costs.
Accurate quantification of microbial populations in situ is crucial for understanding ecosystem function.

Purpose of the Study:

To develop a quantitative SMRT (qSMRT) approach for simultaneous classification and enumeration of in situ microbial populations.
To apply qSMRT for quantifying protistan communities in the Ross Sea.
To establish a rapid and cost-effective method for analyzing microbial diversity.

Main Methods:

Developed a quantitative SMRT (qSMRT) approach using exogenous standards.
Quantified ribosomal amplicons (V7-9 regions of 18S SSU rDNA) from environmental samples.
Utilized three standards of varying lengths and optimized conditions for accurate quantification.

Main Results:

Successfully applied qSMRT to protistan community samples from the Ross Sea.
Achieved accurate quantitative retrieval across a range of amplicon sizes.
Demonstrated the ability to concurrently identify and quantify microorganisms.

Conclusions:

qSMRT is a powerful, rapid, and cost-effective method for microbial community analysis.
This approach enables precise definition of ecosystem diversity and function.
qSMRT advances the characterization of microbial populations in their natural environments.