Making and Sequencing Heavily Multiplexed, High-Throughput 16S Ribosomal RNA Gene Amplicon Libraries Using a...

Area of Science:

• Microbiology
• Molecular Biology
• Bioinformatics

Background:

• Deep sequencing of ribosomal RNA (rRNA) genes is crucial for microbial community analysis.
• Current methods for preparing genomic DNA for sequencing can be complex.

Purpose of the Study:

• To describe a straightforward, two-stage PCR protocol for preparing genomic DNA for Illumina sequencing.
• To enable high-throughput microbial community characterization.

Main Methods:

• A two-stage polymerase chain reaction (PCR) amplification of small subunit (16S or 18S) ribosomal RNA (rRNA) gene fragments.
• Utilizing Illumina sequencing technology.
• Protocol designed for simultaneous processing of up to 384 samples.

Main Results:

• The described protocol simplifies genomic DNA preparation for deep sequencing.
• The method allows for flexible primer selection.
• High-throughput analysis of microbial communities is achievable.

Conclusions:

• The presented two-stage PCR method is an efficient and flexible approach for preparing microbial samples for deep sequencing.
• This protocol facilitates comprehensive microbial community structure and composition studies.