Generating amplicon reads for microbial community assessment with next-generation sequencing.

Area of Science:

• Microbiology
• Genomics
• Bioinformatics

Background:

• Marker gene amplicon sequencing is widely used for microbial community characterization due to cost-effectiveness and ability to study uncultivable microbes.
• The technique involves multiple experimental steps, each introducing potential errors and biases.
• Optimizing these steps is crucial for accurate microbial community assessment.

Purpose of the Study:

• To provide a comprehensive, up-to-date overview of the marker gene amplicon sequencing pipeline.
• To guide researchers in making informed decisions throughout the experimental process.
• To highlight potential pitfalls and suggest mitigation strategies in amplicon sequencing.

Main Methods:

• Review and synthesis of current best practices in marker gene amplicon sequencing.
• Detailed examination of each stage of the experimental pipeline, from sample collection to sequencing data analysis.
• Identification of critical control points and potential sources of bias.

Main Results:

• An in-depth analysis of the entire amplicon sequencing workflow.
• Recommendations for optimizing experimental design and execution.
• Strategies for minimizing bias and errors at each step.

Conclusions:

• Informed choices at each experimental stage are essential for reliable microbial community characterization.
• Understanding and addressing potential pitfalls in amplicon sequencing improves data accuracy.
• This overview serves as a valuable resource for researchers conducting microbial community studies.