A fungal mock community control for amplicon sequencing experiments

Area of Science:

• Microbial Ecology
• Molecular Biology
• Bioinformatics

Background:

• Marker gene sequencing advances microbial ecology but is prone to biases.
• Mock communities with known compositions are essential for assessing sequencing limitations.
• Understanding these biases is critical for accurate environmental microbial community analysis.

Purpose of the Study:

• To evaluate the impact of PCR modifications on amplicon sequencing accuracy for fungal communities.
• To identify key factors contributing to errors and biases in marker gene sequencing.
• To guide improvements in library preparation and data processing for microbial ecology studies.

Main Methods:

• Creation of three fungal mock communities with known compositions (19 taxa).
• Amplicon sequencing of mock communities under varied PCR conditions (cycles, extension time, polymerase fidelity).
• Analysis of chimera formation, error rates, and community structure similarity to known compositions.

Main Results:

• Increased PCR cycles elevated chimera formation and errors.
• Polymerase fidelity significantly impacted error rates.
• A bias-minimizing master mix yielded the most accurate community profiles despite high error rates.
• Amplification bias and size selection affected accuracy, sometimes omitting dominant taxa.

Conclusions:

• PCR conditions and polymerase choice significantly influence amplicon sequencing accuracy.
• Methodological improvements are needed to mitigate biases and ensure reliable microbial community profiling.
• Mock communities are invaluable tools for optimizing sequencing protocols in microbial ecology.