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Pyrosequencing for Microbial Identification and Characterization
Published on: August 22, 2013
Quality control in multi-tag pyrosequencing of microbial communities
Masoumeh Sikaroodi1, Patrick M Gillevet
1Department of Environmental Science and Policy, George Mason University, Manassas, Virginia, USA. msikaroo@gmu.edu
Biotechniques
|December 12, 2012
Summary
This study introduces a new protocol combining length-heterogeneity PCR fingerprinting with pyrosequencing to prevent bias in microbial community analysis. The method ensures accurate amplicon abundance for reliable microbial community profiling.
Area of Science:
- Microbiology
- Molecular Biology
- Bioinformatics
Background:
- Multi-tag pyrosequencing is crucial for microbial community analysis.
- Initial PCR amplification can introduce kinetic bias, distorting microbial community composition data.
- Accurate representation of microbial communities is essential for ecological and health studies.
Purpose of the Study:
- To develop a protocol ensuring linear amplification of microbial communities for accurate pyrosequencing.
- To validate a method that mitigates kinetic bias during PCR amplification.
- To confirm the reliability of pyrosequencing data after implementing the new protocol.
Main Methods:
- A novel protocol combining length-heterogeneity PCR fingerprinting with pyrosequencing.
- Utilizing fluorescently labeled reverse primers and multi-tagged forward primers for initial amplification.
- Fingerprinting, purification, and quantitation of labeled amplicons before emulsion PCR and pyrosequencing.
Main Results:
- The protocol demonstrated linear amplification in pyrosequencing results, validated against initial length-heterogeneity PCR fingerprints.
- Sequence distribution post-emulsion PCR accurately reflected the initial amplification linearity.
- Incorporated tags and labels did not negatively impact the overall microbial community profiles.
Conclusions:
- The developed protocol effectively ensures linear amplification and accurate microbial community composition analysis via pyrosequencing.
- This method provides a reliable approach to overcome kinetic bias in PCR amplification for microbial ecology studies.
- The protocol is a valuable tool for researchers aiming for precise microbial community profiling.
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