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Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry
Published on: June 21, 2018
New generation sequencers as a tool for genotyping of highly polymorphic multilocus MHC system
Wiesław Babik1, Pierre Taberlet, Maciej Jan Ejsmond
1Institute of Environmental Sciences, Jagiellonian University, ul. Gronostajowa 7, 30-387 Kraków, Poland, Laboratoire d'Ecologie Alpine, CNRS-UMR 5553, Université Joseph Fourier, Grenoble, France.
Molecular Ecology Resources
|May 14, 2011
Summary
Massively parallel 454 sequencing offers an efficient genotyping platform for complex major histocompatibility complex (MHC) systems in vertebrates. Procedures were developed to minimize genotyping errors, distinguishing true alleles from sequencing artefacts.
Area of Science:
- Genomics
- Molecular Biology
- Bioinformatics
Background:
- Accurate genotyping of complex genetic systems like the major histocompatibility complex (MHC) is crucial for various biological studies.
- Simultaneous analysis of multiple co-amplifying loci is often necessary for comprehensive genotyping.
- Traditional genotyping methods can be challenging for complex systems in nonmodel organisms.
Purpose of the Study:
- To evaluate the utility of 454 sequencing for genotyping complex MHC systems in nonmodel vertebrates.
- To develop and validate quality control procedures for reducing genotyping errors associated with 454 sequencing.
- To establish 454 sequencing as a viable alternative to classical genotyping techniques.
Main Methods:
- Utilized tagged polymerase chain reaction (PCR) primers for multiplex amplicon identification.
- Employed 454 massively parallel sequencing for simultaneous sequencing of multiple amplicons.
- Developed a frequency-based threshold (3% average per-individual frequency) to identify and eliminate artefactual alleles (AA).
- Recommended running independent PCR duplicates for direct assessment of genotyping error.
Main Results:
- Demonstrated that 454 parallel sequencing is an efficient genotyping platform for MHC systems.
- Successfully introduced procedures to effectively reduce genotyping errors by filtering artefactual alleles.
- Identified alleles with an average per-individual frequency below 3% as likely artefacts in the studied dataset.
- Showcased the potential to discriminate between true rare alleles and sequencing errors with appropriate quality control.
Conclusions:
- Massively parallel 454 sequencing provides an efficient and alternative genotyping platform for complex MHC systems.
- The developed quality control procedures, particularly the frequency threshold, are effective in minimizing genotyping errors.
- Implementing experimental controls, such as independent PCR duplicates, is essential for accurate genotyping assessments.
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