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MHC genotyping of non-model organisms using next-generation sequencing: a new methodology to deal with artefacts and allelic dropout
- Source :
- BMC Genomics
- Publisher :
- Springer Nature
-
Abstract
- Background The Major Histocompatibility Complex (MHC) is the most important genetic marker to study patterns of adaptive genetic variation determining pathogen resistance and associated life history decisions. It is used in many different research fields ranging from human medical, molecular evolutionary to functional biodiversity studies. Correct assessment of the individual allelic diversity pattern and the underlying structural sequence variation is the basic requirement to address the functional importance of MHC variability. Next-generation sequencing (NGS) technologies are likely to replace traditional genotyping methods to a great extent in the near future but first empirical studies strongly indicate the need for a rigorous quality control pipeline. Strict approaches for data validation and allele calling to distinguish true alleles from artefacts are required. Results We developed the analytical methodology and validated a data processing procedure which can be applied to any organism. It allows the separation of true alleles from artefacts and the evaluation of genotyping reliability, which in addition to artefacts considers for the first time the possibility of allelic dropout due to unbalanced amplification efficiencies across alleles. Finally, we developed a method to assess the confidence level per genotype a-posteriori, which helps to decide which alleles and individuals should be included in any further downstream analyses. The latter method could also be used for optimizing experiment designs in the future. Conclusions Combining our workflow with the study of amplification efficiency offers the chance for researchers to evaluate enormous amounts of NGS-generated data in great detail, improving confidence over the downstream analyses and subsequent applications.
- Subjects :
- Genotyping Techniques
Major histocompatibility complex
Computational biology
DNA sequencing
Genetic variation
Databases, Genetic
Delomys sublineatus
Genetics
Animals
Allele
Cloning, Molecular
Genotyping
Alleles
Rodent
biology
Arvicolinae
Molecular cloning
High-Throughput Nucleotide Sequencing
PCR and sequencing artefacts
Allelic dropout
Genetic marker
Amplification efficiency
biology.protein
Next-generation sequencing
Pyrosequencing
454 pyrosequencing
Artifacts
Research Article
Biotechnology
Subjects
Details
- Language :
- English
- ISSN :
- 14712164
- Volume :
- 14
- Issue :
- 1
- Database :
- OpenAIRE
- Journal :
- BMC Genomics
- Accession number :
- edsair.doi.dedup.....d6598724c73a7999e55bafed451afd54
- Full Text :
- https://doi.org/10.1186/1471-2164-14-542