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Open Access Research article

MHC genotyping of non-model organisms using next-generation sequencing: a new methodology to deal with artefacts and allelic dropout

Simone Sommer1*, Alexandre Courtiol1 and Camila J Mazzoni12

Author Affiliations

1 Evolutionary Genetics, Leibniz-Institute for Zoo and Wildlife Research (IZW), Alfred-Kowalke-Straße 17, D-10315 Berlin, Germany

2 Berlin Center for Genomics in Biodiversity Research, Koenigin-Luise-Straße 6-8, D-14195 Berlin, Germany

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BMC Genomics 2013, 14:542  doi:10.1186/1471-2164-14-542

Published: 9 August 2013

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.

Keywords:
Major histocompatibility complex; Next-generation sequencing; 454 pyrosequencing; Molecular cloning; PCR and sequencing artefacts; Amplification efficiency; Allelic dropout; Rodent; Delomys sublineatus