Evaluation of two approaches to genotyping major histocompatibility complex class I in a passerine-CE-SSCP and 454 pyrosequencing

Genes of the highly dynamic major histocompatibility complex (MHC) are directly linked to individual fitness and are of high interest in evolutionary ecology and conservation genetics. Gene duplication and positive selection usually lead to high levels of polymorphism in the MHC region, making genot...

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Bibliographic Details
Published in:Molecular ecology resources 2012-03, Vol.12 (2), p.285-292
Main Authors: PROMEROVÁ, MARTA, BABIK, WIESŁAW, BRYJA, JOSEF, ALBRECHT, TOMÁŠ, STUGLIK, MICHAŁ, RADWAN, JACEK
Format: Article
Language:English
Subjects:
Animals
avian
Avian Proteins - genetics
Carpodacus erythrinus
Electrophoresis, Capillary - methods
Evolution
Genes
Genes, MHC Class II
Genotype
major histocompatibility complex
Molecular Sequence Data
next-generation sequencing
Passeriformes - classification
Passeriformes - genetics
Phylogeny
Polymorphism, Single-Stranded Conformational
scarlet rosefinch
Sequence Analysis, DNA - methods
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Summary:Genes of the highly dynamic major histocompatibility complex (MHC) are directly linked to individual fitness and are of high interest in evolutionary ecology and conservation genetics. Gene duplication and positive selection usually lead to high levels of polymorphism in the MHC region, making genotyping of MHC a challenging task. Here, we compare the performance of two methods for MHC class I genotyping in a passerine with highly duplicated MHC class I genes: capillary electrophoresis‐single‐strand conformation polymorphism (CE‐SSCP) analysis and 454 GS FLX Titanium pyrosequencing. According to our findings, the number of MHC variants (called alleles for simplicity) detected by CE‐SSCP is significantly lower than detected by 454. To resolve discrepancies between the two methods, we cloned and Sanger sequenced a MHC class I amplicon for an individual with high number of alleles. We found a perfect congruence between cloning/Sanger sequencing results and 454. Thus, in case of multi‐locus amplification, CE‐SSCP considerably underestimates individual MHC diversity. However, numbers of alleles detected by both methods are significantly correlated, although the correlation is weak (r = 0.32). Thus, in systems with highly duplicated MHC, 454 provides more reliable information on individual diversity than CE‐SSCP.
ISSN:1755-098X
1755-0998
DOI:10.1111/j.1755-0998.2011.03082.x