Background: High-throughput sequencing enables high-resolution genotyping of extremely duplicated genes.
454 amplicon sequencing (454) has become the standard technique for genotyping the major histocompatibility
complex (MHC) genes in non-model organisms. However, illumina MiSeq amplicon sequencing (MiSeq), which offers
a much higher read depth, is now superseding 454. The aim of this study was to quantitatively and qualitatively
evaluate the performance of MiSeq in relation to 454 for genotyping MHC class I alleles using a house sparrow (Passer
domesticus) dataset with pedigree information. House sparrows provide a good study system for this comparison as
their MHC class I genes have been studied previously and, consequently, we had prior expectations concerning the
number of alleles per individual.
Results: We found that 454 and MiSeq performed equally well in genotyping amplicons with low diversity, i.e. amplicons
from individuals that had fewer than 6 alleles. Although there was a higher rate of failure in the 454 dataset in
resolving amplicons with higher diversity (6–9 alleles), the same genotypes were identified by both 454 and MiSeq in
98% of cases.
Conclusions: We conclude that low diversity amplicons are equally well genotyped using either 454 or MiSeq,
but the higher coverage afforded by MiSeq can lead to this approach outperforming 454 in amplicons with higher
- Biochemistry and Molecular Biology
- MHC genes
- Amplicon sequencing