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A quantitative and qualitative comparison of illumina MiSeq and 454 amplicon sequencing for genotyping the highly polymorphic major histocompatibility complex (MHC) in a non‑model species

  • Haslina Razali
  • Emily O'Connor
  • Anna Drews
  • Terry Burke
  • Helena Westerdahl
Publishing year: 2017
Language: English
Pages: 346-356
Publication/Series: BMC Research Notes
Volume: 10
Issue: 1
Document type: Journal article
Publisher: BioMed Central

Abstract english

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
  • MiSeq
  • Amplicon sequencing


  • ISSN: 1756-0500
Helena Westerdahl
E-mail: helena [dot] westerdahl [at] biol [dot] lu [dot] se

Senior lecturer


+46 46 222 36 69



Research group

Molecular Ecology and Evolution Lab


Doctoral students and postdocs

Research fellows


Luz Garcia-Longoria

PhD students, main supervisor

Samantha Mellinger

PhD students, assistant supervisor

Gustaf Ekelund Ugge

Helena Westerdahl w house sparrow