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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

Razali, Haslina; O'Connor, Emily LU ; Drews, Anna LU ; Burke, Terry and Westerdahl, Helena LU (2017) In BMC Research Notes 10(1). p.346-356
Abstract
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,... (More)
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
diversity. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
MHC genes, MiSeq, Amplicon sequencing
in
BMC Research Notes
volume
10
issue
1
pages
10 pages
publisher
BioMed Central
external identifiers
  • scopus:85026399418
ISSN
1756-0500
DOI
10.1186/s13104-017-2654-1
language
English
LU publication?
yes
id
28debfbc-a894-40c2-b618-54bd2fb22bd3
date added to LUP
2017-09-01 15:16:23
date last changed
2017-09-05 03:00:12
@article{28debfbc-a894-40c2-b618-54bd2fb22bd3,
  abstract     = {Background: High-throughput sequencing enables high-resolution genotyping of extremely duplicated genes.<br/>454 amplicon sequencing (454) has become the standard technique for genotyping the major histocompatibility<br/>complex (MHC) genes in non-model organisms. However, illumina MiSeq amplicon sequencing (MiSeq), which offers<br/>a much higher read depth, is now superseding 454. The aim of this study was to quantitatively and qualitatively<br/>evaluate the performance of MiSeq in relation to 454 for genotyping MHC class I alleles using a house sparrow (Passer<br/>domesticus) dataset with pedigree information. House sparrows provide a good study system for this comparison as<br/>their MHC class I genes have been studied previously and, consequently, we had prior expectations concerning the<br/>number of alleles per individual.<br/>Results: We found that 454 and MiSeq performed equally well in genotyping amplicons with low diversity, i.e. amplicons<br/>from individuals that had fewer than 6 alleles. Although there was a higher rate of failure in the 454 dataset in<br/>resolving amplicons with higher diversity (6–9 alleles), the same genotypes were identified by both 454 and MiSeq in<br/>98% of cases.<br/>Conclusions: We conclude that low diversity amplicons are equally well genotyped using either 454 or MiSeq,<br/>but the higher coverage afforded by MiSeq can lead to this approach outperforming 454 in amplicons with higher<br/>diversity.},
  author       = {Razali, Haslina and O'Connor, Emily and Drews, Anna and Burke, Terry and Westerdahl, Helena},
  issn         = {1756-0500},
  keyword      = {MHC genes,MiSeq, Amplicon sequencing},
  language     = {eng},
  number       = {1},
  pages        = {346--356},
  publisher    = {BioMed Central},
  series       = {BMC Research Notes},
  title        = {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},
  url          = {http://dx.doi.org/10.1186/s13104-017-2654-1},
  volume       = {10},
  year         = {2017},
}