Data from: Comparison of 454 pyrosequencing methods for characterizing the major histocompatibility complex of nonmodel species and the advantages of ultra deep coverage
Characterization and population genetic analysis of multilocus genes, such as those found in the major histocompatibility complex (MHC) is challenging in nonmodel vertebrates. The traditional method of extensive cloning and Sanger sequencing is costly and time-intensive and indirect methods of asses...
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ftdryad:oai:v1.datadryad.org:10255/dryad.41252 2023-05-15T16:32:20+02:00 Data from: Comparison of 454 pyrosequencing methods for characterizing the major histocompatibility complex of nonmodel species and the advantages of ultra deep coverage Oomen, Rebekah A. Gillett, Roxanne M. Kyle, Christopher J. Ontario Manitoba British Columbia Northwest Territories Yukon Holocene 2017-06-26T15:22:46Z http://hdl.handle.net/10255/dryad.41252 https://doi.org/10.5061/dryad.s5b40 unknown doi:10.5061/dryad.s5b40/1 doi:10.5061/dryad.s5b40/2 doi:10.5061/dryad.s5b40/3 doi:10.5061/dryad.s5b40/4 doi:10.5061/dryad.s5b40/5 doi:10.5061/dryad.s5b40/6 doi:10.5061/dryad.s5b40/7 doi:10.5061/dryad.s5b40/8 doi:10.1111/1755-0998.12027 doi:10.5061/dryad.s5b40 Oomen RA, Gillett RM, Kyle CJ (2013) Comparison of 454 pyrosequencing methods for characterizing the major histocompatibility complex of nonmodel species and the advantages of ultra deep coverage. Molecular Ecology Resources 13(1): 103-116. http://hdl.handle.net/10255/dryad.41252 wolverine major histocompatibility complex multi-locus genes 454 sequencing pyrosequencing Article 2017 ftdryad https://doi.org/10.5061/dryad.s5b40 https://doi.org/10.5061/dryad.s5b40/1 https://doi.org/10.5061/dryad.s5b40/2 https://doi.org/10.5061/dryad.s5b40/3 https://doi.org/10.5061/dryad.s5b40/4 https://doi.org/10.5061/dryad.s5b40/5 https://doi.org/1 2020-01-01T14:57:19Z Characterization and population genetic analysis of multilocus genes, such as those found in the major histocompatibility complex (MHC) is challenging in nonmodel vertebrates. The traditional method of extensive cloning and Sanger sequencing is costly and time-intensive and indirect methods of assessment often underestimate total variation. Here, we explored the suitability of 454 pyrosequencing for characterizing multilocus genes for use in population genetic studies. We compared two sample tagging protocols and two bioinformatic procedures for 454 sequencing through characterization of a 185-bp fragment of MHC DRB exon 2 in wolverines (Gulo gulo) and further compared the results with those from cloning and Sanger sequencing. We found 10 putative DRB alleles in the 88 individuals screened with between two and four alleles per individual, suggesting amplification of a duplicated DRB gene. In addition to the putative alleles, all individuals possessed an easily identifiable pseudogene. In our system, sequence variants with a frequency below 6% in an individual sample were usually artefacts. However, we found that sample preparation and data processing procedures can greatly affect variant frequencies in addition to the complexity of the multilocus system. Therefore, we recommend determining a per-amplicon-variant frequency threshold for each unique system. The extremely deep coverage obtained in our study (approximately 5000×) coupled with the semi-quantitative nature of pyrosequencing enabled us to assign all putative alleles to the two DRB loci, which is generally not possible using traditional methods. Our method of obtaining locus-specific MHC genotypes will enhance population genetic analyses and studies on disease susceptibility in nonmodel wildlife species. Article in Journal/Newspaper Gulo gulo Northwest Territories Yukon Dryad Digital Repository (Duke University) Northwest Territories Yukon |
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Open Polar |
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Dryad Digital Repository (Duke University) |
op_collection_id |
ftdryad |
language |
unknown |
topic |
wolverine major histocompatibility complex multi-locus genes 454 sequencing pyrosequencing |
spellingShingle |
wolverine major histocompatibility complex multi-locus genes 454 sequencing pyrosequencing Oomen, Rebekah A. Gillett, Roxanne M. Kyle, Christopher J. Data from: Comparison of 454 pyrosequencing methods for characterizing the major histocompatibility complex of nonmodel species and the advantages of ultra deep coverage |
topic_facet |
wolverine major histocompatibility complex multi-locus genes 454 sequencing pyrosequencing |
description |
Characterization and population genetic analysis of multilocus genes, such as those found in the major histocompatibility complex (MHC) is challenging in nonmodel vertebrates. The traditional method of extensive cloning and Sanger sequencing is costly and time-intensive and indirect methods of assessment often underestimate total variation. Here, we explored the suitability of 454 pyrosequencing for characterizing multilocus genes for use in population genetic studies. We compared two sample tagging protocols and two bioinformatic procedures for 454 sequencing through characterization of a 185-bp fragment of MHC DRB exon 2 in wolverines (Gulo gulo) and further compared the results with those from cloning and Sanger sequencing. We found 10 putative DRB alleles in the 88 individuals screened with between two and four alleles per individual, suggesting amplification of a duplicated DRB gene. In addition to the putative alleles, all individuals possessed an easily identifiable pseudogene. In our system, sequence variants with a frequency below 6% in an individual sample were usually artefacts. However, we found that sample preparation and data processing procedures can greatly affect variant frequencies in addition to the complexity of the multilocus system. Therefore, we recommend determining a per-amplicon-variant frequency threshold for each unique system. The extremely deep coverage obtained in our study (approximately 5000×) coupled with the semi-quantitative nature of pyrosequencing enabled us to assign all putative alleles to the two DRB loci, which is generally not possible using traditional methods. Our method of obtaining locus-specific MHC genotypes will enhance population genetic analyses and studies on disease susceptibility in nonmodel wildlife species. |
format |
Article in Journal/Newspaper |
author |
Oomen, Rebekah A. Gillett, Roxanne M. Kyle, Christopher J. |
author_facet |
Oomen, Rebekah A. Gillett, Roxanne M. Kyle, Christopher J. |
author_sort |
Oomen, Rebekah A. |
title |
Data from: Comparison of 454 pyrosequencing methods for characterizing the major histocompatibility complex of nonmodel species and the advantages of ultra deep coverage |
title_short |
Data from: Comparison of 454 pyrosequencing methods for characterizing the major histocompatibility complex of nonmodel species and the advantages of ultra deep coverage |
title_full |
Data from: Comparison of 454 pyrosequencing methods for characterizing the major histocompatibility complex of nonmodel species and the advantages of ultra deep coverage |
title_fullStr |
Data from: Comparison of 454 pyrosequencing methods for characterizing the major histocompatibility complex of nonmodel species and the advantages of ultra deep coverage |
title_full_unstemmed |
Data from: Comparison of 454 pyrosequencing methods for characterizing the major histocompatibility complex of nonmodel species and the advantages of ultra deep coverage |
title_sort |
data from: comparison of 454 pyrosequencing methods for characterizing the major histocompatibility complex of nonmodel species and the advantages of ultra deep coverage |
publishDate |
2017 |
url |
http://hdl.handle.net/10255/dryad.41252 https://doi.org/10.5061/dryad.s5b40 |
op_coverage |
Ontario Manitoba British Columbia Northwest Territories Yukon Holocene |
geographic |
Northwest Territories Yukon |
geographic_facet |
Northwest Territories Yukon |
genre |
Gulo gulo Northwest Territories Yukon |
genre_facet |
Gulo gulo Northwest Territories Yukon |
op_relation |
doi:10.5061/dryad.s5b40/1 doi:10.5061/dryad.s5b40/2 doi:10.5061/dryad.s5b40/3 doi:10.5061/dryad.s5b40/4 doi:10.5061/dryad.s5b40/5 doi:10.5061/dryad.s5b40/6 doi:10.5061/dryad.s5b40/7 doi:10.5061/dryad.s5b40/8 doi:10.1111/1755-0998.12027 doi:10.5061/dryad.s5b40 Oomen RA, Gillett RM, Kyle CJ (2013) Comparison of 454 pyrosequencing methods for characterizing the major histocompatibility complex of nonmodel species and the advantages of ultra deep coverage. Molecular Ecology Resources 13(1): 103-116. http://hdl.handle.net/10255/dryad.41252 |
op_doi |
https://doi.org/10.5061/dryad.s5b40 https://doi.org/10.5061/dryad.s5b40/1 https://doi.org/10.5061/dryad.s5b40/2 https://doi.org/10.5061/dryad.s5b40/3 https://doi.org/10.5061/dryad.s5b40/4 https://doi.org/10.5061/dryad.s5b40/5 https://doi.org/1 |
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