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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2021
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Online Access: | https://doi.org/10.5061/dryad.s5b40 |
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fttriple:oai:gotriple.eu:50|dedup_wf_001::ac44986e1c74a98b37a8f6a8cc07090e 2023-05-15T16:32:16+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. 2021-06-20 https://doi.org/10.5061/dryad.s5b40 en eng Dryad http://dx.doi.org/10.5061/dryad.s5b40 https://dx.doi.org/10.5061/dryad.s5b40 lic_creative-commons 10.5061/dryad.s5b40 oai:services.nod.dans.knaw.nl:Products/dans:oai:easy.dans.knaw.nl:easy-dataset:81183 oai:easy.dans.knaw.nl:easy-dataset:81183 10|openaire____::9e3be59865b2c1c335d32dae2fe7b254 10|re3data_____::94816e6421eeb072e7742ce6a9decc5f 10|eurocrisdris::fe4903425d9040f680d8610d9079ea14 10|re3data_____::84e123776089ce3c7a33db98d9cd15a8 re3data_____::r3d100000044 10|openaire____::081b82f96300b6a6e3d282bad31cb6e2 454 sequencing wolverine Gulo gulo multi-locus genes pyrosequencing Major histocompatibility complex Holocene Life sciences medicine and health care Ontario Manitoba British Columbia Northwest Territories Yukon envir stat Dataset https://vocabularies.coar-repositories.org/resource_types/c_ddb1/ 2021 fttriple https://doi.org/10.5061/dryad.s5b40 2023-01-22T16:53:31Z 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. Reads from run 163 of Gulo gulo MHC DRB exon 2 fragmentReads from run 245 of Gulo gulo MHC DRB exon 2 fragmentReads from run 329-1 of Gulo gulo MHC DRB exon 2 fragmentReads from run 329-2 of Gulo gulo MHC DRB exon 2 fragmentReads from run 329-3 of Gulo ... Dataset Gulo gulo Northwest Territories Yukon Unknown Yukon Northwest Territories |
institution |
Open Polar |
collection |
Unknown |
op_collection_id |
fttriple |
language |
English |
topic |
454 sequencing wolverine Gulo gulo multi-locus genes pyrosequencing Major histocompatibility complex Holocene Life sciences medicine and health care Ontario Manitoba British Columbia Northwest Territories Yukon envir stat |
spellingShingle |
454 sequencing wolverine Gulo gulo multi-locus genes pyrosequencing Major histocompatibility complex Holocene Life sciences medicine and health care Ontario Manitoba British Columbia Northwest Territories Yukon envir stat 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 |
454 sequencing wolverine Gulo gulo multi-locus genes pyrosequencing Major histocompatibility complex Holocene Life sciences medicine and health care Ontario Manitoba British Columbia Northwest Territories Yukon envir stat |
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. Reads from run 163 of Gulo gulo MHC DRB exon 2 fragmentReads from run 245 of Gulo gulo MHC DRB exon 2 fragmentReads from run 329-1 of Gulo gulo MHC DRB exon 2 fragmentReads from run 329-2 of Gulo gulo MHC DRB exon 2 fragmentReads from run 329-3 of Gulo ... |
format |
Dataset |
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 |
publisher |
Dryad |
publishDate |
2021 |
url |
https://doi.org/10.5061/dryad.s5b40 |
geographic |
Yukon Northwest Territories |
geographic_facet |
Yukon Northwest Territories |
genre |
Gulo gulo Northwest Territories Yukon |
genre_facet |
Gulo gulo Northwest Territories Yukon |
op_source |
10.5061/dryad.s5b40 oai:services.nod.dans.knaw.nl:Products/dans:oai:easy.dans.knaw.nl:easy-dataset:81183 oai:easy.dans.knaw.nl:easy-dataset:81183 10|openaire____::9e3be59865b2c1c335d32dae2fe7b254 10|re3data_____::94816e6421eeb072e7742ce6a9decc5f 10|eurocrisdris::fe4903425d9040f680d8610d9079ea14 10|re3data_____::84e123776089ce3c7a33db98d9cd15a8 re3data_____::r3d100000044 10|openaire____::081b82f96300b6a6e3d282bad31cb6e2 |
op_relation |
http://dx.doi.org/10.5061/dryad.s5b40 https://dx.doi.org/10.5061/dryad.s5b40 |
op_rights |
lic_creative-commons |
op_doi |
https://doi.org/10.5061/dryad.s5b40 |
_version_ |
1766022023913406464 |