Data from: Rapid identification of chloroplast haplotypes using High-Resolution Melting analysis
We have evaluated High Resolution Melt (HRM) analysis as a method for one-step haplotype identification in phylogeographic analysis. Using two adjoined internal amplicons (c.360 and 390bp) at the chloroplast rps16 intron (c.750bp) we applied HRM to identify haplotypes in 21 populations of two Europe...
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ftdryad:oai:v1.datadryad.org:10255/dryad.39904 2023-05-15T15:10:23+02:00 Data from: Rapid identification of chloroplast haplotypes using High-Resolution Melting analysis Dang, Xiao-Dong Howard-Williams, Emma Kelleher, Colin T. Meade, Conor V. Europe Pleistocene 2012-05-23T19:34:13Z http://hdl.handle.net/10255/dryad.39904 https://doi.org/10.5061/dryad.s904k unknown doi:10.5061/dryad.s904k/1 doi:10.1111/j.1755-0998.2012.03164.x PMID:22783911 doi:10.5061/dryad.s904k Dang X, Howard-Williams E, Kelleher CT, Meade CV (2012) Rapid identification of chloroplast haplotypes using High-Resolution Melting analysis. Molecular Ecology Resources 12(5): 894-908. http://hdl.handle.net/10255/dryad.39904 Angiosperms Phylogeography Population Genetics - Empirical Speciation Conservation Genetics Article 2012 ftdryad https://doi.org/10.5061/dryad.s904k https://doi.org/10.5061/dryad.s904k/1 https://doi.org/10.1111/j.1755-0998.2012.03164.x 2020-01-01T14:56:39Z We have evaluated High Resolution Melt (HRM) analysis as a method for one-step haplotype identification in phylogeographic analysis. Using two adjoined internal amplicons (c.360 and 390bp) at the chloroplast rps16 intron (c.750bp) we applied HRM to identify haplotypes in 21 populations of two European arctic-alpine herb species Arenaria ciliata and A. norvegica (Caryophyllaceae). From 446 accessions studied, 20 composite rps16 haplotypes were identified by the melt-curve protocol, 18 of which could be identified uniquely. In a comparative sensitivity analysis with in silico PCR-RFLP, only 7 of these 20 haplotypes could be identified uniquely. Observed in vitro experimental HRM profiles were corroborated by in silico HRM analysis generated on uMeltSM. In silico mutation analysis carried out on a 360bp wild-type rps16I amplicon determined that the expected rate of missed SNP detection in vitro was similar to existing evaluations of HRM sensitivity, with transversion SNPs being more likely to go undetected compared to transition SNPs. Overall, pairwise melt peak differences between haplotypes were significantly correlated with genetic distance, and in vitro HRM successfully discriminated between all amplicon templates differing by 2 or more base changes (352 cases) and between 11 pairs of amplicons where the only difference was a single transition or transversion SNP. Only one pairwise comparison yielded no discernable HRM curve difference between haplotypes, these differed by one transversion (C/G) SNP. HRM analysis represents an untapped resource in phylogeographic analysis, and with appropriate primer design any polymorphic locus is potentially amenable to this single-reaction method for haplotype identification. Article in Journal/Newspaper Arctic Dryad Digital Repository (Duke University) Arctic |
institution |
Open Polar |
collection |
Dryad Digital Repository (Duke University) |
op_collection_id |
ftdryad |
language |
unknown |
topic |
Angiosperms Phylogeography Population Genetics - Empirical Speciation Conservation Genetics |
spellingShingle |
Angiosperms Phylogeography Population Genetics - Empirical Speciation Conservation Genetics Dang, Xiao-Dong Howard-Williams, Emma Kelleher, Colin T. Meade, Conor V. Data from: Rapid identification of chloroplast haplotypes using High-Resolution Melting analysis |
topic_facet |
Angiosperms Phylogeography Population Genetics - Empirical Speciation Conservation Genetics |
description |
We have evaluated High Resolution Melt (HRM) analysis as a method for one-step haplotype identification in phylogeographic analysis. Using two adjoined internal amplicons (c.360 and 390bp) at the chloroplast rps16 intron (c.750bp) we applied HRM to identify haplotypes in 21 populations of two European arctic-alpine herb species Arenaria ciliata and A. norvegica (Caryophyllaceae). From 446 accessions studied, 20 composite rps16 haplotypes were identified by the melt-curve protocol, 18 of which could be identified uniquely. In a comparative sensitivity analysis with in silico PCR-RFLP, only 7 of these 20 haplotypes could be identified uniquely. Observed in vitro experimental HRM profiles were corroborated by in silico HRM analysis generated on uMeltSM. In silico mutation analysis carried out on a 360bp wild-type rps16I amplicon determined that the expected rate of missed SNP detection in vitro was similar to existing evaluations of HRM sensitivity, with transversion SNPs being more likely to go undetected compared to transition SNPs. Overall, pairwise melt peak differences between haplotypes were significantly correlated with genetic distance, and in vitro HRM successfully discriminated between all amplicon templates differing by 2 or more base changes (352 cases) and between 11 pairs of amplicons where the only difference was a single transition or transversion SNP. Only one pairwise comparison yielded no discernable HRM curve difference between haplotypes, these differed by one transversion (C/G) SNP. HRM analysis represents an untapped resource in phylogeographic analysis, and with appropriate primer design any polymorphic locus is potentially amenable to this single-reaction method for haplotype identification. |
format |
Article in Journal/Newspaper |
author |
Dang, Xiao-Dong Howard-Williams, Emma Kelleher, Colin T. Meade, Conor V. |
author_facet |
Dang, Xiao-Dong Howard-Williams, Emma Kelleher, Colin T. Meade, Conor V. |
author_sort |
Dang, Xiao-Dong |
title |
Data from: Rapid identification of chloroplast haplotypes using High-Resolution Melting analysis |
title_short |
Data from: Rapid identification of chloroplast haplotypes using High-Resolution Melting analysis |
title_full |
Data from: Rapid identification of chloroplast haplotypes using High-Resolution Melting analysis |
title_fullStr |
Data from: Rapid identification of chloroplast haplotypes using High-Resolution Melting analysis |
title_full_unstemmed |
Data from: Rapid identification of chloroplast haplotypes using High-Resolution Melting analysis |
title_sort |
data from: rapid identification of chloroplast haplotypes using high-resolution melting analysis |
publishDate |
2012 |
url |
http://hdl.handle.net/10255/dryad.39904 https://doi.org/10.5061/dryad.s904k |
op_coverage |
Europe Pleistocene |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_relation |
doi:10.5061/dryad.s904k/1 doi:10.1111/j.1755-0998.2012.03164.x PMID:22783911 doi:10.5061/dryad.s904k Dang X, Howard-Williams E, Kelleher CT, Meade CV (2012) Rapid identification of chloroplast haplotypes using High-Resolution Melting analysis. Molecular Ecology Resources 12(5): 894-908. http://hdl.handle.net/10255/dryad.39904 |
op_doi |
https://doi.org/10.5061/dryad.s904k https://doi.org/10.5061/dryad.s904k/1 https://doi.org/10.1111/j.1755-0998.2012.03164.x |
_version_ |
1766341431169908736 |