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Quantitative high resolution melting: two methods to determine SNP allele frequencies from pooled samples.

Capper RL, Jin YK, Lundgren PB, Peplow LM, Matz MV, van Oppen MJ - BMC Genet. (2015)

Bottom Line: We further demonstrate advantages of each method over previously published methods; specifically, the "peaks" method can be rapidly scaled to screen several hundred SNPs at once, whereas the "curves" method is better suited for smaller numbers of SNPs.Compared to genotyping individual samples, these methods can save considerable effort and genotyping costs when relatively few candidate SNPs must be profiled across a large number of populations.One of the main applications of this method could be validation of SNPs of interest identified in population genomic studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, University of Texas at Austin, Austin, TX, 78712, USA. roxana.capper@gmail.com.

ABSTRACT

Background: The advent of next-generation sequencing has brought about an explosion of single nucleotide polymorphism (SNP) data in non-model organisms; however, profiling these SNPs across multiple natural populations still requires substantial time and resources.

Results: Here, we introduce two cost-efficient quantitative High Resolution Melting (qHRM) methods for measuring allele frequencies at known SNP loci in pooled DNA samples: the "peaks" method, which can be applied to large numbers of SNPs, and the "curves" method, which is more labor intensive but also slightly more accurate. Using the reef-building coral Acropora millepora, we show that both qHRM methods can recover the allele proportions from mixtures prepared using two or more individuals of known genotype. We further demonstrate advantages of each method over previously published methods; specifically, the "peaks" method can be rapidly scaled to screen several hundred SNPs at once, whereas the "curves" method is better suited for smaller numbers of SNPs.

Conclusions: Compared to genotyping individual samples, these methods can save considerable effort and genotyping costs when relatively few candidate SNPs must be profiled across a large number of populations. One of the main applications of this method could be validation of SNPs of interest identified in population genomic studies.

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Related in: MedlinePlus

DNA titrations using the peaks method. a) Probe melting peaks for different allele titrations demonstrating clear resolution among allele frequencies (15 %, 25 %, 50 %). b) Two homozygotes were mixed in varying proportions to test qHRM using SNP C45133S676 (Pearson’s r = 0.97, regression slope = 0.82) and SNP C22162S248 (Pearson’s r = 0.99, regression slope = 0.80). The qHRM estimates were tightly correlated to expected frequencies; however, qHRM appears to underestimate the true allele frequency for the low-melting allele for both SNPs considered.
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Fig3: DNA titrations using the peaks method. a) Probe melting peaks for different allele titrations demonstrating clear resolution among allele frequencies (15 %, 25 %, 50 %). b) Two homozygotes were mixed in varying proportions to test qHRM using SNP C45133S676 (Pearson’s r = 0.97, regression slope = 0.82) and SNP C22162S248 (Pearson’s r = 0.99, regression slope = 0.80). The qHRM estimates were tightly correlated to expected frequencies; however, qHRM appears to underestimate the true allele frequency for the low-melting allele for both SNPs considered.

Mentions: As an initial proof of concept, we analyzed DNA from two adult individuals determined to be homozygous for different alleles of the same SNP, mixed in varying proportions to represent a spectrum of allele frequencies. The probe peaks for three allele frequency examples (50 %, 25 %, 15 %) are presented in Fig. 3a. For the two SNPs analyzed in this way, there was a strong linear correlation between the qHRM estimations and the true proportion (Pearson r = 0.97-0.99). However, the slope of the regression for both SNPs was 0.80 (Fig. 3b).Fig. 3


Quantitative high resolution melting: two methods to determine SNP allele frequencies from pooled samples.

Capper RL, Jin YK, Lundgren PB, Peplow LM, Matz MV, van Oppen MJ - BMC Genet. (2015)

DNA titrations using the peaks method. a) Probe melting peaks for different allele titrations demonstrating clear resolution among allele frequencies (15 %, 25 %, 50 %). b) Two homozygotes were mixed in varying proportions to test qHRM using SNP C45133S676 (Pearson’s r = 0.97, regression slope = 0.82) and SNP C22162S248 (Pearson’s r = 0.99, regression slope = 0.80). The qHRM estimates were tightly correlated to expected frequencies; however, qHRM appears to underestimate the true allele frequency for the low-melting allele for both SNPs considered.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4465018&req=5

Fig3: DNA titrations using the peaks method. a) Probe melting peaks for different allele titrations demonstrating clear resolution among allele frequencies (15 %, 25 %, 50 %). b) Two homozygotes were mixed in varying proportions to test qHRM using SNP C45133S676 (Pearson’s r = 0.97, regression slope = 0.82) and SNP C22162S248 (Pearson’s r = 0.99, regression slope = 0.80). The qHRM estimates were tightly correlated to expected frequencies; however, qHRM appears to underestimate the true allele frequency for the low-melting allele for both SNPs considered.
Mentions: As an initial proof of concept, we analyzed DNA from two adult individuals determined to be homozygous for different alleles of the same SNP, mixed in varying proportions to represent a spectrum of allele frequencies. The probe peaks for three allele frequency examples (50 %, 25 %, 15 %) are presented in Fig. 3a. For the two SNPs analyzed in this way, there was a strong linear correlation between the qHRM estimations and the true proportion (Pearson r = 0.97-0.99). However, the slope of the regression for both SNPs was 0.80 (Fig. 3b).Fig. 3

Bottom Line: We further demonstrate advantages of each method over previously published methods; specifically, the "peaks" method can be rapidly scaled to screen several hundred SNPs at once, whereas the "curves" method is better suited for smaller numbers of SNPs.Compared to genotyping individual samples, these methods can save considerable effort and genotyping costs when relatively few candidate SNPs must be profiled across a large number of populations.One of the main applications of this method could be validation of SNPs of interest identified in population genomic studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, University of Texas at Austin, Austin, TX, 78712, USA. roxana.capper@gmail.com.

ABSTRACT

Background: The advent of next-generation sequencing has brought about an explosion of single nucleotide polymorphism (SNP) data in non-model organisms; however, profiling these SNPs across multiple natural populations still requires substantial time and resources.

Results: Here, we introduce two cost-efficient quantitative High Resolution Melting (qHRM) methods for measuring allele frequencies at known SNP loci in pooled DNA samples: the "peaks" method, which can be applied to large numbers of SNPs, and the "curves" method, which is more labor intensive but also slightly more accurate. Using the reef-building coral Acropora millepora, we show that both qHRM methods can recover the allele proportions from mixtures prepared using two or more individuals of known genotype. We further demonstrate advantages of each method over previously published methods; specifically, the "peaks" method can be rapidly scaled to screen several hundred SNPs at once, whereas the "curves" method is better suited for smaller numbers of SNPs.

Conclusions: Compared to genotyping individual samples, these methods can save considerable effort and genotyping costs when relatively few candidate SNPs must be profiled across a large number of populations. One of the main applications of this method could be validation of SNPs of interest identified in population genomic studies.

Show MeSH
Related in: MedlinePlus