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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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Schematic diagram of qHRM. An individual heterozygous for a particular SNP locus is shown as an example. a For both methods, the locus is amplified from genomic DNA using asymmetric PCR to generate an excess of the reverse strand. Next, an oligonucleotide probe is added to the reaction. b The mixture is heated to melt all duplexes apart, and subsequently cooled rapidly to promote unbiased duplex reformation of all possible duplex DNA species. For a biallelic SNP site, this produces six distinct types of duplexes: two perfect-match amplicon duplexes, two mismatch amplicon duplexes, one perfect-match probe duplex, and one mismatch probe duplex. c For the peaks method, the reaction is heated again to denature all duplexes while the fluorescence of the reaction, which quantifies double stranded DNA, is monitored. d Next, the negative first derivative of the decreasing fluorescence curve is calculated to transform the curve into a peaks profile. The height of the lowest-melting, mismatched probe duplex peak is divided by the total heights of both probe peaks to yield the frequency of the mismatched allele in the sample. e For the curves method, the same process was repeated as described for panel c. Three known samples that consist of each genotype (i.e. heterozygote, high-melting homozygote and low-melting homozygote) were used as references. f Melt curves from the raw channel were normalized by averaging fluorescence value outside a melting phase and forcing the values to be the same for each sample
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Fig2: Schematic diagram of qHRM. An individual heterozygous for a particular SNP locus is shown as an example. a For both methods, the locus is amplified from genomic DNA using asymmetric PCR to generate an excess of the reverse strand. Next, an oligonucleotide probe is added to the reaction. b The mixture is heated to melt all duplexes apart, and subsequently cooled rapidly to promote unbiased duplex reformation of all possible duplex DNA species. For a biallelic SNP site, this produces six distinct types of duplexes: two perfect-match amplicon duplexes, two mismatch amplicon duplexes, one perfect-match probe duplex, and one mismatch probe duplex. c For the peaks method, the reaction is heated again to denature all duplexes while the fluorescence of the reaction, which quantifies double stranded DNA, is monitored. d Next, the negative first derivative of the decreasing fluorescence curve is calculated to transform the curve into a peaks profile. The height of the lowest-melting, mismatched probe duplex peak is divided by the total heights of both probe peaks to yield the frequency of the mismatched allele in the sample. e For the curves method, the same process was repeated as described for panel c. Three known samples that consist of each genotype (i.e. heterozygote, high-melting homozygote and low-melting homozygote) were used as references. f Melt curves from the raw channel were normalized by averaging fluorescence value outside a melting phase and forcing the values to be the same for each sample

Mentions: Though within-plate variation among replicates was low for the peaks method, we noticed that between-plate variation could shift the melting profile of all duplex DNA species (i.e. for an analyzed heterozygote, such species include two perfect-match amplicons, two mismatched amplicons, one perfect-match probe-SNP duplex, and one mismatch probe-SNP duplex; Fig. 2a,b) by as much as 2 °C. To accommodate this phenomenon, which occurred only in the peaks method, we included standard internal melting calibrator primers with each reaction in order to provide landmarks in the melting profile. Four oligonucleotide calibrators were designed to form two complementary DNA duplexes with known melting temperatures. They were based on sequences from Gundry et al. [29] but were adjusted to melt at the slightly lower and higher temperatures of 47.5 °C and 90 °C than the published sequences in order to melt well outside of the range of the probe and amplicon duplexes. The 3′ terminal ends of each of the four oligos were modified with an inverted T to block any potential primer extension from occurring. The forward sequence of the low-melting calibrator used is 5′- ATT TTA TAT TTA TAT ATT TAT ATA TTT TT/3InvdT/ -3′, while the forward sequence of the high-melting calibrator is 5′- GCG CGG CCG GCA CTG ACC CGA GAC TCT GAG CGG CTG CTG GAG GTG CGG AAG CGG AGG GGC GGG/3InvdT/. The calibrator oligos were included in the initial asymmetric PCR reaction at a concentration of 0.05 μM for each of the high-melting oligos and 0.5 μM for the low-melting oligos; addition of the calibrators did not interfere with amplification of the target products.Fig. 2


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)

Schematic diagram of qHRM. An individual heterozygous for a particular SNP locus is shown as an example. a For both methods, the locus is amplified from genomic DNA using asymmetric PCR to generate an excess of the reverse strand. Next, an oligonucleotide probe is added to the reaction. b The mixture is heated to melt all duplexes apart, and subsequently cooled rapidly to promote unbiased duplex reformation of all possible duplex DNA species. For a biallelic SNP site, this produces six distinct types of duplexes: two perfect-match amplicon duplexes, two mismatch amplicon duplexes, one perfect-match probe duplex, and one mismatch probe duplex. c For the peaks method, the reaction is heated again to denature all duplexes while the fluorescence of the reaction, which quantifies double stranded DNA, is monitored. d Next, the negative first derivative of the decreasing fluorescence curve is calculated to transform the curve into a peaks profile. The height of the lowest-melting, mismatched probe duplex peak is divided by the total heights of both probe peaks to yield the frequency of the mismatched allele in the sample. e For the curves method, the same process was repeated as described for panel c. Three known samples that consist of each genotype (i.e. heterozygote, high-melting homozygote and low-melting homozygote) were used as references. f Melt curves from the raw channel were normalized by averaging fluorescence value outside a melting phase and forcing the values to be the same for each sample
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Schematic diagram of qHRM. An individual heterozygous for a particular SNP locus is shown as an example. a For both methods, the locus is amplified from genomic DNA using asymmetric PCR to generate an excess of the reverse strand. Next, an oligonucleotide probe is added to the reaction. b The mixture is heated to melt all duplexes apart, and subsequently cooled rapidly to promote unbiased duplex reformation of all possible duplex DNA species. For a biallelic SNP site, this produces six distinct types of duplexes: two perfect-match amplicon duplexes, two mismatch amplicon duplexes, one perfect-match probe duplex, and one mismatch probe duplex. c For the peaks method, the reaction is heated again to denature all duplexes while the fluorescence of the reaction, which quantifies double stranded DNA, is monitored. d Next, the negative first derivative of the decreasing fluorescence curve is calculated to transform the curve into a peaks profile. The height of the lowest-melting, mismatched probe duplex peak is divided by the total heights of both probe peaks to yield the frequency of the mismatched allele in the sample. e For the curves method, the same process was repeated as described for panel c. Three known samples that consist of each genotype (i.e. heterozygote, high-melting homozygote and low-melting homozygote) were used as references. f Melt curves from the raw channel were normalized by averaging fluorescence value outside a melting phase and forcing the values to be the same for each sample
Mentions: Though within-plate variation among replicates was low for the peaks method, we noticed that between-plate variation could shift the melting profile of all duplex DNA species (i.e. for an analyzed heterozygote, such species include two perfect-match amplicons, two mismatched amplicons, one perfect-match probe-SNP duplex, and one mismatch probe-SNP duplex; Fig. 2a,b) by as much as 2 °C. To accommodate this phenomenon, which occurred only in the peaks method, we included standard internal melting calibrator primers with each reaction in order to provide landmarks in the melting profile. Four oligonucleotide calibrators were designed to form two complementary DNA duplexes with known melting temperatures. They were based on sequences from Gundry et al. [29] but were adjusted to melt at the slightly lower and higher temperatures of 47.5 °C and 90 °C than the published sequences in order to melt well outside of the range of the probe and amplicon duplexes. The 3′ terminal ends of each of the four oligos were modified with an inverted T to block any potential primer extension from occurring. The forward sequence of the low-melting calibrator used is 5′- ATT TTA TAT TTA TAT ATT TAT ATA TTT TT/3InvdT/ -3′, while the forward sequence of the high-melting calibrator is 5′- GCG CGG CCG GCA CTG ACC CGA GAC TCT GAG CGG CTG CTG GAG GTG CGG AAG CGG AGG GGC GGG/3InvdT/. The calibrator oligos were included in the initial asymmetric PCR reaction at a concentration of 0.05 μM for each of the high-melting oligos and 0.5 μM for the low-melting oligos; addition of the calibrators did not interfere with amplification of the target products.Fig. 2

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