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Universal digital high-resolution melt: a novel approach to broad-based profiling of heterogeneous biological samples.

Fraley SI, Hardick J, Jo Masek B, Athamanolap P, Rothman RE, Gaydos CA, Carroll KC, Wakefield T, Wang TH, Yang S - Nucleic Acids Res. (2013)

Bottom Line: The U-dHRM approach uses broad-based primers or ligated adapter sequences to universally amplify all nucleic acid molecules in a heterogeneous sample, which have been partitioned, as in digital PCR.We show that single-molecule detection and single nucleotide sensitivity is possible.U-dHRM using broad-based 16S rRNA gene primers demonstrates universal single cell detection of bacterial pathogens, even in the presence of larger amounts of contaminating bacteria; U-dHRM using universally adapted Lethal-7 miRNAs in a heterogeneous mixture showcases the single copy sensitivity and single nucleotide specificity of this approach.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, The Johns Hopkins University, Baltimore, MD 21218, USA, Department of Emergency Medicine, The Johns Hopkins University, Baltimore, MD 21218, USA, Division of Infectious Disease, Department of Medicine, The Johns Hopkins University, Baltimore, MD 21218, USA, Division of Medical Microbiology, Department of Pathology, The Johns Hopkins University, Baltimore, MD 21218, USA, The Johns Hopkins Hospital, Baltimore, MD 21287, USA and Department of Mechanical Engineering, The Johns Hopkins University, Baltimore, MD 21218, USA.

ABSTRACT
Comprehensive profiling of nucleic acids in genetically heterogeneous samples is important for clinical and basic research applications. Universal digital high-resolution melt (U-dHRM) is a new approach to broad-based PCR diagnostics and profiling technologies that can overcome issues of poor sensitivity due to contaminating nucleic acids and poor specificity due to primer or probe hybridization inaccuracies for single nucleotide variations. The U-dHRM approach uses broad-based primers or ligated adapter sequences to universally amplify all nucleic acid molecules in a heterogeneous sample, which have been partitioned, as in digital PCR. Extensive assay optimization enables direct sequence identification by algorithm-based matching of melt curve shape and Tm to a database of known sequence-specific melt curves. We show that single-molecule detection and single nucleotide sensitivity is possible. The feasibility and utility of U-dHRM is demonstrated through detection of bacteria associated with polymicrobial blood infection and microRNAs (miRNAs) associated with host response to infection. U-dHRM using broad-based 16S rRNA gene primers demonstrates universal single cell detection of bacterial pathogens, even in the presence of larger amounts of contaminating bacteria; U-dHRM using universally adapted Lethal-7 miRNAs in a heterogeneous mixture showcases the single copy sensitivity and single nucleotide specificity of this approach.

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Lethal-7 and related miRNA are resolvable by HRM. (a) Raw HRM data, before calibration and normalization, combined from two experiments showing the loss of fluorescence in individual wells containing standard dilutions of Let-7a, b, c or miR-29 (unmixed). Flat lines are negative for amplified product. (b) A derivative plot of the fluorescence data from a, which has been temperature shifted by alignment of the temperature calibrator curve peaks, vertical dotted lines. Multiple melt curves from various dilutions of each sequence now overlap. Negative wells (gray lines) give only temperature calibrator melt curves. (c) Helicity plot of data from b after normalization and temperature calibration showing highly reproducible, unique melt curves for each tagged miRNA sequence and negative controls are clearly distinguishable (gray curves). (d) Normalized, temperature calibrated standard curves generated from 5 ten-fold dilutions across two independent experiments using each Let-7 family and related miRNA sequence from Table 1 give database references for future U-dHRM target identification.
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gkt684-F2: Lethal-7 and related miRNA are resolvable by HRM. (a) Raw HRM data, before calibration and normalization, combined from two experiments showing the loss of fluorescence in individual wells containing standard dilutions of Let-7a, b, c or miR-29 (unmixed). Flat lines are negative for amplified product. (b) A derivative plot of the fluorescence data from a, which has been temperature shifted by alignment of the temperature calibrator curve peaks, vertical dotted lines. Multiple melt curves from various dilutions of each sequence now overlap. Negative wells (gray lines) give only temperature calibrator melt curves. (c) Helicity plot of data from b after normalization and temperature calibration showing highly reproducible, unique melt curves for each tagged miRNA sequence and negative controls are clearly distinguishable (gray curves). (d) Normalized, temperature calibrated standard curves generated from 5 ten-fold dilutions across two independent experiments using each Let-7 family and related miRNA sequence from Table 1 give database references for future U-dHRM target identification.

Mentions: Here, we demonstrate that the most difficult miRNAs to correctly identify by current microarray and qPCR methods (4,42), members of the Let-7 family differing by only 1–4 nt in sequence (Table 1, differences in bold), can be identified by U-dHRM. Likewise, miRNAs having drastically different sequences can also be identified (e.g. miR-29 and miR-98, Table 1). To accomplish unbiased universal amplification, Let-7a, b, c, d, e, f, g, i, miR-98 and miR-29 sequences were tagged for universal priming (see ‘Materials and Methods’ section). In database generation experiments, each tagged cDNA sequence was serially diluted down to the digital level, universally amplified with tag primers, and HRM was performed on each homogeneous reaction product. Figure 2A shows the raw fluorescence melt data from multiple runs of standard dilutions of Let-7a, Let-7b, Let-7c and miR-29. Wells negative for amplification are clearly distinguishable as the flat lines in Figure 2A and gray lines in Figure 2B and C. A derivative plot of the fluorescence data was generated, and alignment and normalization according to the low and high temperature calibrator sequences were performed (Figure 2B). Figure 2C shows the calibrated and normalized melt data as a percentage of the highest fluorescence, i.e. when amplicons are fully annealed in a helical structure. Optimization of reaction conditions resulted in highly reproducible melt curves for each sequence in Table 1, and these were readily distinguishable using the LightScanner’s small amplicon genotyping algorithm (Figure 2D). The melt curves in Figure 2 were collected over multiple dilutions and multiple days of experimentation, demonstrating the reliability of the optimized assay. Based on known amounts of Let-7a, b, c and miR-29, a spiked mixture was prepared, digitized and universally amplified, and U-dHRM analysis was performed. The calculated input gave a theoretical occupancy, λoverall = 0.4 copies of miRNA. Figure 3 shows the results of universal U-dHRM for the mixture of miRNA. Normalized melt curves were reliably matched to a previously generated database of temperature calibrated melt curves for each miRNA (Figure 3B). From the mixture, 14 Let-7a miRNA, 12 Let-7b, 2 Let-7c and 8 miR-29 sequences were detected. Of 96 reactions, 11 were unidentified, meaning the melt curve resulted from a combination of two or more of the input sequences, and 49 reactions were negative. Digitization was confirmed by fitting these values to a Poisson distribution. Figure 3C shows the Poisson distribution for different values of λ, including that expected for λ = 0.4. Experimental quantification gave a Poisson distribution with λoverall = 0.65 instead of the calculated 0.4 (Figure 3C). The overall concentration of the input mixture is then (0.65 copies/reaction)/(2 µl of input mixture/reaction) = 0.325 copies/µl miRNA. With an increased number of digital reactions, absolute quantitation of each miRNA species can be achieved (see ‘Discussion’ section).Figure 2.


Universal digital high-resolution melt: a novel approach to broad-based profiling of heterogeneous biological samples.

Fraley SI, Hardick J, Jo Masek B, Athamanolap P, Rothman RE, Gaydos CA, Carroll KC, Wakefield T, Wang TH, Yang S - Nucleic Acids Res. (2013)

Lethal-7 and related miRNA are resolvable by HRM. (a) Raw HRM data, before calibration and normalization, combined from two experiments showing the loss of fluorescence in individual wells containing standard dilutions of Let-7a, b, c or miR-29 (unmixed). Flat lines are negative for amplified product. (b) A derivative plot of the fluorescence data from a, which has been temperature shifted by alignment of the temperature calibrator curve peaks, vertical dotted lines. Multiple melt curves from various dilutions of each sequence now overlap. Negative wells (gray lines) give only temperature calibrator melt curves. (c) Helicity plot of data from b after normalization and temperature calibration showing highly reproducible, unique melt curves for each tagged miRNA sequence and negative controls are clearly distinguishable (gray curves). (d) Normalized, temperature calibrated standard curves generated from 5 ten-fold dilutions across two independent experiments using each Let-7 family and related miRNA sequence from Table 1 give database references for future U-dHRM target identification.
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Related In: Results  -  Collection

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gkt684-F2: Lethal-7 and related miRNA are resolvable by HRM. (a) Raw HRM data, before calibration and normalization, combined from two experiments showing the loss of fluorescence in individual wells containing standard dilutions of Let-7a, b, c or miR-29 (unmixed). Flat lines are negative for amplified product. (b) A derivative plot of the fluorescence data from a, which has been temperature shifted by alignment of the temperature calibrator curve peaks, vertical dotted lines. Multiple melt curves from various dilutions of each sequence now overlap. Negative wells (gray lines) give only temperature calibrator melt curves. (c) Helicity plot of data from b after normalization and temperature calibration showing highly reproducible, unique melt curves for each tagged miRNA sequence and negative controls are clearly distinguishable (gray curves). (d) Normalized, temperature calibrated standard curves generated from 5 ten-fold dilutions across two independent experiments using each Let-7 family and related miRNA sequence from Table 1 give database references for future U-dHRM target identification.
Mentions: Here, we demonstrate that the most difficult miRNAs to correctly identify by current microarray and qPCR methods (4,42), members of the Let-7 family differing by only 1–4 nt in sequence (Table 1, differences in bold), can be identified by U-dHRM. Likewise, miRNAs having drastically different sequences can also be identified (e.g. miR-29 and miR-98, Table 1). To accomplish unbiased universal amplification, Let-7a, b, c, d, e, f, g, i, miR-98 and miR-29 sequences were tagged for universal priming (see ‘Materials and Methods’ section). In database generation experiments, each tagged cDNA sequence was serially diluted down to the digital level, universally amplified with tag primers, and HRM was performed on each homogeneous reaction product. Figure 2A shows the raw fluorescence melt data from multiple runs of standard dilutions of Let-7a, Let-7b, Let-7c and miR-29. Wells negative for amplification are clearly distinguishable as the flat lines in Figure 2A and gray lines in Figure 2B and C. A derivative plot of the fluorescence data was generated, and alignment and normalization according to the low and high temperature calibrator sequences were performed (Figure 2B). Figure 2C shows the calibrated and normalized melt data as a percentage of the highest fluorescence, i.e. when amplicons are fully annealed in a helical structure. Optimization of reaction conditions resulted in highly reproducible melt curves for each sequence in Table 1, and these were readily distinguishable using the LightScanner’s small amplicon genotyping algorithm (Figure 2D). The melt curves in Figure 2 were collected over multiple dilutions and multiple days of experimentation, demonstrating the reliability of the optimized assay. Based on known amounts of Let-7a, b, c and miR-29, a spiked mixture was prepared, digitized and universally amplified, and U-dHRM analysis was performed. The calculated input gave a theoretical occupancy, λoverall = 0.4 copies of miRNA. Figure 3 shows the results of universal U-dHRM for the mixture of miRNA. Normalized melt curves were reliably matched to a previously generated database of temperature calibrated melt curves for each miRNA (Figure 3B). From the mixture, 14 Let-7a miRNA, 12 Let-7b, 2 Let-7c and 8 miR-29 sequences were detected. Of 96 reactions, 11 were unidentified, meaning the melt curve resulted from a combination of two or more of the input sequences, and 49 reactions were negative. Digitization was confirmed by fitting these values to a Poisson distribution. Figure 3C shows the Poisson distribution for different values of λ, including that expected for λ = 0.4. Experimental quantification gave a Poisson distribution with λoverall = 0.65 instead of the calculated 0.4 (Figure 3C). The overall concentration of the input mixture is then (0.65 copies/reaction)/(2 µl of input mixture/reaction) = 0.325 copies/µl miRNA. With an increased number of digital reactions, absolute quantitation of each miRNA species can be achieved (see ‘Discussion’ section).Figure 2.

Bottom Line: The U-dHRM approach uses broad-based primers or ligated adapter sequences to universally amplify all nucleic acid molecules in a heterogeneous sample, which have been partitioned, as in digital PCR.We show that single-molecule detection and single nucleotide sensitivity is possible.U-dHRM using broad-based 16S rRNA gene primers demonstrates universal single cell detection of bacterial pathogens, even in the presence of larger amounts of contaminating bacteria; U-dHRM using universally adapted Lethal-7 miRNAs in a heterogeneous mixture showcases the single copy sensitivity and single nucleotide specificity of this approach.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, The Johns Hopkins University, Baltimore, MD 21218, USA, Department of Emergency Medicine, The Johns Hopkins University, Baltimore, MD 21218, USA, Division of Infectious Disease, Department of Medicine, The Johns Hopkins University, Baltimore, MD 21218, USA, Division of Medical Microbiology, Department of Pathology, The Johns Hopkins University, Baltimore, MD 21218, USA, The Johns Hopkins Hospital, Baltimore, MD 21287, USA and Department of Mechanical Engineering, The Johns Hopkins University, Baltimore, MD 21218, USA.

ABSTRACT
Comprehensive profiling of nucleic acids in genetically heterogeneous samples is important for clinical and basic research applications. Universal digital high-resolution melt (U-dHRM) is a new approach to broad-based PCR diagnostics and profiling technologies that can overcome issues of poor sensitivity due to contaminating nucleic acids and poor specificity due to primer or probe hybridization inaccuracies for single nucleotide variations. The U-dHRM approach uses broad-based primers or ligated adapter sequences to universally amplify all nucleic acid molecules in a heterogeneous sample, which have been partitioned, as in digital PCR. Extensive assay optimization enables direct sequence identification by algorithm-based matching of melt curve shape and Tm to a database of known sequence-specific melt curves. We show that single-molecule detection and single nucleotide sensitivity is possible. The feasibility and utility of U-dHRM is demonstrated through detection of bacteria associated with polymicrobial blood infection and microRNAs (miRNAs) associated with host response to infection. U-dHRM using broad-based 16S rRNA gene primers demonstrates universal single cell detection of bacterial pathogens, even in the presence of larger amounts of contaminating bacteria; U-dHRM using universally adapted Lethal-7 miRNAs in a heterogeneous mixture showcases the single copy sensitivity and single nucleotide specificity of this approach.

Show MeSH
Related in: MedlinePlus