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Optimized detection of sequence variation in heterozygous genomes using DNA microarrays with isothermal-melting probes.

ABSTRACT: The use of DNA microarrays to identify nucleotide variation is almost 20 years old. A variety of improvements in probe design and experimental conditions have brought this technology to the point that single-nucleotide differences can be efficiently detected in unmixed samples, although developing reliable methods for detection of mixed sequences (e.g., heterozygotes) remains challenging. Surprisingly, a comprehensive study of the probe design parameters and experimental conditions that optimize discrimination of single-nucleotide polymorphisms (SNPs) has yet to be reported, so the limits of this technology remain uncertain. By targeting 24,549 SNPs that differ between two Saccharomyces cerevisiae strains, we studied the effect of SNPs on hybridization efficiency to DNA microarray probes of different lengths under different hybridization conditions. We found that the critical parameter for optimization of sequence discrimination is the relationship between probe melting temperature (T(m)) and the temperature at which the hybridization reaction is performed. This relationship can be exploited through the design of microarrays containing probes of equal T(m) by varying the length of probes. We demonstrate using such a microarray that we detect >90% homozygous SNPs and >80% heterozygous SNPs using the SNPScanner algorithm. The optimized design and experimental parameters determined in this study should guide DNA microarray designs for applications that require sequence discrimination such as mutation detection, genotyping of unmixed and mixed samples, and allele-specific gene expression. Moreover, designing microarray probes with optimized sensitivity to mismatches should increase the accuracy of standard microarray applications such as copy-number variation detection and gene expression analysis.

SUBMITTER: Gresham D

PROVIDER: S-EPMC2824413 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Optimized detection of sequence variation in heterozygous genomes using DNA microarrays with isothermal-melting probes.

Gresham David D Curry Bo B Ward Alexandra A Gordon D Benjamin DB Brizuela Leonardo L Kruglyak Leonid L Botstein David D

Proceedings of the National Academy of Sciences of the United States of America 20100108 4

The use of DNA microarrays to identify nucleotide variation is almost 20 years old. A variety of improvements in probe design and experimental conditions have brought this technology to the point that single-nucleotide differences can be efficiently detected in unmixed samples, although developing reliable methods for detection of mixed sequences (e.g., heterozygotes) remains challenging. Surprisingly, a comprehensive study of the probe design parameters and experimental conditions that optimize ...[more]

PMID: 20080586

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Project description:Goal: To identify copy number variation in normal individuals using high density, non-polymorphic oligonucleotide probes Background DNA sequence diversity within the human genome may be more greatly affected by copy number variations (CNVs) than single nucleotide polymorphisms (SNPs). Although the importance of CNVs in genome wide association studies (GWAS) is becoming widely accepted, the optimal methods for identifying these variants are still under evaluation. We have previously reported a comprehensive view of CNVs in the HapMap DNA collection using high density 500K EA (Early Access) SNP genotyping arrays which revealed greater than 1,000 CNVs ranging in size from 1kb to over 3Mb. Although the arrays used most commonly for GWAS predominantly interrogate SNPs, CNV identification and detection does not necessarily require the use of DNA probes centered on polymorphic nucleotides and may even be hindered by the dependence on a successful SNP genotyping assay. Results In this study, we have designed and evaluated a high density array predicated on the use of non-polymorphic oligonucleotide probes for CNV detection. This approach effectively uncouples copy number detection from SNP genotyping and thus has the potential to significantly improve probe coverage for genome-wide CNV identification. This array, in conjunction with PCR-based, complexity-reduced DNA target, queries over 1.3M independent NspI restriction enzyme fragments in the 200bp to 1100bp size range, which is a several fold increase in marker density as compared to the 500K EA array. In addition, a novel algorithm was developed and validated to extract CNV regions and boundaries. Conclusions Using a well-characterized pair of DNA samples, close to 200 CNVs were identified, of which nearly 50% appear novel yet were independently validated using quantitative PCR. The results indicate that non-polymorphic probes provide a robust approach for CNV identification, and the increasing precision of CNV boundary delineation should allow a more complete analysis of their genomic organization. Keywords: Copy number variation (CNV) detection

Project description:With looming resistance to fluoroquinolones in Mycoplasma genitalium, public health control strategies require effective antimicrobial resistance (AMR) diagnostic methods for clinical and phenotypic AMR surveillance. We developed a novel AMR detection method, MGparC-AsyHRM, based on the combination of asymmetric high-resolution melting (HRM) technology and unlabeled probes, which simultaneously performs M. genitalium identification and genotypes eight mutations in the parC gene that are responsible for most cases of fluoroquinolone resistance. These enhancements expand the traditional HRM from the conventional detection of single-position mutations to a method capable of detecting short fragments with closely located AMR positions with a high diversity of mutations. Based on the results of clinical sample testing, this method produces an accordance of 98.7% with the Sanger sequencing method. Furthermore, the specificity for detecting S83I, S83N, S83R, and D87Y variants, the most frequently detected mutations in fluoroquinolone resistance, was 100%. This method maintained a stable and accurate performance for genomic copies at rates of ≥20 copies per reaction, demonstrating high sensitivity. Additionally, no specific cross-reactions were observed when testing eight common sexually transmitted infection (STI)-related agents. Notably, this work highlights the significant potential of our method in the field of AMR testing, with the results suggesting that our method can be applied in a range of scenarios and to additional pathogens. In summary, our method enables high throughput, provides excellent specificity and sensitivity, and is cost-effective, suggesting that this method can be used to rapidly monitor the molecular AMR status and complement current AMR surveillance. IMPORTANCE Mycoplasma genitalium was recently added to the antimicrobial-resistant (AMR) threats "watch list" of the U.S. Centers for Disease Control and Prevention because this pathogen has become extremely difficult to treat as a result of increased resistance. M. genitalium is also difficult to culture, and therefore, molecule detection is the only method available for AMR testing. In this work, we developed a novel AMR detection method, MGparC-AsyHRM, based on the combination of asymmetrical HRM technology and unlabeled probes, and it simultaneously performs M. genitalium identification and genotypes eight mutations in the parC gene that are responsible for most cases of fluoroquinolone resistance. The MGparC-AsyHRM method is a high-throughput, low-cost, simple, and culture-free procedure that can enhance public health and management of M. genitalium infections and AMR control, providing a strong complement to phenotypic AMR surveillance to address the spread of fluoroquinolone resistance.

Dataset Information

Optimized detection of sequence variation in heterozygous genomes using DNA microarrays with isothermal-melting probes.

Publications

Optimized detection of sequence variation in heterozygous genomes using DNA microarrays with isothermal-melting probes.

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