Biomedical subjects

Richard Humbert

Publications and source records attributed to Richard Humbert.

6 recordsLinked to original sources

PubMed2006

Genome-scale mapping of DNase I sensitivity in vivo using tiling DNA microarrays.

Localized accessibility of critical DNA sequences to the regulatory machinery is a key requirement for regulation of human genes. Here we describe a high-resolution, genome-scale approach for quantifying chromatin accessibility by measuring DNase I sensitivity as a continuous function of genome position using tiling DNA microarrays (DNase-array). We demonstrate this approach across 1% ( approximately 30 Mb) of the human genome, wherein we localized 2,690 classical DNase I hypersensitive sites with high sensitivity and specificity, and also mapped larger-scale patterns of chromatin architecture. DNase I hypersensitive sites exhibit marked aggregation around transcriptional start sites (TSSs), though the majority mark nonpromoter functional elements. We also developed a computational approach for visualizing higher-order features of chromatin structure. This revealed that human chromatin organization is dominated by large (100-500 kb) 'superclusters' of DNase I hypersensitive sites, which encompass both gene-rich and gene-poor regions. DNase-array is a powerful and straightforward approach for systematic exposition of the cis-regulatory architecture of complex genomes.

Chromatin↗

PubMed2006

Automated validation of polymerase chain reaction amplicon melting curves.

The polymerase chain reaction (PCR) is a fundamental tool of molecular biology. Quantitative PCR is the gold-standard methodology for determination of DNA copy numbers, quantitating transcription, and numerous other applications. A major barrier to large-scale application of PCR for quantitative genomic analyses is the current requirement for manual validation of individual PCRs to ensure generation of a single product. This typically requires visual inspection either of gel electrophoreses or temperature dissociation ("melting") curves of individual PCRs--a time-consuming and costly process. Here we describe a robust computational solution to this fundamental problem. Using a training set of 10 080 reactions comprising multiple quantitative PCRs from each of 1728 unique human genomic amplicons, we developed a support vector machine classifier capable of discriminating single-product PCRs with better than 99% accuracy. This approach has broad utility, and eliminates a major bottleneck to widespread application of PCR for high-throughput genomic applications.

Algorithms↗

PubMed2005

Automated validation of polymerase chain reactions using amplicon melting curves.

PCR, the polymerase chain reaction, is a fundamental tool of molecular biology. Quantitative PCR is the gold-standard methodology for determination of DNA copy numbers, quantitating transcription, and numerous other applications. A major barrier to large-scale application of PCR for quantitative genomic analyses is the current requirement for manual validation of individual PCR reactions to ensure generation of a single product. This typically requires visual inspection either of gel electrophoreses or temperature dissociation ("melting") curves of individual PCR reactions - a time-consuming and costly process. Here we describe a robust computational solution to this fundamental problem. Using a training set of 10,080 reactions comprising multiple quantitative PCR reactions from each of 1,728 unique human genomic amplicons, we developed a support vector machine classifier capable of discriminating single-product PCR reactions with better than 99% accuracy. This approach has broad utility, and eliminates a major bottleneck to widespread application of PCR for high-throughput genomic applications.

Algorithms↗

PubMed2004

High-throughput localization of functional elements by quantitative chromatin profiling.

Identification of functional, noncoding elements that regulate transcription in the context of complex genomes is a major goal of modern biology. Localization of functionality to specific sequences is a requirement for genetic and computational studies. Here, we describe a generic approach, quantitative chromatin profiling, that uses quantitative analysis of in vivo chromatin structure over entire gene loci to rapidly and precisely localize cis-regulatory sequences and other functional modalities encoded by DNase I hypersensitive sites. To demonstrate the accuracy of this approach, we analyzed approximately 300 kilobases of human genome sequence from diverse gene loci and cleanly delineated functional elements corresponding to a spectrum of classical cis-regulatory activities including enhancers, promoters, locus control regions and insulators as well as novel elements. Systematic, high-throughput identification of functional elements coinciding with DNase I hypersensitive sites will substantially expand our knowledge of transcriptional regulation and should simplify the search for noncoding genetic variation with phenotypic consequences.

Algorithms↗

PubMed2004

Discovery of functional noncoding elements by digital analysis of chromatin structure.

We developed a quantitative methodology, digital analysis of chromatin structure (DACS), for high-throughput, automated mapping of DNase I-hypersensitive sites and associated cis-regulatory sequences in the human and other complex genomes. We used 19/20-bp genomic DNA tags to localize individual DNase I cutting events in nuclear chromatin and produced approximately 257,000 tags from erythroid cells. Tags were mapped to the human genome, and a quantitative algorithm was applied to discriminate statistically significant clusters of independent DNase I cutting events. We show that such clusters identify both known regulatory sequences and previously unrecognized functional elements across the genome. We used in silico simulation to demonstrate that DACS is capable of efficient and accurate localization of the majority of DNase I-hypersensitive sites in the human genome without requiring an independent validation step. A unique feature of DACS is that it permits unbiased evaluation of the chromatin state of regulatory sequences from widely separated genomic loci. We found surprisingly large differences in the accessibility of distant regulatory sequences, suggesting the existence of a hierarchy of nuclear organization that escapes detection by conventional chromatin assays.

Chromatin↗

PubMed2004

Genome-wide identification of DNaseI hypersensitive sites using active chromatin sequence libraries.

Comprehensive identification of sequences that regulate transcription is one of the major goals of genome biology. Focal alteration in chromatin structure in vivo, detectable through hypersensitivity to DNaseI and other nucleases, is the sine qua non of a diverse cast of transcriptional regulatory elements including enhancers, promoters, insulators, and locus control regions. We developed an approach for genome-scale identification of DNaseI hypersensitive sites (HSs) via isolation and cloning of in vivo DNaseI cleavage sites to create libraries of active chromatin sequences (ACSs). Here, we describe analysis of >61,000 ACSs derived from erythroid cells. We observed peaks in the density of ACSs at the transcriptional start sites of known genes at non-gene-associated CpG islands, and, to a lesser degree, at evolutionarily conserved noncoding sequences. Peaks in ACS density paralleled the distribution of DNaseI HSs. ACSs and DNaseI HSs were distributed between both expressed and nonexpressed genes, suggesting that a large proportion of genes reside within open chromatin domains. The results permit a quantitative approximation of the distribution of HSs and classical cis-regulatory sequences in the human genome.

Chromatin↗