Biomedical subjects

Todd A Richmond

Publications and source records attributed to Todd A Richmond.

10 recordsLinked to original sources

PubMed2021

Evaluating the analytical validity of circulating tumor DNA sequencing assays for precision oncology.

Circulating tumor DNA (ctDNA) sequencing is being rapidly adopted in precision oncology, but the accuracy, sensitivity and reproducibility of ctDNA assays is poorly understood. Here we report the findings of a multi-site, cross-platform evaluation of the analytical performance of five industry-leading ctDNA assays. We evaluated each stage of the ctDNA sequencing workflow with simulations, synthetic DNA spike-in experiments and proficiency testing on standardized, cell-line-derived reference samples. Above 0.5% variant allele frequency, ctDNA mutations were detected with high sensitivity, precision and reproducibility by all five assays, whereas, below this limit, detection became unreliable and varied widely between assays, especially when input material was limited. Missed mutations (false negatives) were more common than erroneous candidates (false positives), indicating that the reliable sampling of rare ctDNA fragments is the key challenge for ctDNA assays. This comprehensive evaluation of the analytical performance of ctDNA assays serves to inform best practice guidelines and provides a resource for precision oncology.

Circulating Tumor DNA↗

PubMed2006

A high-resolution map of segmental DNA copy number variation in the mouse genome.

Submicroscopic (less than 2 Mb) segmental DNA copy number changes are a recently recognized source of genetic variability between individuals. The biological consequences of copy number variants (CNVs) are largely undefined. In some cases, CNVs that cause gene dosage effects have been implicated in phenotypic variation. CNVs have been detected in diverse species, including mice and humans. Published studies in mice have been limited by resolution and strain selection. We chose to study 21 well-characterized inbred mouse strains that are the focus of an international effort to measure, catalog, and disseminate phenotype data. We performed comparative genomic hybridization using long oligomer arrays to characterize CNVs in these strains. This technique increased the resolution of CNV detection by more than an order of magnitude over previous methodologies. The CNVs range in size from 21 to 2,002 kb. Clustering strains by CNV profile recapitulates aspects of the known ancestry of these strains. Most of the CNVs (77.5%) contain annotated genes, and many (47.5%) colocalize with previously mapped segmental duplications in the mouse genome. We demonstrate that this technique can identify copy number differences associated with known polymorphic traits. The phenotype of previously uncharacterized strains can be predicted based on their copy number at these loci. Annotation of CNVs in the mouse genome combined with sequence-based analysis provides an important resource that will help define the genetic basis of complex traits.

Animals↗

PubMed2006

Chromosome Conformation Capture Carbon Copy (5C): a massively parallel solution for mapping interactions between genomic elements.

Physical interactions between genetic elements located throughout the genome play important roles in gene regulation and can be identified with the Chromosome Conformation Capture (3C) methodology. 3C converts physical chromatin interactions into specific ligation products, which are quantified individually by PCR. Here we present a high-throughput 3C approach, 3C-Carbon Copy (5C), that employs microarrays or quantitative DNA sequencing using 454-technology as detection methods. We applied 5C to analyze a 400-kb region containing the human beta-globin locus and a 100-kb conserved gene desert region. We validated 5C by detection of several previously identified looping interactions in the beta-globin locus. We also identified a new looping interaction in K562 cells between the beta-globin Locus Control Region and the gamma-beta-globin intergenic region. Interestingly, this region has been implicated in the control of developmental globin gene switching. 5C should be widely applicable for large-scale mapping of cis- and trans- interaction networks of genomic elements and for the study of higher-order chromosome structure.

Base Sequence↗

PubMed2006

Discovery of previously unidentified genomic disorders from the duplication architecture of the human genome.

Genomic disorders are characterized by the presence of flanking segmental duplications that predispose these regions to recurrent rearrangement. Based on the duplication architecture of the genome, we investigated 130 regions that we hypothesized as candidates for previously undescribed genomic disorders. We tested 290 individuals with mental retardation by BAC array comparative genomic hybridization and identified 16 pathogenic rearrangements, including de novo microdeletions of 17q21.31 found in four individuals. Using oligonucleotide arrays, we refined the breakpoints of this microdeletion, defining a 478-kb critical region containing six genes that were deleted in all four individuals. We mapped the breakpoints of this deletion and of four other pathogenic rearrangements in 1q21.1, 15q13, 15q24 and 17q12 to flanking segmental duplications, suggesting that these are also sites of recurrent rearrangement. In common with the 17q21.31 deletion, these breakpoint regions are sites of copy number polymorphism in controls, indicating that these may be inherently unstable genomic regions.

Chromosome Breakage↗

PubMed2006

Comparative isoschizomer profiling of cytosine methylation: the HELP assay.

The distribution of cytosine methylation in 6.2 Mb of the mouse genome was tested using cohybridization of genomic representations from a methylation-sensitive restriction enzyme and its methylation-insensitive isoschizomer. This assay, termed HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR), allows both intragenomic profiling and intergenomic comparisons of cytosine methylation. The intragenomic profile shows most of the genome to be contiguous methylated sequence with occasional clusters of hypomethylated loci, usually but not exclusively at promoters and CpG islands. Intergenomic comparison found marked differences in cytosine methylation between spermatogenic and brain cells, identifying 223 new candidate tissue-specific differentially methylated regions (T-DMRs). Bisulfite pyrosequencing confirmed the four candidates tested to be T-DMRs, while quantitative RT-PCR for two genes with T-DMRs located at their promoters showed the HELP data to be correlated with gene activity at these loci. The HELP assay is robust, quantitative, and accurate and is providing new insights into the distribution and dynamic nature of cytosine methylation in the genome.

Animals↗

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↗

PubMed2005

Mutation discovery in bacterial genomes: metronidazole resistance in Helicobacter pylori.

We developed a microarray hybridization-based method, 'comparative genome sequencing' (CGS), to find mutations in bacterial genomes and used it to study metronidazole resistance in H. pylori. CGS identified mutations in several genes, most likely affecting metronidazole activation, and produced no false positives in analysis of three megabases. We conclude that CGS identifies mutations in bacterial genomes efficiently, should enrich understanding of systems biology and genome evolution, and help track pathogens during outbreaks.

Anti-Infective Agents↗

PubMed2005

A high-resolution map of active promoters in the human genome.

In eukaryotic cells, transcription of every protein-coding gene begins with the assembly of an RNA polymerase II preinitiation complex (PIC) on the promoter. The promoters, in conjunction with enhancers, silencers and insulators, define the combinatorial codes that specify gene expression patterns. Our ability to analyse the control logic encoded in the human genome is currently limited by a lack of accurate information regarding the promoters for most genes. Here we describe a genome-wide map of active promoters in human fibroblast cells, determined by experimentally locating the sites of PIC binding throughout the human genome. This map defines 10,567 active promoters corresponding to 6,763 known genes and at least 1,196 un-annotated transcriptional units. Features of the map suggest extensive use of multiple promoters by the human genes and widespread clustering of active promoters in the genome. In addition, examination of the genome-wide expression profile reveals four general classes of promoters that define the transcriptome of the cell. These results provide a global view of the functional relationships among transcriptional machinery, chromatin structure and gene expression in human cells.

Chromatin↗

PubMed2005

Analysis of chromosome breakpoints in neuroblastoma at sub-kilobase resolution using fine-tiling oligonucleotide array CGH.

Understanding the genes and genetic pathways targeted by recurrent chromosomal imbalances in malignancy, along with the molecular mechanisms that generate the imbalances, are important problems in cancer biology. In this report, we demonstrate that oligonucleotide array CGH (oaCGH) analysis can routinely map chromosomal imbalance breakpoints at exon-level resolution, including imbalances that are single copy number genomic alterations. Different tiling-path array designs were used in this study: a whole-genome array with a 6-kb median probe spacing and fine-tiling arrays for selected genomic regions with either 50- or 140-bp median probe spacing. In both array formats, oligonucleotide probes were of isothermal design and were tiled through genic and inter-genic regions. Whole-genome oaCGH analysis of two neuroblastoma cell lines and three primary tumors led to the identification of 58 chromosomal breakpoints that generated 45 large-scale partial chromosomal imbalances (> 2 Mb). An unexpectedly high proportion (34%) of these breakpoint intervals mapped to regions containing segmental duplications. In addition, 88 smaller-sized regions (< 2 Mb) of imbalance were detected, the majority of which mapped to segmentally duplicated regions and may reflect constitutional copy number polymorphisms. The chromosomal breakpoints for 12 recurrent abnormalities exhibited in neuroblastoma tumors and cell lines, including MYCN amplicon boundaries, loss of 3p, loss of 11q, and gain of 17q, could be mapped to intervals ranging from 50 bp to 10 kb in size using high-density fine-tiling oligonucleotide microarrays. Fine-tiling oaCGH analysis provides an unprecedented level of resolution, allowing detailed mapping of recurrent unbalanced chromosomal abnormalities. Supplementary material for this article can be found on the Genes, Chromosomes, and Cancer website at http://www.interscience.wiley.com/jpages/1045-2257/suppmat/index.html.

Chromosome Breakage↗

PubMed2004

Tracking the evolution of the SARS coronavirus using high-throughput, high-density resequencing arrays.

Mutations in the SARS-Coronavirus (SARS-CoV) can alter its clinical presentation, and the study of its mutation patterns in human populations can facilitate contact tracing. Here, we describe the development and validation of an oligonucleotide resequencing array for interrogating the entire 30-kb SARS-CoV genome in a rapid, cost-effective fashion. Using this platform, we sequenced SARS-CoV genomes from Vero cell culture isolates of 12 patients and directly from four patient tissues. The sequence obtained from the array is highly reproducible, accurate (>99.99% accuracy) and capable of identifying known and novel variants of SARS-CoV. Notably, we applied this technology to a field specimen of probable SARS and rapidly deduced its infectious source. We demonstrate that array-based resequencing-by-hybridization is a fast, reliable, and economical alternative to capillary sequencing for obtaining SARS-CoV genomic sequence on a population scale, making this an ideal platform for the global monitoring of SARS-CoV and other small-genome pathogens.

Animals↗