Browsing by Author "Mittelman, David"

Now showing 1 - 7 of 7
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    Accurate human microsatellite genotypes from high-throughput resequencing data using informed error profiles
    Highnam, Gareth; Franck, Christopher T.; Martin, Andy; Stephens, Calvin; Puthige, Ashwin; Mittelman, David (Oxford University Press, 2013-01)
    Repetitive sequences are biologically and clinically important because they can influence traits and disease, but repeats are challenging to analyse using short-read sequencing technology. We present a tool for genotyping microsatellite repeats called RepeatSeq, which uses Bayesian model selection guided by an empirically derived error model that incorporates sequence and read properties. Next, we apply RepeatSeq to high-coverage genomes from the 1000 Genomes Project to evaluate performance and accuracy. The software uses common formats, such as VCF, for compatibility with existing genome analysis pipelines. Source code and binaries are available at http://github.com/adaptivegenome/repeatseq.
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    Analysis of Microsatellite Variation in Drosophila melanogaster with Population-Scale Genome Sequencing
    Fondon, John W. III; Martin, Andy; Richards, Stephen; Gibbs, Richard A.; Mittelman, David (PLoS, 2012-03-12)
    Genome sequencing technologies promise to revolutionize our understanding of genetics, evolution, and disease by making it feasible to survey a broad spectrum of sequence variation on a population scale. However, this potential can only be realized to the extent that methods for extracting and interpreting distinct forms of variation can be established. The error profiles and read length limitations of early versions of next-generation sequencing technologies rendered them ineffective for some sequence variant types, particularly microsatellites and other tandem repeats, and fostered the general misconception that such variants are inherently inaccessible to these platforms. At the same time, tandem repeats have emerged as important sources of functional variation. Tandem repeats are often located in and around genes, and frequent mutations in their lengths exert quantitative effects on gene function and phenotype, rapidly degrading linkage disequilibrium between markers and traits. Sensitive identification of these variants in large-scale next-gen sequencing efforts will enable more comprehensive association studies capable of revealing previously invisible associations. We present a population-scale analysis of microsatellite repeats using whole-genome data from 158 inbred isolates from the Drosophila Genetics Reference Panel, a collection of over 200 extensively phenotypically characterized isolates from a single natural population, to uncover processes underlying repeat mutation and to enable associations with behavioral, morphological, and life-history traits. Analysis of repeat variation from next-generation sequence data will also enhance studies of genome stability and neurodegenerative diseases.
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    An analytical framework for optimizing variant discovery from personal genomes
    Highnam, Gareth; Wang, Jason J.; Kusler, Dean; Zook, Justin; Vijayan, Vinaya; Leibovich, Nir; Mittelman, David (Springer Nature, 2015-02)
    The standardization and performance testing of analysis tools is a prerequisite to widespread adoption of genome-wide sequencing, particularly in the clinic. However, performance testing is currently complicated by the paucity of standards and comparison metrics, as well as by the heterogeneity in sequencing platforms, applications and protocols. Here we present the genome comparison and analytic testing (GCAT) platform to facilitate development of performance metrics and comparisons of analysis tools across these metrics. Performance is reported through interactive visualizations of benchmark and performance testing data, with support for data slicing and filtering. The platform is freely accessible at http://www.bioplanet.com/gcat.
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    Focusing of mammalian cells under an ultrahigh pH gradient created by unidirectional electropulsation in a confined microchamber
    Loufakis, Despina N.; Cao, Zhenning; Ma, Sai; Mittelman, David; Lu, Chang (The Royal Society of Chemistry, 2014-06-09)
    The transport and manipulation of cells in microfluidic structures are often critically required in cellular analysis. Cells typically make consistent movement in a dc electric field in a single direction, due to their electrophoretic mobility or electroosmotic flow or the combination of the two. Here we demonstrate that mammalian cells focus to the middle of a closed microfluidic chamber under the application of unidirectional direct current pulses. With experimental and computational data, we show that under the pulses electrochemical reactions take place in the confined microscale space and create an ultrahigh and nonlinear pH gradient (~2 orders of magnitude higher than the ones in protein isoelectric focusing) at the middle of the chamber. The varying local pH affects the cell surface charge and the electrophoretic mobility, leading to focusing in free solution. Our approach provides a new and simple method for focusing and concentrating mammalian cells at the microscale.
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    GFP-Based Fluorescence Assay for CAG Repeat Instability in Cultured Human Cells
    Santillan, Beatriz A.; Moye, Christopher; Mittelman, David; Wilson, John H. (PLOS, 2014-11-25)
    Trinucleotide repeats can be highly unstable, mutating far more frequently than point mutations. Repeats typically mutate by addition or loss of units of the repeat. CAG repeat expansions in humans trigger neurological diseases that include myotonic dystrophy, Huntington disease, and several spinocerebellar ataxias. In human cells, diverse mechanisms promote CAG repeat instability, and in mice, the mechanisms of instability are varied and tissue-dependent. Dissection of mechanistic complexity and discovery of potential therapeutics necessitates quantitative and scalable screens for repeat mutation. We describe a GFP-based assay for screening modifiers of CAG repeat instability in human cells. The assay exploits an engineered intronic CAG repeat tract that interferes with expression of an inducible GFP minigene. Like the phenotypes of many trinucleotide repeat disorders, we find that GFP function is impaired by repeat expansion, in a length-dependent manner. The intensity of fluorescence varies inversely with repeat length, allowing estimates of repeat tract changes in live cells. We validate the assay using transcription through the repeat and engineered CAG-specific nucleases, which have previously been reported to induce CAG repeat instability. The assay is relatively fast and should be adaptable to large-scale screens of chemical and shRNA libraries.
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    Lentiviral and targeted cellular barcoding reveals ongoing clonal dynamics of cell lines in vitro and in vivo
    Porter, Shaina N.; Baker, Lee C.; Mittelman, David; Porteus, Matthew H. (2014-05-30)
    Background Cell lines are often regarded as clonal, even though this simplifies what is known about mutagenesis, transformation and other processes that destabilize them over time. Monitoring these clonal dynamics is important for multiple areas of biomedical research, including stem cell and cancer biology. Tracking the contributions of individual cells to large populations, however, has been constrained by limitations in sensitivity and complexity. Results We utilize cellular barcoding methods to simultaneously track the clonal contributions of tens of thousands of cells. We demonstrate that even with optimal culturing conditions, common cell lines including HeLa, K562 and HEK-293 T exhibit ongoing clonal dynamics. Starting a population with a single clone diminishes but does not eradicate this phenomenon. Next, we compare lentiviral and zinc-finger nuclease barcode insertion approaches, finding that the zinc-finger nuclease protocol surprisingly results in reduced clonal diversity. We also document the expected reduction in clonal complexity when cells are challenged with genotoxic stress. Finally, we demonstrate that xenografts maintain clonal diversity to a greater extent than in vitro culturing of the human non-small-cell lung cancer cell line HCC827. Conclusions We demonstrate the feasibility of tracking and quantifying the clonal dynamics of entire cell populations within multiple cultured cell lines. Our results suggest that cell heterogeneity should be considered in the design and interpretation of in vitro culture experiments. Aside from clonal cell lines, we propose that cellular barcoding could prove valuable in modeling the clonal behavior of heterogeneous cell populations over time, including tumor populations treated with chemotherapeutic agents.
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    Polymorphic tandem repeats within gene promoters act as modifiers of gene expression and DNA methylation in humans
    Quilez, Javier; Guilmatre, Audrey; Garg, Paras; Highnam, Gareth; Gymrek, Melissa; Erlich, Yaniv; Joshi, Ricky S.; Mittelman, David; Sharp, Andrew J. (2016-05-05)
    Despite representing an important source of genetic variation, tandem repeats (TRs) remain poorly studied due to technical difficulties. We hypothesized that TRs can operate as expression (eQTLs) and methylation (mQTLs) quantitative trait loci. To test this we analyzed the effect of variation at 4849 promoter-associated TRs, genotyped in 120 individuals, on neighboring gene expression and DNA methylation. Polymorphic promoter TRs were associated with increased variance in local gene expression and DNA methylation, suggesting functional consequences related to TR variation. We identified >100 TRs associated with expression/methylation levels of adjacent genes. These potential eQTL/mQTL TRs were enriched for overlaps with transcription factor binding and DNaseI hypersensitivity sites, providing a rationale for their effects. Moreover, we showed that most TR variants are poorly tagged by nearby single nucleotide polymorphisms (SNPs) markers, indicating that many functional TR variants are not effectively assayed by SNP-based approaches. Our study assigns biological significance to TR variations in the human genome, and suggests that a significant fraction of TR variations exert functional effects via alterations of local gene expression or epigenetics. We conclude that targeted studies that focus on genotyping TR variants are required to fully ascertain functional variation in the genome.