Person: Kharchenko, Peter
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Kharchenko
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Peter
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Kharchenko, Peter
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Publication dropEst: pipeline for accurate estimation of molecular counts in droplet-based single-cell RNA-seq experiments(BioMed Central, 2018) Petukhov, Viktor; Guo, Jimin; Baryawno, Ninib; Severe, Nicolas; Scadden, David; Samsonova, Maria G.; Kharchenko, PeterRecent single-cell RNA-seq protocols based on droplet microfluidics use massively multiplexed barcoding to enable simultaneous measurements of transcriptomes for thousands of individual cells. The increasing complexity of such data creates challenges for subsequent computational processing and troubleshooting of these experiments, with few software options currently available. Here, we describe a flexible pipeline for processing droplet-based transcriptome data that implements barcode corrections, classification of cell quality, and diagnostic information about the droplet libraries. We introduce advanced methods for correcting composition bias and sequencing errors affecting cellular and molecular barcodes to provide more accurate estimates of molecular counts in individual cells. Electronic supplementary material The online version of this article (10.1186/s13059-018-1449-6) contains supplementary material, which is available to authorized users.Publication Integrative single-cell analysis of transcriptional and epigenetic states in the human adult brain(2017) Lake, Blue B.; Chen, Song; Sos, Brandon C.; Fan, Jean; Kaeser, Gwendolyn E.; Yung, Yun C.; Duong, Thu E.; Gao, Derek; Chun, Jerold; Kharchenko, Peter; Zhang, KunDetailed characterization of the cell types in the human brain requires scalable experimental approaches to examine multiple aspects of the molecular state of individual cells, and computational integration of the data to produce unified cell-state annotations. Here we report improved high-throughput methods for single-nucleus Droplet-based sequencing (snDrop-seq) and single-cell transposome hypersensitive-site sequencing (scTHS-seq). We used each method to acquire nuclear transcriptomic and DNA accessibility maps for >60,000 single cells from the human adult visual cortex, frontal cortex, and cerebellum. Integration of these data revealed regulatory elements and transcription factors that underlie cell-type distinctions, providing a basis for studying complex processes in the brain, such as genetic programs coordinating adult remyelination. We also mapped disease-associated risk variants to specific cellular populations, providing insights into normal and pathogenic cellular processes in the human brain. This integrative multi-omics approach permits more detailed single-cell interrogation of complex organs and tissues.Publication RNA Velocity of Single Cells(Springer Science and Business Media LLC, 2018-08) La Manno, Gioele; Soldatov, Ruslan; Zeisel, Amit; Braun, Emelie; Hochgerner, Hannah; Petukhov, Viktor; Lidschreiber, Katja; Kastriti, Maria E.; Lönnerberg, Peter; Furlan, Alessandro; Fan, Jean; Borm, Lars E.; Liu, Zehua; van Bruggen, David; Guo, Jimin; He, Xiaoling; Barker, Roger; Sundström, Erik; Castelo-Branco, Gonçalo; Cramer, Patrick; Adameyko, Igor; Linnarsson, Sten; Kharchenko, PeterRNA abundance is a powerful indicator of the state of individual cells. Single-cell RNA sequencing can reveal RNA abundance with high quantitative accuracy, sensitivity and throughput1. However, this approach captures only a static snapshot at a point in time, posing a challenge for the analysis of time-resolved phenomena, such as embryogenesis or tissue regeneration. Here we show that RNA velocity—the time derivative of the gene expression state—can be directly estimated by distinguishing unspliced and spliced mRNAs in common single-cell RNA sequencing protocols. RNA velocity is a high-dimensional vector that predicts the future state of individual cells on a timescale of hours. We validate its accuracy in the neural crest lineage, demonstrate its use on multiple published datasets and technical platforms, reveal the branching lineage tree of the developing mouse hippocampus, and examine the kinetics of transcription in human embryonic brain. We expect RNA velocity to greatly aid the analysis of developmental lineages and cellular dynamics, particularly in humans.Publication Characterizing transcriptional heterogeneity through pathway and gene set overdispersion analysis(2016) Fan, Jean; Salathia, Neeraj; Liu, Rui; Kaeser, Gwendolyn E.; Yung, Yun C.; Herman, Joseph L.; Kaper, Fiona; Fan, Jian-Bing; Zhang, Kun; Chun, Jerold; Kharchenko, PeterThe transcriptional state of a cell reflects a variety of biological factors, from persistent cell-type specific features to transient processes such as cell cycle. Depending on biological context, all such aspects of transcriptional heterogeneity may be of interest, but detecting them from noisy single-cell RNA-seq data remains challenging. We developed PAGODA to resolve multiple, potentially overlapping aspects of transcriptional heterogeneity by testing gene sets for coordinated variability amongst measured cells.Publication Bayesian approach to single-cell differential expression analysis(Springer Nature, 2014) Kharchenko, Peter; Silberstein, Lev; Scadden, DavidSingle-cell data provide a means to dissect the composition of complex tissues and specialized cellular environments. However, the analysis of such measurements is complicated by high levels of technical noise and intrinsic biological variability. We describe a probabilistic model of expression-magnitude distortions typical of single-cell RNA-sequencing measurements, which enables detection of differential expression signatures and identification of subpopulations of cells in a way that is more tolerant of noise.Publication Challenges and emerging directions in single-cell analysis(BioMed Central, 2017) Yuan, Guo-Cheng; Cai, Long; Elowitz, Michael; Enver, Tariq; Fan, Guoping; Guo, Guoji; Irizarry, Rafael; Kharchenko, Peter; Kim, Junhyong; Orkin, Stuart; Quackenbush, John; Saadatpour, Assieh; Schroeder, Timm; Shivdasani, Ramesh; Tirosh, ItaySingle-cell analysis is a rapidly evolving approach to characterize genome-scale molecular information at the individual cell level. Development of single-cell technologies and computational methods has enabled systematic investigation of cellular heterogeneity in a wide range of tissues and cell populations, yielding fresh insights into the composition, dynamics, and regulatory mechanisms of cell states in development and disease. Despite substantial advances, significant challenges remain in the analysis, integration, and interpretation of single-cell omics data. Here, we discuss the state of the field and recent advances and look to future opportunities.Publication Circadian Kinetics of Cell Cycle Progression in Adult Neurogenic Niches of a Diurnal Vertebrate(Society for Neuroscience, 2017) Akle, Veronica; Stankiewicz, Alexander J.; Kharchenko, Vasili; Yu, Lili; Kharchenko, Peter; Zhdanova, Irina V.The circadian system may regulate adult neurogenesis via intracellular molecular clock mechanisms or by modifying the environment of neurogenic niches, with daily variation in growth factors or nutrients depending on the animal's diurnal or nocturnal lifestyle. In a diurnal vertebrate, zebrafish, we studied circadian distribution of immunohistochemical markers of the cell division cycle (CDC) in 5 of the 16 neurogenic niches of adult brain, the dorsal telencephalon, habenula, preoptic area, hypothalamus, and cerebellum. We find that common to all niches is the morning initiation of G1/S transition and daytime S-phase progression, overnight increase in G2/M, and cycle completion by late night. This is supported by the timing of gene expression for critical cell cycle regulators cyclins D, A2, and B2 and cyclin-dependent kinase inhibitor p20 in brain tissue. The early-night peak in p20, limiting G1/S transition, and its phase angle with the expression of core clock genes, Clock1 and Per1, are preserved in constant darkness, suggesting intrinsic circadian patterns of cell cycle progression. The statistical modeling of CDC kinetics reveals the significant circadian variation in cell proliferation rates across all of the examined niches, but interniche differences in the magnitude of circadian variation in CDC, S-phase length, phase angle of entrainment to light or clock, and its dispersion. We conclude that, in neurogenic niches of an adult diurnal vertebrate, the circadian modulation of cell cycle progression involves both systemic and niche-specific factors. SIGNIFICANCE STATEMENT This study establishes that in neurogenic niches of an adult diurnal vertebrate, the cell cycle progression displays a robust circadian pattern. Common to neurogenic niches located in diverse brain regions is daytime progression of DNA replication and nighttime mitosis, suggesting systemic regulation. Differences between neurogenic niches in the phase and degree of S-phase entrainment to the clock suggest additional roles for niche-specific regulatory mechanisms. Understanding the circadian regulation of adult neurogenesis can help optimize the timing of therapeutic approaches in patients with brain traumas or neurodegenerative disorders and preserve neural stem cells during cytostatic cancer therapies.Publication Enrichment of HP1a on Drosophila Chromosome 4 Genes Creates an Alternate Chromatin Structure Critical for Regulation in this Heterochromatic Domain(Public Library of Science, 2012) Riddle, Nicole C.; Jung, Lucy; Gu, Tingting; Alekseyenko, Artyom A.; Asker, Dalal; Gui, Hongxing; Kharchenko, Peter; Minoda, Aki; Plachetka, Annette; Schwartz, Yuri B.; Tolstorukov, Michael Y.; Kuroda, Mitzi; Pirrotta, Vincenzo; Karpen, Gary H.; Park, Peter; Elgin, Sarah C. R.Chromatin environments differ greatly within a eukaryotic genome, depending on expression state, chromosomal location, and nuclear position. In genomic regions characterized by high repeat content and high gene density, chromatin structure must silence transposable elements but permit expression of embedded genes. We have investigated one such region, chromosome 4 of Drosophila melanogaster. Using chromatin-immunoprecipitation followed by microarray (ChIP–chip) analysis, we examined enrichment patterns of 20 histone modifications and 25 chromosomal proteins in S2 and BG3 cells, as well as the changes in several marks resulting from mutations in key proteins. Active genes on chromosome 4 are distinct from those in euchromatin or pericentric heterochromatin: while there is a depletion of silencing marks at the transcription start sites (TSSs), HP1a and H3K9me3, but not H3K9me2, are enriched strongly over gene bodies. Intriguingly, genes on chromosome 4 are less frequently associated with paused polymerase. However, when the chromatin is altered by depleting HP1a or POF, the RNA pol II enrichment patterns of many chromosome 4 genes shift, showing a significant decrease over gene bodies but not at TSSs, accompanied by lower expression of those genes. Chromosome 4 genes have a low incidence of TRL/GAGA factor binding sites and a low Tm downstream of the TSS, characteristics that could contribute to a low incidence of RNA polymerase pausing. Our data also indicate that EGG and POF jointly regulate H3K9 methylation and promote HP1a binding over gene bodies, while HP1a targeting and H3K9 methylation are maintained at the repeats by an independent mechanism. The HP1a-enriched, POF-associated chromatin structure over the gene bodies may represent one type of adaptation for genes embedded in repetitive DNA.Publication Sequence-Specific Targeting of Dosage Compensation in Drosophila Favors an Active Chromatin Context(Public Library of Science, 2012) Alekseyenko, Artyom A.; Gelbart, Marnie; Tolstorukov, Michael Y.; Gorchakov, Andrey A.; Larschan, Erica; Gu, Tingting; Minoda, Aki; Riddle, Nicole C.; Schwartz, Yuri B.; Elgin, Sarah C. R.; Karpen, Gary H.; Pirrotta, Vincenzo; Ho, Joshua Wing Kei; Peng, Shouyong; Plachetka, Annette; Kharchenko, Peter; Jung, Lucy; Kuroda, Mitzi; Park, PeterThe Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at “entry sites” that contain a consensus sequence motif (“MSL recognition element” or MRE). However, this motif is only \(\sim\)2 fold enriched on X, and only a fraction of the motifs on X are initially targeted. Here we ask whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells (which contain MSL complex) and female Kc cells (which lack the complex), we find that the presence of active chromatin modifications, together with an elevated local GC content in the surrounding sequences, has strong predictive value for functional MSL entry sites, independent of MSL binding. We tested these sites for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. We show that ectopic MSL expression in Kc cells leads to H4K16 acetylation around these sites and a relative increase in X chromosome transcription. Collectively, our results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. Our analysis can serve as a model for identifying chromatin and local sequence features that may contribute to selection of functional protein binding sites in the genome.Publication Identification of regions in the HOX cluster that can confer repression in a Polycomb-dependent manner(BioMed Central, 2013) Woo, Caroline J; Kharchenko, Peter; Daheron, Laurence; Park, Peter; Kingston, Robert