Our lab works at the interface of biotechnology, computational biology, cellular biology, and clinical medicine to develop and apply new tools for characterizing genetic variation across single cells within a tissue with unparalleled sensitivity and accuracy. We are focused on applying these technologies to study cancer clonal evolution while patients are undergoing treatment with the aim of identifying cancer clonotypes that are associated with resistance to specific drugs so as to better understand and predict treatment response. We are also applying these methods to understand how more virulent pathogens emerge from a population of bacteria or viruses with an emphasis on developing a deeper understanding of how antibiotic resistance develops.
- Pediatric Hematology-Oncology
- Hematologic Malignancies
Honors & Awards
Investigator, Chan Zuckerberg Biohub (2019-2024)
Taube Distinguished Scholar for Pediatric Oncology, Stanford (2019-2024)
New Innovator Award, Office of Director, National Institutes of Health (2018-2023)
Career Award for Medical Scientists, Burroughs Wellcome Fund (2015-2020)
Special Fellow, Leukemia and Lymphoma Society (2013-2016)
Fellow Basic Research Scholar, American Society of Hematology (2013-2015)
Advanced Residency Training Program, Stanford (2011-present)
Fellowship for Medical Students Continued Support Award, HHMI (2003-2005)
Cloister Scholar, HHMI-NIH (2002-2003)
Boards, Advisory Committees, Professional Organizations
Founder, Board DIrector, BioSkryb, Inc. (2019 - Present)
Editorial Board Member, Nucleic Acids Research Cancer (2019 - Present)
Residency: UCLA Pediatric Residency (2009) CA
PhD Training: Stanford University School of Medicine - Office of Graduate Affairs - Postdoctoral Affairs (2015) CA
Board Certification: American Board of Pediatrics, Pediatric Hematology-Oncology (2013)
Fellowship: Stanford University Pediatric Hematology Oncology Fellowship (2012) CA
Board Certification: American Board of Pediatrics, Pediatrics (2010)
Medical Education: University of Arizona College of Medicine Office of the Registrar (2006) AZ
PhD, Stanford University, Cancer Biology (2015)
MD, University of Arizona, Medicine (2006)
BS, Arizona State University, Chemistry and Microbiology (2001)
Charles Gawad, Veronica Gonzalez-Pena, Robert Carter, Sivaraman Natarajan, Jason West. "United States Patent 62/881,180 Genetic Mutation Analysis"
Charles Gawad, Jason West. "United States Patent 62/881,183 Single Cell Analysis"
Charles Gawad, Jason West, Paul McEwan. "United States Patent 62/972,557 Phi29 Mutants and Use Thereof"
Charles Gawad, Veronica Gonzalez-Pena, John Easton. "United States Patent WO2019148119A1 Method for Nucleic Acid Amplification"
Charles Gawad, Siddhartha Kadia, Jason West. "United StatesDetection of Low Abundance Nucleic Acids", Apr 28, 2020
Charles Gawad, Jason West, Jon Zawistowski. "United States Patent 55461-706.101 Detection of Low Abundance Viruses", Mar 31, 2020
Genome, Proteome and Tissue Microarray in Childhood Acute Leukemia
We will study gene and protein expression in leukemia cells of children diagnosed with acute leukemia. We hope to identify genes or proteins which can help us grade leukemia at diagnosis in order to: (a) develop better means of diagnosis and (b) more accurately choose the best therapy for each patient.
Stanford is currently not accepting patients for this trial. For more information, please contact Norman J Lacayo, 650-723-5535.
Bringing precision oncology to cellular resolution with single-cell genomics.
Clinical & experimental metastasis
Single-cell sequencing technologies have undergone rapid development and adoption by the scientific community in the past 5years, fueling discoveries about the etiology, pathogenesis, and treatment responsiveness of individual tumor cells within cancer ecosystems. Most of the advancements in our understanding of cancer with these new technologies have focused on basic tumor biology. However, the knowledge produced by these and other studies are beginning to provide biomarkers and drug targets for clinically-relevant subpopulations within a tumor, creating opportunities for the development of biologically-informed, clone-specific combination treatment strategies. Here we provide an overview of the development of the field of single-cell cancer sequencing and provide a roadmap for shepherding these technologies from research tools to diagnostic instruments that provide high-resolution, treatment-directing details of tumors to clinical oncologists.
View details for DOI 10.1007/s10585-021-10129-4
View details for PubMedID 34807338
Accurate genomic variant detection in single cells with primary template-directed amplification.
Proceedings of the National Academy of Sciences of the United States of America
2021; 118 (24)
Improvements in whole genome amplification (WGA) would enable new types of basic and applied biomedical research, including studies of intratissue genetic diversity that require more accurate single-cell genotyping. Here, we present primary template-directed amplification (PTA), an isothermal WGA method that reproducibly captures >95% of the genomes of single cells in a more uniform and accurate manner than existing approaches, resulting in significantly improved variant calling sensitivity and precision. To illustrate the types of studies that are enabled by PTA, we developed direct measurement of environmental mutagenicity (DMEM), a tool for mapping genome-wide interactions of mutagens with single living human cells at base-pair resolution. In addition, we utilized PTA for genome-wide off-target indel and structural variant detection in cells that had undergone CRISPR-mediated genome editing, establishing the feasibility for performing single-cell evaluations of biopsies from edited tissues. The improved precision and accuracy of variant detection with PTA overcomes the current limitations of accurate WGA, which is the major obstacle to studying genetic diversity and evolution at cellular resolution.
View details for DOI 10.1073/pnas.2024176118
View details for PubMedID 34099548
Integrative genomic analyses reveal mechanisms of glucocorticoid resistance in acute lymphoblastic leukemia.
2020; 1 (3): 329–44
Identification of genomic and epigenomic determinants of drug resistance provides important insights for improving cancer treatment. Using agnostic genome-wide interrogation of mRNA and miRNA expression, DNA methylation, SNPs, CNAs and SNVs/Indels in primary human acute lymphoblastic leukemia cells, we identified 463 genomic features associated with glucocorticoid resistance. Gene-level aggregation identified 118 overlapping genes, 15 of which were confirmed by genome-wide CRISPR screen. Collectively, this identified 30 of 38 (79%) known glucocorticoid-resistance genes/miRNAs and all 38 known resistance pathways, while revealing 14 genes not previously associated with glucocorticoid-resistance. Single cell RNAseq and network-based transcriptomic modelling corroborated the top previously undiscovered gene, CELSR2. Manipulation of CELSR2 recapitulated glucocorticoid resistance in human leukemia cell lines and revealed a synergistic drug combination (prednisolone and venetoclax) that mitigated resistance in mouse xenograft models. These findings illustrate the power of an integrative genomic strategy for elucidating genes and pathways conferring drug resistance in cancer cells.
View details for DOI 10.1038/s43018-020-0037-3
View details for PubMedID 32885175
View details for PubMedCentralID PMC7467080
Evaluation of Plasma Microbial Cell-Free DNA Sequencing to Predict Bloodstream Infection in Pediatric Patients With Relapsed or Refractory Cancer.
Bloodstream infection (BSI) is a common, life-threatening complication of treatment for cancer. Predicting BSI before onset of clinical symptoms would enable preemptive therapy, but there is no reliable screening test.To estimate sensitivity and specificity of plasma microbial cell-free DNA sequencing (mcfDNA-seq) for predicting BSI in patients at high risk of life-threatening infection.A prospective pilot cohort study of mcfDNA-seq for predicting BSI in pediatric patients (<25 years of age) with relapsed or refractory cancers at St Jude Children's Research Hospital, a specialist quaternary pediatric hematology-oncology referral center. Remnant clinical blood samples were collected during chemotherapy and hematopoietic cell transplantation. Samples collected during the 7 days before and at onset of BSI episodes, along with negative control samples from study participants, underwent blinded testing using a mcfDNA-seq test in a Clinical Laboratory Improvement Amendments/College of American Pathologists-approved laboratory.The primary outcomes were sensitivity of mcfDNA-seq for detecting a BSI pathogen during the 3 days before BSI onset and specificity of mcfDNA-seq in the absence of fever or infection in the preceding or subsequent 7 days.Between August 9, 2017, and June 4, 2018, 47 participants (27 [57%] male; median age [IQR], 10 [5-14] years) were enrolled; 19 BSI episodes occurred in 12 participants, and predictive samples were available for 16 episodes, including 15 bacterial BSI episodes. In the 3 days before the onset of infection, predictive sensitivity of mcfDNA-seq was 75% for all BSIs (12 of 16; 95% CI, 51%-90%) and 80% (12 of 15; 95% CI, 55%-93%) for bacterial BSIs. The specificity of mcfDNA-seq, evaluated on 33 negative control samples from enrolled participants, was 82% (27 of 33; 95% CI, 66%-91%) for any bacterial or fungal organism and 91% (30 of 33; 95% CI, 76%-97%) for any common BSI pathogen, and the concentration of pathogen DNA was lower in control than predictive samples.A clinically relevant pathogen can be identified by mcfDNA-seq days before the onset of BSI in a majority of episodes, potentially enabling preemptive treatment. Clinical application appears feasible pending further study.ClinicalTrials.gov identifier: NCT03226158.
View details for DOI 10.1001/jamaoncol.2019.4120
View details for PubMedID 31855231
Sequencing the Genomes of Single Cells.
Methods in molecular biology (Clifton, N.J.)
2019; 1979: 227–34
Single-cell genome sequencing can detect low-frequency genetic alterations present in complex tissues. However, the experimental procedures are technically challenging. This includes dissociation of the tissue, isolation of single cells, whole-genome amplification, sequencing library preparation, and an optional target enrichment. Here we describe how to perform each of these processes to obtain high-quality single-cell genome sequencing data.
View details for DOI 10.1007/978-1-4939-9240-9_14
View details for PubMedID 31028641
Single-cell RNA sequencing reveals the impact of chromosomal instability on glioblastoma cancer stem cells.
BMC medical genomics
2019; 12 (1): 79
Intra-tumor heterogeneity stems from genetic, epigenetic, functional, and environmental differences among tumor cells. A major source of genetic heterogeneity comes from DNA sequence differences and/or whole chromosome and focal copy number variations (CNVs). Whole chromosome CNVs are caused by chromosomal instability (CIN) that is defined by a persistently high rate of chromosome mis-segregation. Accordingly, CIN causes constantly changing karyotypes that result in extensive cell-to-cell genetic heterogeneity. How the genetic heterogeneity caused by CIN influences gene expression in individual cells remains unknown.We performed single-cell RNA sequencing on a chromosomally unstable glioblastoma cancer stem cell (CSC) line and a control normal, diploid neural stem cell (NSC) line to investigate the impact of CNV due to CIN on gene expression. From the gene expression data, we computationally inferred large-scale CNVs in single cells. Also, we performed copy number adjusted differential gene expression analysis between NSCs and glioblastoma CSCs to identify copy number dependent and independent differentially expressed genes.Here, we demonstrate that gene expression across large genomic regions scales proportionally to whole chromosome copy number in chromosomally unstable CSCs. Also, we show that the differential expression of most genes between normal NSCs and glioblastoma CSCs is largely accounted for by copy number alterations. However, we identify 269 genes whose differential expression in glioblastoma CSCs relative to normal NSCs is independent of copy number. Moreover, a gene signature derived from the subset of genes that are differential expressed independent of copy number in glioblastoma CSCs correlates with tumor grade and is prognostic for patient survival.These results demonstrate that CIN is directly responsible for gene expression changes and contributes to both genetic and transcriptional heterogeneity among glioblastoma CSCs. These results also demonstrate that the expression of some genes is buffered against changes in copy number, thus preserving some consistency in gene expression levels from cell-to-cell despite the continuous change in karyotype driven by CIN. Importantly, a gene signature derived from the subset of genes whose expression is buffered against copy number alterations correlates with tumor grade and is prognostic for patient survival that could facilitate patient diagnosis and treatment.
View details for DOI 10.1186/s12920-019-0532-5
View details for PubMedID 31151460
View details for PubMedCentralID PMC6545015
Resolving medulloblastoma cellular architecture by single-cell genomics.
2019; 572 (7767): 74–79
Medulloblastoma is a malignant childhood cerebellar tumour type that comprises distinct molecular subgroups. Whereas genomic characteristics of these subgroups are well defined, the extent to which cellular diversity underlies their divergent biology and clinical behaviour remains largely unexplored. Here we used single-cell transcriptomics to investigate intra- and intertumoral heterogeneity in 25 medulloblastomas spanning all molecular subgroups. WNT, SHH and Group 3 tumours comprised subgroup-specific undifferentiated and differentiated neuronal-like malignant populations, whereas Group 4 tumours consisted exclusively of differentiated neuronal-like neoplastic cells. SHH tumours closely resembled granule neurons of varying differentiation states that correlated with patient age. Group 3 and Group 4 tumours exhibited a developmental trajectory from primitive progenitor-like to more mature neuronal-like cells, the relative proportions of which distinguished these subgroups. Cross-species transcriptomics defined distinct glutamatergic populations as putative cells-of-origin for SHH and Group 4 subtypes. Collectively, these data provide insights into the cellular and developmental states underlying subtype-specific medulloblastoma biology.
View details for DOI 10.1038/s41586-019-1434-6
View details for PubMedID 31341285
Murine hematopoietic stem cell activity is derived from pre-circulation embryos but not yolk sacs.
2018; 9 (1): 5405
The embryonic site of definitive hematopoietic stem cell (dHSC) origination has been debated for decades. Although an intra-embryonic origin is well supported, the yolk sac (YS) contribution to adult hematopoiesis remains controversial. The same developmental origin makes it difficult to identify specific markers that discern between an intraembryonic versus YS-origin using a lineage trace approach. Additionally, the highly migratory nature of blood cells and the inability of pre-circulatory embryonic cells (i.e., 5-7 somite pairs (sp)) to robustly engraft in transplantation, even after culture, has precluded scientists from properly answering these questions. Here we report robust, multi-lineage and serially transplantable dHSC activity from cultured 2-7sp murine embryonic explants (Em-Ex). dHSC are undetectable in 2-7sp YS explants. Additionally, the engraftment from Em-Ex is confined to an emerging CD31+CD45+c-Kit+CD41- population. In sum, our work supports a model in which the embryo, not the YS, is the major source of lifelong definitive hematopoiesis.
View details for DOI 10.1038/s41467-018-07769-8
View details for PubMedID 30573729
View details for PubMedCentralID PMC6302089
High-resolution transcriptional dissection of in vivo Atoh1-mediated hair cell conversion in mature cochleae identifies Isl1 as a co-reprogramming factor.
2018; 14 (7): e1007552
In vivo direct conversion of differentiated cells holds promise for regenerative medicine; however, improving the conversion efficiency and producing functional target cells remain challenging. Ectopic Atoh1 expression in non-sensory supporting cells (SCs) in mouse cochleae induces their partial conversion to hair cells (HCs) at low efficiency. Here, we performed single-cell RNA sequencing of whole mouse sensory epithelia harvested at multiple time points after conditional overexpression of Atoh1. Pseudotemporal ordering revealed that converted HCs (cHCs) are present along a conversion continuum that correlates with both endogenous and exogenous Atoh1 expression. Bulk sequencing of isolated cell populations and single-cell qPCR confirmed 51 transcription factors, including Isl1, are differentially expressed among cHCs, SCs and HCs. In transgenic mice, co-overexpression of Atoh1 and Isl1 enhanced the HC conversion efficiency. Together, our study shows how high-resolution transcriptional profiling of direct cell conversion can identify co-reprogramming factors required for efficient conversion.
View details for DOI 10.1371/journal.pgen.1007552
View details for PubMedID 30063705
View details for PubMedCentralID PMC6086484
A Single-Cell Transcriptional Atlas of the Developing Murine Cerebellum.
Current biology : CB
2018; 28 (18): 2910–20.e2
The cerebellum develops from a restricted number of cell types that precisely organize to form the circuitry that controls sensory-motor coordination and some higher-order cognitive processes. To acquire an enhanced understanding of the molecular processes that mediate cerebellar development, we performed single-cell RNA-sequencing of 39,245 murine cerebellar cells at twelve critical developmental time points. Using recognized lineage markers, we confirmed that the single-cell data accurately recapitulate cerebellar development. We then followed distinct populations from emergence through migration and differentiation, and determined the associated transcriptional cascades. After identifying key lineage commitment decisions, focused analyses uncovered waves of transcription factor expression at those branching points. Finally, we created Cell Seek, a flexible online interface that facilitates exploration of the dataset. Our study provides a transcriptional summarization of cerebellar development at single-cell resolution that will serve as a valuable resource for future investigations of cerebellar development, neurobiology, and disease.
View details for DOI 10.1016/j.cub.2018.07.062
View details for PubMedID 30220501
Pan-cancer genome and transcriptome analyses of 1,699 paediatric leukaemias and solid tumours.
2018; 555 (7696): 371–76
Analysis of molecular aberrations across multiple cancer types, known as pan-cancer analysis, identifies commonalities and differences in key biological processes that are dysregulated in cancer cells from diverse lineages. Pan-cancer analyses have been performed for adult but not paediatric cancers, which commonly occur in developing mesodermic rather than adult epithelial tissues. Here we present a pan-cancer study of somatic alterations, including single nucleotide variants, small insertions or deletions, structural variations, copy number alterations, gene fusions and internal tandem duplications in 1,699 paediatric leukaemias and solid tumours across six histotypes, with whole-genome, whole-exome and transcriptome sequencing data processed under a uniform analytical framework. We report 142 driver genes in paediatric cancers, of which only 45% match those found in adult pan-cancer studies; copy number alterations and structural variants constituted the majority (62%) of events. Eleven genome-wide mutational signatures were identified, including one attributed to ultraviolet-light exposure in eight aneuploid leukaemias. Transcription of the mutant allele was detectable for 34% of protein-coding mutations, and 20% exhibited allele-specific expression. These data provide a comprehensive genomic architecture for paediatric cancers and emphasize the need for paediatric cancer-specific development of precision therapies.
View details for DOI 10.1038/nature25795
View details for PubMedID 29489755
View details for PubMedCentralID PMC5854542
Measurable residual disease detection by high-throughput sequencing improves risk stratification for pediatric B-ALL.
2018; 131 (12): 1350–59
Early response to induction chemotherapy is an important prognostic factor in B-lymphoblastic leukemia (B-ALL). Here, we compare high-throughput sequencing (HTS) of IGH and TRG genes vs flow cytometry (FC) for measurable residual disease (MRD) detection at the end of induction chemotherapy in pediatric patients with newly diagnosed B-ALL. Six hundred nineteen paired pretreatment and end-of-induction bone marrow samples from Children's Oncology Group studies AALL0331 (clinicaltrials.gov #NCT00103285) (standard risk [SR]; with MRD by FC at any level) and AALL0232 (clinicaltrials.gov #NCT00075725) (high risk; with day 29 MRD <0.1% by FC) were evaluated by HTS and FC for event-free (EFS) and overall survival (OS). HTS and FC showed similar 5-year EFS and OS for MRD-positive and -negative patients using an MRD threshold of 0.01%. However, there was a high discordant rate with HTS identifying 55 (38.7%) more patients MRD positive at this threshold. These discrepant patients have worse outcomes than FC MRD-negative patients. In addition, the increased analytic sensitivity of HTS permitted identification of 19.9% of SR patients without MRD at any detectable level who had excellent 5-year EFS (98.1%) and OS (100%). The higher analytic sensitivity and lower false-negative rate of HTS improves upon FC for MRD detection in pediatric B-ALL by identifying a novel subset of patients at end of induction who are essentially cured using current chemotherapy and identifying MRD at 0.01% in up to one-third of patients who are missed at the same threshold by FC.
View details for DOI 10.1182/blood-2017-09-806521
View details for PubMedID 29284596
View details for PubMedCentralID PMC5865233
LC3-Associated Phagocytosis in Myeloid Cells Promotes Tumor Immune Tolerance.
2018; 175 (2): 429–41.e16
Targeting autophagy in cancer cells and in the tumor microenvironment are current goals of cancer therapy. However, components of canonical autophagy play roles in other biological processes, adding complexity to this goal. One such alternative function of autophagy proteins is LC3-associated phagocytosis (LAP), which functions in phagosome maturation and subsequent signaling events. Here, we show that impairment of LAP in the myeloid compartment, rather than canonical autophagy, induces control of tumor growth by tumor-associated macrophages (TAM) upon phagocytosis of dying tumor cells. Single-cell RNA sequencing (RNA-seq) analysis revealed that defects in LAP induce pro-inflammatory gene expression and trigger STING-mediated type I interferon responses in TAM. We found that the anti-tumor effects of LAP impairment require tumor-infiltrating T cells, dependent upon STING and the type I interferon response. Therefore, autophagy proteins in the myeloid cells of the tumor microenvironment contribute to immune suppression of T lymphocytes by effecting LAP.
View details for DOI 10.1016/j.cell.2018.08.061
View details for PubMedID 30245008
View details for PubMedCentralID PMC6201245
Genome-wide segregation of single nucleotide and structural variants into single cancer cells.
2017; 18 (1): 906
Single-cell genome sequencing provides high-resolution details of the clonal genomic modifications that occur during cancer initiation, progression, and ongoing evolution as patients undergo treatment. One limitation of current single-cell sequencing strategies is a suboptimal capacity to detect all classes of single-nucleotide and structural variants in the same cells.Here we present a new approach for determining comprehensive variant profiles of single cells using a microfluidic amplicon-based strategy to detect structural variant breakpoint sequences instead of using relative read depth to infer copy number changes. This method can reconstruct the clonal architecture and mutational history of a malignancy using all classes and sizes of somatic variants, providing more complete details of the temporal changes in mutational classes and processes that led to the development of a malignant neoplasm. Using this approach, we interrogated cells from a patient with leukemia, determining that processes producing structural variation preceded single nucleotide changes in the development of that malignancy.All classes and sizes of genomic variants can be efficiently detected in single cancer cells using our new method, enabling the ordering of distinct classes of mutations during tumor evolution.
View details for DOI 10.1186/s12864-017-4286-1
View details for PubMedID 29178827
View details for PubMedCentralID PMC5702214
Early somatic mosaicism is a rare cause of long-QT syndrome
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2016; 113 (41): 11555-11560
Somatic mosaicism, the occurrence and propagation of genetic variation in cell lineages after fertilization, is increasingly recognized to play a causal role in a variety of human diseases. We investigated the case of life-threatening arrhythmia in a 10-day-old infant with long QT syndrome (LQTS). Rapid genome sequencing suggested a variant in the sodium channel NaV1.5 encoded by SCN5A, NM_000335:c.5284G > T predicting p.(V1762L), but read depth was insufficient to be diagnostic. Exome sequencing of the trio confirmed read ratios inconsistent with Mendelian inheritance only in the proband. Genotyping of single circulating leukocytes demonstrated the mutation in the genomes of 8% of patient cells, and RNA sequencing of cardiac tissue from the infant confirmed the expression of the mutant allele at mosaic ratios. Heterologous expression of the mutant channel revealed significantly delayed sodium current with a dominant negative effect. To investigate the mechanism by which mosaicism might cause arrhythmia, we built a finite element simulation model incorporating Purkinje fiber activation. This model confirmed the pathogenic consequences of cardiac cellular mosaicism and, under the presenting conditions of this case, recapitulated 2:1 AV block and arrhythmia. To investigate the extent to which mosaicism might explain undiagnosed arrhythmia, we studied 7,500 affected probands undergoing commercial gene-panel testing. Four individuals with pathogenic variants arising from early somatic mutation events were found. Here we establish cardiac mosaicism as a causal mechanism for LQTS and present methods by which the general phenomenon, likely to be relevant for all genetic diseases, can be detected through single-cell analysis and next-generation sequencing.
View details for DOI 10.1073/pnas.1607187113
View details for PubMedID 27681629
Single-cell genome sequencing: current state of the science
NATURE REVIEWS GENETICS
2016; 17 (3): 175-188
The field of single-cell genomics is advancing rapidly and is generating many new insights into complex biological systems, ranging from the diversity of microbial ecosystems to the genomics of human cancer. In this Review, we provide an overview of the current state of the field of single-cell genome sequencing. First, we focus on the technical challenges of making measurements that start from a single molecule of DNA, and then explore how some of these recent methodological advancements have enabled the discovery of unexpected new biology. Areas highlighted include the application of single-cell genomics to interrogate microbial dark matter and to evaluate the pathogenic roles of genetic mosaicism in multicellular organisms, with a focus on cancer. We then attempt to predict advances we expect to see in the next few years.
View details for DOI 10.1038/nrg.2015.16
View details for Web of Science ID 000371739200012
View details for PubMedID 26806412
Dynamic ASXL1 Exon Skipping and Alternative Circular Splicing in Single Human Cells.
2016; 11 (10)
Circular RNAs comprise a poorly understood new class of noncoding RNA. In this study, we used a combination of targeted deletion, high-resolution splicing detection, and single-cell sequencing to deeply probe ASXL1 circular splicing. We found that efficient circular splicing required the canonical transcriptional start site and inverted AluSx elements. Sequencing-based interrogation of isoforms after ASXL1 overexpression identified promiscuous linear splicing between all exons, with the two most abundant non-canonical linear products skipping the exons that produced the circular isoforms. Single-cell sequencing revealed a strong preference for either the linear or circular ASXL1 isoforms in each cell, and found the predominant exon skipping product is frequently co-expressed with its reciprocal circular isoform. Finally, absolute quantification of ASXL1 isoforms confirmed our findings and suggests that standard methods overestimate circRNA abundance. Taken together, these data reveal a dynamic new view of circRNA genesis, providing additional framework for studying their roles in cellular biology.
View details for DOI 10.1371/journal.pone.0164085
View details for PubMedID 27736885
View details for PubMedCentralID PMC5063410
Dissecting the clonal origins of childhood acute lymphoblastic leukemia by single-cell genomics.
Proceedings of the National Academy of Sciences of the United States of America
2014; 111 (50): 17947-17952
Many cancers have substantial genomic heterogeneity within a given tumor, and to fully understand that diversity requires the ability to perform single cell analysis. We performed targeted sequencing of a panel of single nucleotide variants (SNVs), deletions, and IgH sequences in 1,479 single tumor cells from six acute lymphoblastic leukemia (ALL) patients. By accurately segregating groups of cooccurring mutations into distinct clonal populations, we identified codominant clones in the majority of patients. Evaluation of intraclonal mutation patterns identified clone-specific punctuated cytosine mutagenesis events, showed that most structural variants are acquired before SNVs, determined that KRAS mutations occur late in disease development but are not sufficient for clonal dominance, and identified clones within the same patient that are arrested at varied stages in B-cell development. Taken together, these data order the sequence of genetic events that underlie childhood ALL and provide a framework for understanding the development of the disease at single-cell resolution.
View details for DOI 10.1073/pnas.1420822111
View details for PubMedID 25425670
Noninvasive in vivo monitoring of tissue-specific global gene expression in humans.
Proceedings of the National Academy of Sciences of the United States of America
2014; 111 (20): 7361-7366
Circulating cell-free RNA in the blood provides a potential window into the health, phenotype, and developmental programs of a variety of human organs. We used high-throughput methods of RNA analysis such as microarrays and next-generation sequencing to characterize the global landscape circulating RNA in a cohort of human subjects. By focusing on genes whose expression is highly specific to certain tissues, we were able to identify the relative contributions of these tissues to circulating RNA and to monitor changes in tissue development and health. As one application of this approach, we performed a longitudinal study on pregnant women and analyzed their combined cell-free RNA transcriptomes across all three trimesters of pregnancy and after delivery. In addition to the analysis of mRNA, we observed and characterized noncoding species such as long noncoding RNA and circular RNA transcripts whose presence had not been previously observed in human plasma. We demonstrate that it is possible to track specific longitudinal phenotypic changes in both the mother and the fetus and that it is possible to directly measure transcripts from a variety of fetal tissues in the maternal blood sample. We also studied the role of neuron-specific transcripts in the blood of healthy adults and those suffering from the neurodegenerative disorder Alzheimer's disease and showed that disease specific neural transcripts are present at increased levels in the blood of affected individuals. Characterization of the cell-free transcriptome in its entirety may thus provide broad insights into human health and development without the need for invasive tissue sampling.
View details for DOI 10.1073/pnas.1405528111
View details for PubMedID 24799715
A quantitative comparison of single-cell whole genome amplification methods.
2014; 9 (8)
Single-cell sequencing is emerging as an important tool for studies of genomic heterogeneity. Whole genome amplification (WGA) is a key step in single-cell sequencing workflows and a multitude of methods have been introduced. Here, we compare three state-of-the-art methods on both bulk and single-cell samples of E. coli DNA: Multiple Displacement Amplification (MDA), Multiple Annealing and Looping Based Amplification Cycles (MALBAC), and the PicoPLEX single-cell WGA kit (NEB-WGA). We considered the effects of reaction gain on coverage uniformity, error rates and the level of background contamination. We compared the suitability of the different WGA methods for the detection of copy-number variations, for the detection of single-nucleotide polymorphisms and for de-novo genome assembly. No single method performed best across all criteria and significant differences in characteristics were observed; the choice of which amplifier to use will depend strongly on the details of the type of question being asked in any given experiment.
View details for DOI 10.1371/journal.pone.0105585
View details for PubMedID 25136831
View details for PubMedCentralID PMC4138190
Massive evolution of the immunoglobulin heavy chain locus in children with B precursor acute lymphoblastic leukemia
2012; 120 (22): 4407-4417
The ability to distinguish clonal B-cell populations based on the sequence of their rearranged immunoglobulin heavy chain (IgH) locus is an important tool for diagnosing B-cell neoplasms and monitoring treatment response. Leukemic precursor B cells may continue to undergo recombination of the IgH gene after malignant transformation; however, the magnitude of evolution at the IgH locus is currently unknown. We used next-generation sequencing to characterize the repertoire of IgH sequences in diagnostic samples of 51 children with B precursor acute lymphoblastic leukemia (B-ALL). We identified clonal IgH rearrangements in 43 of 51 (84%) cases and found that the number of evolved IgH sequences per patient ranged dramatically from 0 to 4024. We demonstrate that the evolved IgH sequences are not the result of amplification artifacts and are unique to leukemic precursor B cells. In addition, the evolution often follows an allelic exclusion pattern, where only 1 of 2 rearranged IgH loci exhibit ongoing recombination. Thus, precursor B-cell leukemias maintain evolution at the IgH locus at levels that were previously underappreciated. This finding sheds light on the mechanisms associated with leukemic clonal evolution and may fundamentally change approaches for monitoring minimal residual disease burden.
View details for DOI 10.1182/blood-2012-05-429811
View details for Web of Science ID 000313111300023
View details for PubMedID 22932801
View details for PubMedCentralID PMC3507147
Circular RNAs Are the Predominant Transcript Isoform from Hundreds of Human Genes in Diverse Cell Types
2012; 7 (2)
Most human pre-mRNAs are spliced into linear molecules that retain the exon order defined by the genomic sequence. By deep sequencing of RNA from a variety of normal and malignant human cells, we found RNA transcripts from many human genes in which the exons were arranged in a non-canonical order. Statistical estimates and biochemical assays provided strong evidence that a substantial fraction of the spliced transcripts from hundreds of genes are circular RNAs. Our results suggest that a non-canonical mode of RNA splicing, resulting in a circular RNA isoform, is a general feature of the gene expression program in human cells.
View details for DOI 10.1371/journal.pone.0030733
View details for Web of Science ID 000301977500016
View details for PubMedID 22319583
View details for PubMedCentralID PMC3270023
- Gene Expression Arrays in Pancreatic Cancer Drug Discovery Research DRUG DISCOVERY IN PANCREATIC CANCER: MODELS AND TECHNIQUES 2010: 113-134
Towards molecular medicine - A case for a biological periodic table
AMERICAN JOURNAL OF PHARMACOGENOMICS
2005; 5 (4): 207-211
The recently amplified pace of development in the technologies to study both normal and aberrant cellular physiology has allowed for a transition from the traditional reductionist approaches to global interrogations of human biology. This transformation has created the anticipation that we will soon more effectively treat or contain most types of diseases through a 'systems-based' approach to understanding and correcting the underlying etiology of these processes. However, to accomplish these goals, we must first have a more comprehensive understanding of all the elements involved in human cellular physiology, as well as why and how they interact. With the vast number of biological components that have and are being discovered, creating methods with modern computational techniques to better organize biological elements is the next requisite step in this process. This article aims to articulate the importance of the organization of chemical elements into a periodic table had on the conversion of chemistry into a quantitative, translatable science, as well as how we can apply the lessons learned in that transition to the current transformation taking place in biology.
View details for Web of Science ID 000231842000001
View details for PubMedID 16078857