Showing papers by "Stephen R. Quake published in 2015"

A survey of human brain transcriptome diversity at the single cell level

Abstract: The human brain is a tissue of vast complexity in terms of the cell types it comprises. Conventional approaches to classifying cell types in the human brain at single cell resolution have been limited to exploring relatively few markers and therefore have provided a limited molecular characterization of any given cell type. We used single cell RNA sequencing on 466 cells to capture the cellular complexity of the adult and fetal human brain at a whole transcriptome level. Healthy adult temporal lobe tissue was obtained during surgical procedures where otherwise normal tissue was removed to gain access to deeper hippocampal pathology in patients with medical refractory seizures. We were able to classify individual cells into all of the major neuronal, glial, and vascular cell types in the brain. We were able to divide neurons into individual communities and show that these communities preserve the categorization of interneuron subtypes that is typically observed with the use of classic interneuron markers. We then used single cell RNA sequencing on fetal human cortical neurons to identify genes that are differentially expressed between fetal and adult neurons and those genes that display an expression gradient that reflects the transition between replicating and quiescent fetal neuronal populations. Finally, we observed the expression of major histocompatibility complex type I genes in a subset of adult neurons, but not fetal neurons. The work presented here demonstrates the applicability of single cell RNA sequencing on the study of the adult human brain and constitutes a first step toward a comprehensive cellular atlas of the human brain.

Noninvasive monitoring of infection and rejection after lung transplantation.

Abstract: The survival rate following lung transplantation is among the lowest of all solid-organ transplants, and current diagnostic tests often fail to distinguish between infection and rejection, the two primary posttransplant clinical complications. We describe a diagnostic assay that simultaneously monitors for rejection and infection in lung transplant recipients by sequencing of cell-free DNA (cfDNA) in plasma. We determined that the levels of donor-derived cfDNA directly correlate with the results of invasive tests of rejection (area under the curve 0.9). We also analyzed the nonhuman cfDNA as a hypothesis-free approach to test for infections. Cytomegalovirus is most frequently assayed clinically, and the levels of CMV-derived sequences in cfDNA are consistent with clinical results. We furthermore show that hypothesis-free monitoring for pathogens using cfDNA reveals undiagnosed cases of infection, and that certain infectious pathogens such as human herpesvirus (HHV) 6, HHV-7, and adenovirus, which are not often tested clinically, occur with high frequency in this cohort.

Brain Tumor Mutations Detected in Cerebral Spinal Fluid

Abstract: Background Detecting tumor-derived cell-free DNA (cfDNA) in the blood of brain tumor patients is challenging, presumably owing to the blood-brain barrier. Cerebral spinal fluid (CSF) may serve as an alternative "liquid biopsy" of brain tumors by enabling measurement of circulating DNA within CSF to characterize tumor-specific mutations. Many aspects about the characteristics and detectability of tumor mutations in CSF remain undetermined. Methods We used digital PCR and targeted amplicon sequencing to quantify tumor mutations in the cfDNA of CSF and plasma collected from 7 patients with solid brain tumors. Also, we applied cancer panel sequencing to globally characterize the somatic mutation profile from the CSF of 1 patient with suspected leptomeningeal disease. Results We detected tumor mutations in CSF samples from 6 of 7 patients with solid brain tumors. The concentration of the tumor mutant alleles varied widely between patients, from Conclusions Tumor mutations were detectable in cfDNA from the CSF of patients with different primary and metastatic brain tumors. We designed 2 strategies to characterize tumor mutations in CSF for potential clinical diagnosis: the targeted detection of known driver mutations to monitor brain metastasis and the global characterization of genomic aberrations to direct personalized cancer care.

The phylogenetic and geographic structure of Y-chromosome haplogroup R1a.

Abstract: R1a-M420 is one of the most widely spread Y-chromosome haplogroups; however, its substructure within Europe and Asia has remained poorly characterized. Using a panel of 16 244 male subjects from 126 populations sampled across Eurasia, we identified 2923 R1a-M420 Y-chromosomes and analyzed them to a highly granular phylogeographic resolution. Whole Y-chromosome sequence analysis of eight R1a and five R1b individuals suggests a divergence time of ∼25 000 (95% CI: 21 300–29 000) years ago and a coalescence time within R1a-M417 of ∼5800 (95% CI: 4800–6800) years. The spatial frequency distributions of R1a sub-haplogroups conclusively indicate two major groups, one found primarily in Europe and the other confined to Central and South Asia. Beyond the major European versus Asian dichotomy, we describe several younger sub-haplogroups. Based on spatial distributions and diversity patterns within the R1a-M420 clade, particularly rare basal branches detected primarily within Iran and eastern Turkey, we conclude that the initial episodes of haplogroup R1a diversification likely occurred in the vicinity of present-day Iran.

Dissecting noncoding and pathogen RNA-protein interactomes.

Abstract: RNA-protein interactions are central to biological regulation. Cross-linking immunoprecipitation (CLIP)-seq is a powerful tool for genome-wide interrogation of RNA-protein interactomes, but current CLIP methods are limited by challenging biochemical steps and fail to detect many classes of noncoding and nonhuman RNAs. Here we present FAST-iCLIP, an integrated pipeline with improved CLIP biochemistry and an automated informatic pipeline for comprehensive analysis across protein coding, noncoding, repetitive, retroviral, and nonhuman transcriptomes. FAST-iCLIP of Poly-C binding protein 2 (PCBP2) showed that PCBP2-bound CU-rich motifs in different topologies to recognize mRNAs and noncoding RNAs with distinct biological functions. FAST-iCLIP of PCBP2 in hepatitis C virus-infected cells enabled a joint analysis of the PCBP2 interactome with host and viral RNAs and their interplay. These results show that FAST-iCLIP can be used to rapidly discover and decipher mechanisms of RNA-protein recognition across the diversity of human and pathogen RNAs.

Digital signaling decouples activation probability and population heterogeneity.

Abstract: Digital signaling enhances robustness of cellular decisions in noisy environments, but it is unclear how digital systems transmit temporal information about a stimulus. To understand how temporal input information is encoded and decoded by the NF-κB system, we studied transcription factor dynamics and gene regulation under dose- and duration-modulated inflammatory inputs. Mathematical modeling predicted and microfluidic single-cell experiments confirmed that integral of the stimulus (or area, concentration × duration) controls the fraction of cells that activate NF-κB in the population. However, stimulus temporal profile determined NF-κB dynamics, cell-to-cell variability, and gene expression phenotype. A sustained, weak stimulation lead to heterogeneous activation and delayed timing that is transmitted to gene expression. In contrast, a transient, strong stimulus with the same area caused rapid and uniform dynamics. These results show that digital NF-κB signaling enables multidimensional control of cellular phenotype via input profile, allowing parallel and independent control of single-cell activation probability and population heterogeneity. DOI: http://dx.doi.org/10.7554/eLife.08931.001

Multiplexed locus-specific analysis of DNA methylation in single cells

Abstract: Cheow et al. provide a protocol for SCRAM, a method for determining DNA methylation status at defined target sites in single cells. It is reliable, low in cost and relatively fast, making it a good option when full genome coverage is not required. This protocol details a method for measuring the DNA methylation state of multiple target sites in single cells, otherwise known as single-cell restriction analysis of methylation (SCRAM). The basic steps include isolating and lysing single cells, digesting genomic DNA with a methylation-sensitive restriction endonuclease (MSRE) and amplification of multiple targets by two rounds of PCR to determine the methylation status of target sites. The method can reliably and accurately detect the methylation status of multiple target sites in each single cell, and it can be completed in a relatively short time (<2 d) at low cost. Consequently, the method may be preferable over whole-genome methods in applications requiring highly reliable and cost-effective coverage of specific target sites in all cells from a sample and in cases when the DNA methylation states of single CpG sites are representative of the methylation status of corresponding regions of interest.

A Biotic Game Design Project for Integrated Life Science and Engineering Education

Abstract: Engaging, hands-on design experiences are key for formal and informal Science, Technology, Engineering, and Mathematics (STEM) education. Robotic and video game design challenges have been particularly effective in stimulating student interest, but equivalent experiences for the life sciences are not as developed. Here we present the concept of a "biotic game design project" to motivate student learning at the interface of life sciences and device engineering (as part of a cornerstone bioengineering devices course). We provide all course material and also present efforts in adapting the project's complexity to serve other time frames, age groups, learning focuses, and budgets. Students self-reported that they found the biotic game project fun and motivating, resulting in increased effort. Hence this type of design project could generate excitement and educational impact similar to robotics and video games.

Compositions and methods to treat latent viral infections

Abstract: Viral infection is a persistent cause of human disease. Guided nuclease systems target the genomes of viral infections, rendering the viruses incapacitated.

High Temporal Resolution Detection of Patient-Specific Glucose Uptake from Human ex Vivo Adipose Tissue On-Chip

Abstract: Human tissue in vitro models on-chip are highly desirable to dissect the complexity of a physio-pathological in vivo response because of their advantages compared to traditional static culture systems in terms of high control of microenvironmental conditions, including accurate perturbations and high temporal resolution analyses of medium outflow. Human adipose tissue (hAT) is a key player in metabolic disorders, such as Type 2 Diabetes Mellitus (T2DM). It is involved in the overall energy homeostasis not only as passive energy storage but also as an important metabolic regulator. Here, we aim at developing a large scale microfluidic platform for generating high temporal resolution of glucose uptake profiles, and consequently insulin sensitivity, under physio-pathological stimulations in ex vivo adipose tissues from nondiabetic and T2DM individuals. A multiscale mathematical model that integrates fluid dynamics and an intracellular insulin signaling pathway description was used for assisting microfluidic ...

Monitoring pharmacologically induced immunosuppression by immune repertoire sequencing to detect acute allograft rejection in heart transplant patients: a proof-of-concept diagnostic accuracy study.

Abstract: Background It remains difficult to predict and to measure the efficacy of pharmacological immunosuppression. We hypothesized that measuring the B-cell repertoire would enable assessment of the overall level of immunosuppression after heart transplantation. Methods and Findings In this proof-of-concept study, we implemented a molecular-barcode-based immune repertoire sequencing assay that sensitively and accurately measures the isotype and clonal composition of the circulating B cell repertoire. We used this assay to measure the temporal response of the B cell repertoire to immunosuppression after heart transplantation. We selected a subset of 12 participants from a larger prospective cohort study (ClinicalTrials.gov NCT01985412) that is ongoing at Stanford Medical Center and for which enrollment started in March 2010. This subset of 12 participants was selected to represent post-heart-transplant events, with and without acute rejection (six participants with moderate-to-severe rejection and six without). We analyzed 130 samples from these patients, with an average follow-up period of 15 mo. Immune repertoire sequencing enables the measurement of a patient’s net state of immunosuppression (correlation with tacrolimus level, r = −0.867, 95% CI −0.968 to −0.523, p = 0.0014), as well as the diagnosis of acute allograft rejection, which is preceded by increased immune activity with a sensitivity of 71.4% (95% CI 30.3% to 94.9%) and a specificity of 82.0% (95% CI 72.1% to 89.1%) (cell-free donor-derived DNA as noninvasive gold standard). To illustrate the potential of immune repertoire sequencing to monitor atypical post-transplant trajectories, we analyzed two more patients, one with chronic infections and one with amyloidosis. A larger, prospective study will be needed to validate the power of immune repertoire sequencing to predict rejection events, as this proof-of-concept study is limited to a small number of patients who were selected based on several criteria including the availability of a large number of samples and the absence or presence of rejection events. Conclusions If confirmed in larger, prospective studies, the method described here has potential applications in the tailored management of post-transplant immunosuppression and, more broadly, as a method for assessing the overall activity of the immune system.

Novel exons and splice variants in the human antibody heavy chain identified by single cell and single molecule sequencing.

Abstract: Antibody heavy chains contain a variable and a constant region. The constant region of the antibody heavy chain is encoded by multiple groups of exons which define the isotype and therefore many functional characteristics of the antibody. We performed both single B cell RNAseq and long read single molecule sequencing of antibody heavy chain transcripts and were able to identify novel exons for IGHA1 and IGHA2 as well as novel isoforms for IGHM antibody heavy chain.

Tools for the Microbiome: Nano and Beyond

Abstract: The microbiome presents great opportunities for understanding and improving the world around us and elucidating the interactions that compose it. The microbiome also poses tremendous challenges for mapping and manipulating the entangled networks of interactions among myriad diverse organisms. Here, we describe the opportunities, technical needs, and potential approaches to address these challenges, based on recent and upcoming advances in measurement and control at the nanoscale and beyond. These technical needs will provide the basis for advancing the largely descriptive studies of the microbiome to the theoretical and mechanistic understandings that will underpin the discipline of microbiome engineering. We anticipate that the new tools and methods developed will also be more broadly useful in environmental monitoring, medicine, forensics, and other areas.

Compositions and methods of delivering treatments for latent viral infections

Abstract: The invention provides delivery methods and compositions for antiviral therapeutics. Methods and compositions are provided for targeted delivery of antiviral therapeutics into cells of interest using, for example, viral vectors such as adenovirus, AAV, and replication incompetent HSV. These and other delivery systems can be used as vehicles to deliver DNA vectors encoding a nuclease or a cell-killing gene. These delivery methods can also be used to deliver naked DNA or RNA, protein products, plasmids containing a promoter that is active only in a latent viral state which drives a cell-killing gene, or other therapeutic agents.

Implantable pressure sensors for telemetric measurements through bodily tissues

Abstract: Continuous pressure sensing is important for patients with several different conditions. We provide an implantable sensor, based on microfluidic principles, which in one example has 1 mmHg limit of detection, high sensitivity and excellent reproducibility. This sensor has an optical interface, which enables pressure to be read with, for example, a cell phone camera. The design and fabrication, along with the option of self-monitoring are promising steps toward better patient care and treatment.

Bmet-15tumor cell free dna in cerebral spinal fluid reflects treatment response in leptomeningeal metastasis

Abstract: Cerebral Spinal Fluid (CSF) from brain tumor patients contains tumor cell free DNA (cfDNA), which may provide a non-invasive and routinely accessible method to obtain tumor genomic information. This "liquid biopsy" method would prove considerably less morbid compared to an open removal of tumor tissue from the brain. The opportunities of cfDNA in CSF have not been systematically studied in CSF, particularly in relation to how metastatic brain tumor cfDNA may respond to intrathecal therapy. METHODS: A patient with metastatic melanoma underwent serial lumbar punctures upon development of leptomeningeal metastases. CSF was obtained for research purposes through an informed consent, IRB approved protocol. The primary melanoma tumor was known to have a BRAF mutation 1799T > A (V600E). cfDNA was isolated from the CSF, and sequenced to confirm the presence of this same tumor-specific BRAF mutation. Droplet Digital PCR (ddPCR) quantified the mutant DNA amount and fraction in CSF. RESULTS: The total tumor DNA and mutant DNA fraction in CSF decreased as the patient responded to radiation therapy, correlating with a decrease in his previously debilitating symptoms. When the patient was asymptomatic, the mutant tumor DNA fraction was likewise undetectable. When the patient's symptoms recurred, the tumor cellular and cell free DNA mutation fraction was also elevated, similar to prior pre-treatment levels. CONCLUSION: Tumor cellular and cfDNA was found in CSF from a cancer patient with leptomeningeal metastases. Both the mutant fraction and total copies of mutant DNA corresponded with the patient's response to radiation therapy. This non-invasive method may provide a powerful tool for identifying and monitoring the genetic fingerprint of brain metastases.

Read count-based method for high-throughput allelic genotyping of transposable elements and structural variants

Abstract: Like other structural variants, transposable element insertions can be highly polymorphic across individuals. Their functional impact, however, remains poorly understood. Current genome-wide approaches for genotyping insertion-site polymorphisms based on targeted or whole-genome sequencing remain very expensive and can lack accuracy, hence new large-scale genotyping methods are needed. We describe a high-throughput method for genotyping transposable element insertions and other types of structural variants that can be assayed by breakpoint PCR. The method relies on next-generation sequencing of multiplex, site-specific PCR amplification products and read count-based genotype calls. We show that this method is flexible, efficient (it does not require rounds of optimization), cost-effective and highly accurate. This method can benefit a wide range of applications from the routine genotyping of animal and plant populations to the functional study of structural variants in humans.

Monitoring immunocompetance using immune repertoire sequencing

Abstract: Methods, machine readable media and kits are provided for analyzing an antibody repertoire of a subject to determine the immunocompetence of the subject. The method may include analyzing the subject's antibody repertoire. Analyzing may include identifying the composition of abundant related IgH sequences of the subject's antibody repertoire. The method may further include providing an assessment of the immunocompetence of the subject, based on the analysis.

Genomic Approaches to the Analysis of Cell Free Nucleic Acids

Abstract: Recent advances in high throughput DNA sequencing and microarray technologies have revolutionized the field of genomics and also opened up many opportunities for the analysis of cell-free nucleic acids. These genomic approaches have not only provided a more comprehensive portrait of the landscape of cell-free nucleic acids, but also enabled a number of non-invasive genome-wide diagnostic methods. In this chapter, we introduce the basic mechanism of high throughput DNA sequencing and discuss some unique characteristics of cell-free nucleic acids that make their experimental procedure for high throughput analysis different from ordinary cellular nucleic acids. We describe different DNA sequencing protocols that have been used for cell-free DNA, including whole genome sequencing, exome sequencing and targeted amplicon sequencing. We explain the statistical model underlying the detection of copy number variation and point mutation from cell-free DNA. We also review recent clinical applications of sequencing cell-free DNA, from the non-invasive diagnosis of fetal genetic defects, to detection of tumor mutations from plasma and monitoring rejection of organ transplantation. In addition, we outline the perspective of profiling cell-free mRNA and cell-free microRNA using RNA-seq and microarray, and their potential applications. Finally, we conclude with discussions of the current challenges and possible future advances for genomic analysis of cell-free nucleic acids.