Showing papers by "Stephen R. Quake published in 2013"

Lineage Structure of the Human Antibody Repertoire in Response to Influenza Vaccination

Abstract: The human antibody repertoire is one of the most important defenses against infectious disease, and the development of vaccines has enabled the conferral of targeted protection to specific pathogens. However, there are many challenges to measuring and analyzing the immunoglobulin sequence repertoire, including that each B cell’s genome encodes a distinct antibody sequence, that the antibody repertoire changes over time, and the high similarity between antibody sequences. We have addressed these challenges by using high-throughput long read sequencing to perform immunogenomic characterization of expressed human antibody repertoires in the context of influenza vaccination. Informatic analysis of 5 million antibody heavy chain sequences from healthy individuals allowed us to perform global characterizations of isotype distributions, determine the lineage structure of the repertoire, and measure age- and antigen-related mutational activity. Our analysis of the clonal structure and mutational distribution of individuals’ repertoires shows that elderly subjects have a decreased number of lineages but an increased prevaccination mutation load in their repertoire and that some of these subjects have an oligoclonal character to their repertoire in which the diversity of the lineages is greatly reduced relative to younger subjects. We have thus shown that global analysis of the immune system’s clonal structure provides direct insight into the effects of vaccination and provides a detailed molecular portrait of age-related effects.

The genome sequence of the colonial chordate, Botryllus schlosseri

Abstract: Botryllus schlosseri is a colonial urochordate that follows the chordate plan of development following sexual reproduction, but invokes a stem cell-mediated budding program during subsequent rounds of asexual reproduction. As urochordates are considered to be the closest living invertebrate relatives of vertebrates, they are ideal subjects for whole genome sequence analyses. Using a novel method for high-throughput sequencing of eukaryotic genomes, we sequenced and assembled 580 Mbp of the B. schlosseri genome. The genome assembly is comprised of nearly 14,000 intron-containing predicted genes, and 13,500 intron-less predicted genes, 40% of which could be confidently parceled into 13 (of 16 haploid) chromosomes. A comparison of homologous genes between B. schlosseri and other diverse taxonomic groups revealed genomic events underlying the evolution of vertebrates and lymphoid-mediated immunity. The B. schlosseri genome is a community resource for studying alternative modes of reproduction, natural transplantation reactions, and stem cell-mediated regeneration.

Genetic measurement of memory B-cell recall using antibody repertoire sequencing.

Abstract: Annual influenza vaccinations aim to protect against seasonal infections, and vaccine strain compositions are updated every year. This protection is based on antibodies that are produced by either newly activated or memory B cells recalled from previous encounters with influenza vaccination or infection. The extent to which the B-cell repertoire responds to vaccination and recalls antibodies has so far not been analyzed at a genetic level—which is to say, at the level of antibody sequences. Here, we developed a consensus read sequencing approach that incorporates unique barcode labels on each starting RNA molecule. These labels allow one to combine multiple sequencing reads covering the same RNA molecule to reduce the error rate to a desired level, and they also enable accurate quantification of RNA and isotype levels. We validated this approach and analyzed the differential response of the antibody repertoire to live-attenuated or trivalent-inactivated influenza vaccination. Additionally, we analyzed the antibody repertoire in response to repeated yearly vaccinations with trivalent-inactivated influenza vaccination. We found antibody sequences that were present in both years, providing a direct genetic measurement of B-cell recall.

Single-cell and metagenomic analyses indicate a fermentative and saccharolytic lifestyle for members of the OP9 lineage

Abstract: OP9 is a yet-uncultivated bacterial lineage found in geothermal systems, petroleum reservoirs, anaerobic digesters and wastewater treatment facilities. Here we use single-cell and metagenome sequencing to obtain two distinct, nearly complete OP9 genomes, one constructed from single cells sorted from hot spring sediments and the other derived from binned metagenomic contigs from an in situ-enriched cellulolytic, thermophilic community. Phylogenomic analyses support the designation of OP9 as a candidate phylum for which we propose the name 'Atribacteria'. Although a plurality of predicted proteins is most similar to those from Firmicutes, the presence of key genes suggests a diderm cell envelope. Metabolic reconstruction from the core genome suggests an anaerobic lifestyle based on sugar fermentation by Embden-Meyerhof glycolysis with production of hydrogen, acetate and ethanol. Putative glycohydrolases and an endoglucanase may enable catabolism of (hemi)cellulose in thermal environments. This study lays a foundation for understanding the physiology and ecological role of the 'Atribacteria'.

Nucleic acid amplification using microfluidic devices

Abstract: The present invention provides microfluidic devices and methods using the same in various types of thermal cycling reactions. Certaom devices include a rotary microfluidic channel and a plurality of temperature regions at different locations along the rotary microfluidic channel at which temperature is regulated. Solution can be repeatedly passed through the temperature regions such that the solution is exposed to different temperatures. Other microfluidic devices include an array of reaction chambers formed by intersecting vertical and horizontal flow channels, with the ability to regulate temperature at the reaction chambers. The microfluidic devices can be used to conduct a number of different analyses, including various primer extension reactions and nucleic acid amplification reactions.

Single-Cell DNA-Methylation Analysis Reveals Epigenetic Chimerism in Preimplantation Embryos

Abstract: Epigenetic alterations are increasingly recognized as causes of human cancers and disease. These aberrations are likely to arise during genomic reprogramming in mammalian preimplantation embryos, when their epigenomes are most vulnerable. However, this process is only partially understood because of the experimental inaccessibility of early-stage embryos. Here, we introduce a methodologic advance, probing single cells for various DNA-methylation errors at multiple loci, to reveal failed maintenance of epigenetic mark results in chimeric mice, which display unpredictable phenotypes leading to developmental arrest. Yet we show that mouse pronuclear transfer can be used to ameliorate such reprogramming defects. This study not only details the epigenetic reprogramming dynamics in early mammalian embryos but also suggests diagnostic and potential future therapeutic applications.

Migration of cells in a social context

Abstract: In multicellular organisms and complex ecosystems, cells migrate in a social context. Whereas this is essential for the basic processes of life, the influence of neighboring cells on the individual remains poorly understood. Previous work on isolated cells has observed a stereotypical migratory behavior characterized by short-time directional persistence with long-time random movement. We discovered a much richer dynamic in the social context, with significant variations in directionality, displacement, and speed, which are all modulated by local cell density. We developed a mathematical model based on the experimentally identified “cellular traffic rules” and basic physics that revealed that these emergent behaviors are caused by the interplay of single-cell properties and intercellular interactions, the latter being dominated by a pseudopod formation bias mediated by secreted chemicals and pseudopod collapse following collisions. The model demonstrates how aspects of complex biology can be explained by simple rules of physics and constitutes a rapid test bed for future studies of collective migration of individual cells.

Identification of a Colonial Chordate Histocompatibility Gene

Abstract: Histocompatibility is the basis by which multicellular organisms of the same species distinguish self from nonself. Relatively little is known about the mechanisms underlying histocompatibility reactions in lower organisms. Botryllus schlosseri is a colonial urochordate, a sister group of vertebrates, that exhibits a genetically determined natural transplantation reaction, whereby self-recognition between colonies leads to formation of parabionts with a common vasculature, whereas rejection occurs between incompatible colonies. Using genetically defined lines, whole-transcriptome sequencing, and genomics, we identified a single gene that encodes self-nonself and determines "graft" outcomes in this organism. This gene is significantly up-regulated in colonies poised to undergo fusion and/or rejection, is highly expressed in the vasculature, and is functionally linked to histocompatibility outcomes. These findings establish a platform for advancing the science of allorecognition.

Simultaneous Measurement of Amyloid Fibril Formation by Dynamic Light Scattering and Fluorescence Reveals Complex Aggregation Kinetics

Abstract: An apparatus that combines dynamic light scattering and Thioflavin T fluorescence detection is used to simultaneously probe fibril formation in polyglutamine peptides, the aggregating subunit associated with Huntington's disease, in vitro. Huntington's disease is a neurodegenerative disorder in a class of human pathologies that includes Alzheimer's and Parkinson's disease. These pathologies are all related by the propensity of their associated protein or polypeptide to form insoluble, β-sheet rich, amyloid fibrils. Despite the wide range of amino acid sequence in the aggregation prone polypeptides associated with these diseases, the resulting amyloids display strikingly similar physical structure, an observation which suggests a physical basis for amyloid fibril formation. Thioflavin T fluorescence reports β-sheet fibril content while dynamic light scattering measures particle size distributions. The combined techniques allow elucidation of complex aggregation kinetics and are used to reveal multiple stages of amyloid fibril formation.

Optofluidic Cell Selection from Complex Microbial Communities for Single-Genome Analysis

Abstract: Genetic analysis of single cells is emerging as a powerful approach for studies of heterogeneous cell populations Indeed, the notion of homogeneous cell populations is receding as approaches to resolve genetic and phenotypic variation between single cells are applied throughout the life sciences A key step in single-cell genomic analysis today is the physical isolation of individual cells from heterogeneous populations, particularly microbial populations, which often exhibit high diversity Here, we detail the construction and use of instrumentation for optical trapping inside microfluidic devices to select individual cells for analysis by methods including nucleic acid sequencing This approach has unique advantages for analyses of rare community members, cells with irregular morphologies, small quantity samples, and studies that employ advanced optical microscopy

A microfluidic device for preparing next generation DNA sequencing libraries and for automating other laboratory protocols that require one or more column chromatography steps.

Abstract: Library preparation for next-generation DNA sequencing (NGS) remains a key bottleneck in the sequencing process which can be relieved through improved automation and miniaturization. We describe a microfluidic device for automating laboratory protocols that require one or more column chromatography steps and demonstrate its utility for preparing Next Generation sequencing libraries for the Illumina and Ion Torrent platforms. Sixteen different libraries can be generated simultaneously with significantly reduced reagent cost and hands-on time compared to manual library preparation. Using an appropriate column matrix and buffers, size selection can be performed on-chip following end-repair, dA tailing, and linker ligation, so that the libraries eluted from the chip are ready for sequencing. The core architecture of the device ensures uniform, reproducible column packing without user supervision and accommodates multiple routine protocol steps in any sequence, such as reagent mixing and incubation; column packing, loading, washing, elution, and regeneration; capture of eluted material for use as a substrate in a later step of the protocol; and removal of one column matrix so that two or more column matrices with different functional properties can be used in the same protocol. The microfluidic device is mounted on a plastic carrier so that reagents and products can be aliquoted and recovered using standard pipettors and liquid handling robots. The carrier-mounted device is operated using a benchtop controller that seals and operates the device with programmable temperature control, eliminating any requirement for the user to manually attach tubing or connectors. In addition to NGS library preparation, the device and controller are suitable for automating other time-consuming and error-prone laboratory protocols requiring column chromatography steps, such as chromatin immunoprecipitation.

Proteome-wide protein interaction measurements of bacterial proteins of unknown function

Abstract: Despite the enormous proliferation of bacterial genome data, surprisingly persistent collections of bacterial proteins have resisted functional annotation In a typical genome, roughly 30% of genes have no assigned function Many of these proteins are conserved across a large number of bacterial genomes To assign a putative function to these conserved proteins of unknown function, we created a physical interaction map by measuring biophysical interaction of these proteins Binary protein-–protein interactions in the model organism Streptococcus pneumoniae (TIGR4) are measured with a microfluidic high-throughput assay technology In some cases, informatic analysis was used to restrict the space of potential binding partners In other cases, we performed in vitro proteome-wide interaction screens We were able to assign putative functions to 50 conserved proteins of unknown function that we studied with this approach

Direct molecular diagnosis of fetal aneuploidy

Abstract: Methods and materials for detection of aneuploidy and other chromosomal abnormalities using fetal tissue are disclosed. Results can be obtained rapidly, without cell culture. The method uses digital PCR for amplification and detection of single target sequences, allowing an accurate count of a specific chromosome or chromosomal region. Specific polynucleic acid primers and probes are disclosed for chromosomes 1, 13, 18, 21, X and Y. These polynucleic acid sequences are chosen to be essentially invariant between individuals, so the test is not dependent on sequence differences between fetus and mother.

Microfluidic serial digital to analog pressure converter for arbitrary pressure generation and contamination-free flow control

Abstract: Multilayer microfluidics based on PDMS (polydimethylsiloxane) soft lithography have offered parallelism and integration for biological and chemical sciences, where reduction in reaction volume and consistency of controlled variables across experiments translate into reduced cost, increased quantity and quality of data. One issue with push up or push down microfluidic control concept is the inability to provide multiple control pressures without adding more complex and expensive external pressure controls. We present here a microfluidic serial DAC (Digital to Analog Converter) that can be integrated with any PDMS device to expand the device's functionality by effectively adding an on-chip pressure regulator. The microfluidic serial DAC can be used with any incompressible fluids and operates in a similar fashion compared to an electronic serial DAC. It can be easily incorporated into any existing multilayer microfluidic devices, and the output pressure that the device generates could be held for extensive times. We explore in this paper various factors that affect resolution, speed, and linearity of the DAC output. As an application, we demonstrate microfluidic DAC's ability for on-chip manipulation of flow resistance when integrated with a simple flow network. In addition, we illustrate an added advantage of using the microfluidic serial DAC in preventing back flow and possible contamination.

Methods for profiling and quantitating cell-free rna

Abstract: The invention generally relates to methods for assessing the health of a tissue by characterizing circulating nucleic acids in a biological sample. According to certain embodiments, methods for assessing the health of a tissue include the steps of detecting a sample level of RNA in a biological sample, comparing the sample level of RNA to a reference level of RNA specific to the tissue, determining whether a difference exists between the sample level and the reference level, and characterizing the tissue as abnormal if a difference is detected.

Correlation of Gene Expression and Genome Mutation in Single B-Cells

Abstract: High-throughput measurement of gene-expression and immune receptor repertoires have recently become powerful tools in the study of adaptive immune response. However, despite their now-widespread use, both tend to discard cell identity by treating cell populations in bulk, and therefore lose the correlation between genetic variability and gene-expression at the single cell level. In order to recover this information, we developed a method to simultaneously measure gene expression profiles and genome mutations in single cells. We applied this method by quantifying the relationships between gene expression and antibody mutation in ensembles of individual B-cells from immunized mice. The results reveal correlations reflecting the manner in which information propagates between a B-cell’s antigen receptors, its gene expression, and its mutagenic machinery, and demonstrate the power of this approach to illuminate both heterogeneity and physiology in cell populations.

Single-cell and metagenomic analyses indicate a fermentative and saccharolytic lifestyle for members of the OP9 lineage

Abstract: United States. National Aeronautics and Space Administration (Exobiology Grant EXO-NNX11AR78G)

Designing the statistically optimal drug for cancer therapy

Abstract: Cancer and healthy cells have distinct distributions of molecular properties and thus respond differently to drugs. Cancer drugs ideally kill cancer cells while limiting harm to healthy cells. However, the inherent variance among cells in both cancer and healthy cell populations increases the difficulty of selective drug action. Here we propose a classification framework based on the idea that an ideal cancer drug should maximally discriminate between cancer and healthy cells. We first explore how molecular markers can be used to discriminate cancer cells from healthy cells on a single cell basis, and then how the effects of drugs are statistically predicted by these molecular markers. We then combine these two ideas to show how to optimally match drugs to tumor cells. We find that expression levels of a handful of genes suffice to discriminate well between individual cells in cancer and healthy tissue. We also find that gene expression predicts the efficacy of cancer drugs, suggesting that the cancer drugs act as classifiers using gene profiles. In agreement with our first finding, a small number of genes predict drug efficacy well. Finally, we formulate a framework that defines an optimal drug, and predicts drug cocktails that may target cancer more accurately than the individual drugs alone. Conceptualizing cancer drugs as solving a discrimination problem in the high-dimensional space of molecular markers promises to inform the design of new cancer drugs and drug cocktails.

Analysis of Human Colon Tissue Cell Composition Using Single-Cell Gene-Expression PCR

Abstract: In both health and disease, human tissues are composed of a multiplicity of cellular types, many of which unknown and uncharacterized. Accurate, quantitative measurements of tissue cell composition are difficult to perform, as classic analytical techniques, such as flow cytometry and immunohistochemistry, depend on the availability of antigen-specific monoclonal antibodies and allow only for a few markers to be tested in parallel on each cell. As a result, the full repertoire of cell types contained in a tissue, and their relative abundance in physiological and pathological situations, remain largely unexplored. We developed a novel analytical platform for the study of tissue cell composition, based on single-cell gene-expression RT-PCR (SINCE-PCR). We tested the precision, accuracy and sensitivity of the method, and we used it to investigate the cell composition of human colon epithelia, both normal and cancerous. Starting from primary surgical specimens, we obtained single-cell suspensions of colon epithelial cells and we used flow cytometry to sort them one-by-one, in arrays of hundreds of single cells. We then exploited a microfluidic platform to analyze each individual cell for the expression of 96 genes in parallel, and we used statistical clustering algorithms to associate cells with similar gene-expression profiles. This novel approach led to the discovery of new epithelial cell populations and novel biomarkers to differentially label them. Moreover, it showed that, in human colon tumors, the heterogeneity observed among cancer cells closely mirrors the cellular diversity observed in normal epithelia, where immature progenitor cells intermingle with distinct subsets of mature goblet cells and enterocytes. Finally, SINCE-PCR analysis of monoclonal colon cancer xenografts, obtained from injection of a single (n = 1) “cancer stem cell” into immunodeficient mice, formally proved that this diversity is epigenetic in nature and originates from multi-lineage differentiation processes, reminiscent of physiological stem-cell systems.