Biology subjects

Quake, S. R.

Publications and source records attributed to Quake, S. R..

At least 109 records · Page 6Linked to original sources

bioRxiv2017

Evolutionary Origin of the Mammalian Hematopoietic System Found in a Colonial Chordate

Hematopoiesis is an essential process that evolved in multicellular animals. At the heart of this process are hematopoietic stem cells (HSCs), which are multipotent, self-renewing and generate the entire repertoire of blood and immune cells throughout life. Here we studied the hematopoietic system of Botryllus schlosseri, a colonial tunicate that has vasculature, circulating blood cells, and interesting characteristics of stem cell biology and immunity. Self-recognition between genetically compatible B. schlosseri colonies leads to the formation of natural parabionts with shared circulation, whereas incompatible colonies reject each other. Using flow-cytometry, whole-transcriptome sequencing of defined cell populations, and diverse functional assays, we identified HSCs, progenitors, immune-effector cells, the HSC niche, and demonstrated that self-recognition inhibits cytotoxic reaction. Our study implies that the HSC and myeloid lineages emerged in a common ancestor of tunicates and vertebrates and suggests that hematopoietic bone marrow and the B. schlosseri endostyle niche evolved from the same origin.

immunology↗

bioRxiv2017

Transcriptomic characterization of 20 organs and tissues from mouse at single cell resolution creates a Tabula Muris

The Tabula Muris ConsortiumWe have created a compendium of single cell transcriptome data from the model organism Mus musculus comprising more than 100,000 cells from 20 organs and tissues. These data represent a new resource for cell biology, revealing gene expression in poorly characterized cell populations and allowing for direct and controlled comparison of gene expression in cell types shared between tissues, such as T-lymphocytes and endothelial cells from distinct anatomical locations. Two distinct technical approaches were used for most tissues: one approach, microfluidic droplet-based 3-end counting, enabled the survey of thousands of cells at relatively low coverage, while the other, FACS-based full length transcript analysis, enabled characterization of cell types with high sensitivity and coverage. The cumulative data provide the foundation for an atlas of transcriptomic cell biology.

cell biology↗

bioRxiv2017

Matrix-corrected mass spectrometry enables sensitive detection of food allergens and reveals widespread soy contamination of processed foods

The frequent use of precautionary food allergen labeling statements such as \"may contain\" poses challenges to allergic individuals who rely on such labeling to determine whether a food is safe to consume. We developed a multiplexed liquid chromatography-mass spectrometry assay targeting 14 common allergens in order to survey how frequently these precautionary statements indicate allergen contamination and to assess whether variations in precautionary phrasing affect the likelihood of allergen contamination. A survey of 84 foods revealed how scheduled multiple reaction monitoring (MRM) transition interference derived from complex and heterogeneous sample matrices hinders sensitive analyte detection. As a solution, we developed MAtrix-Dependent Interference Correction (MADIC), an approach to sensitively detect trace peptide quantities through interference identification and stringent peptide quality control criteria. Applying this method, we find frequent contamination of soy in breads and corn flour, and observe additional instances of food contamination with tree nuts, wheat, milk, and egg. In some of these cases, the food had no precautionary labeling for the offending allergen. We also find that only 10% of warning labels are indicative of contamination, and that products with \"same facility\" precautionary labeling are not necessarily less likely to contain trace amounts of allergens than products labeled \"may contain.\"

immunology↗

bioRxiv2017

A Noninvasive Molecular Clock for Fetal Development Predicts Gestational Age and Preterm Delivery

We performed a high time-resolution, longitudinal study of normal pregnancy development by measuring cell-free RNA (cfRNA) in blood from women during each week of pregnancy. Analysis of tissue-specific transcripts in these samples enabled us to follow fetal and placental development with high resolution and sensitivity, and also to detect gene-specific responses of the maternal immune system to pregnancy. We established a \"clock\" for normal pregnancy development and enabled a direct molecular approach to determine expected delivery dates with comparable accuracy to ultrasound, creating the basis for a portable, inexpensive fetal dating method. We also identified a related gene set that accurately discriminated women at risk for spontaneous preterm delivery up to two months in advance of labor, forming the basis of a potential screening test for risk of preterm delivery.

bioengineering↗

bioRxiv2017

Signatures of Selection in the Human Antibody Repertoire: Selective Sweeps, Competing Subclones, and Neutral Drift

Antibodies are created and refined by somatic evolution in B cell populations, which endows the human immune system with the ability to recognize and eliminate diverse pathogens. However, the evolutionary processes that sculpt antibody repertoires remain poorly understood. Here, using an unbiased repertoire-scale approach, we show that the molecular signatures of evolution are evident in human B cell lineages and reveal how antibodies evolve somatically. We measured the dynamics and genetic diversity of B cell responses of five adults longitudinally before and after influenza vaccination using high-throughput antibody repertoire sequencing. We identified vaccine-responsive B cell lineages that carry signatures of selective sweeps driven by positive selection, and discovered that they often display evidence for selective sweeps favoring multiple subclones. We also found persistent B cell lineages that exhibit stable population dynamics and carry signatures of neutral drift. By exploiting the linkage between B cell fitness and antibody binding affinity, we demonstrated the potential for using signatures of selection to identify antibodies with high binding affinity. This quantitative characterization reveals that antibody repertoires are shaped by an unexpectedly broad spectrum of evolutionary processes and shows how signatures of evolutionary history can be harnessed for antibody discovery and engineering.\n\nOne Sentence SummaryMolecular signatures of somatic evolution reveal that diverse evolutionary processes ranging from strong positive selection to neutral drift sculpt human antibodies.

immunology↗

bioRxiv2017

Single-cell transcriptional dynamics of flavivirus infection

Dengue and Zika viral infections affect millions of people annually and can be complicated by hemorrhage or neurological manifestations, respectively. However, a thorough understanding of the host response to these viruses is lacking, partly because conventional approaches ignore heterogeneity in virus abundance across cells. We present viscRNA-Seq (virus-inclusive single cell RNA-Seq), an approach to probe the host transcriptome together with intracellular viral RNA at the single cell level. We applied viscRNA-Seq to monitor dengue and Zika virus infection in cultured cells and discovered extreme heterogeneity in virus abundance. We exploited this variation to identify host factors that show complex dynamics and a high degree of specificity for either virus, including proteins involved in the endoplasmic reticulum translocon, signal peptide processing, and membrane trafficking. We validated the viscRNA-Seq hits and discovered novel proviral and antiviral factors. viscRNA-Seq is a powerful approach to assess the genome-wide virus-host dynamics at single cell level.

microbiology↗

bioRxiv2017

Nonivasive prenatal diagnosis of single-gene disorders using droplet digital PCR

BackgroundPrenatal diagnosis in pregnancies at risk of single-gene disorders is currently performed using invasive methods such as chorionic villus sampling and amniocentesis. This is in contrast with screening for common aneuploidies, for which noninvasive methods with a single maternal blood sample have become standard clinical practice.\n\nMethodsWe developed a protocol for noninvasive prenatal diagnosis of inherited single gene disorders using droplet digital PCR (ddPCR) from circulating cell-free DNA (cfDNA) in maternal plasma. First, the amount of cfDNA and fetal fraction are determined using a panel of Taqman assays targeting high-variability SNPs. Second, the ratio of healthy and diseased alleles in maternal plasma are quantified using Taqman assays targeting the mutations carried by the parents. Two validation approaches of the mutation assay are presented.\n\nResultsWe collected blood samples from 9 pregnancies at risk for different single gene disorders including common conditions and rare metabolic disorders. We measured cases at risk of hemophilia, ornithine transcarbamylase deficiency, cystic fibrosis, {beta}-thalassemia, mevalonate kinase deficiency, acetylcholine receptor deficiency and DFNB1 nonsyndromic hearing loss. We correctly differentiated affected and unaffected pregnancies (2 affected, 7 unaffected), confirmed by neonatal testing. We successfully measured an affected pregnancy as early as week 11, and with a fetal fraction as low as 3.7{+/-}0.3%.\n\nConclusionOur method detects single nucleotide mutations of autosomal recessive diseases as early as the first trimester of pregnancy. This is of importance for metabolic disorders where early diagnosis can affect management of the disease and reduce complications and anxiety related to invasive testing.

genetics↗

bioRxiv2017

Single-cell RNA-seq reveals intrinsic and extrinsic regulatory heterogeneity in yeast responding to stress

From bacteria to humans, individual cells within isogenic populations can show significant variation in stress tolerance, but the nature of this heterogeneity is not clear. To investigate this, we used single-cell RNA sequencing to quantify transcript heterogeneity in single S. cerevisiae cells treated with and without salt stress, to explore population variation and identify cellular covariates that influence the stress-responsive transcriptome. Leveraging the extensive knowledge of yeast transcriptional regulation, we uncovered significant regulatory variation in individual yeast cells, both before and after stress. We also discovered that a subset of cells decouple expression of ribosomal protein genes from the environmental stress response, in a manner partly correlated with the cell cycle but unrelated to the yeast ultradian metabolic cycle. Live-cell imaging of cells expressing pairs of fluorescent regulators, including the transcription factor Msn2 with Dot6, Sfp1, or MAP kinase Hog1, revealed both coordinated and decoupled nucleocytoplasmic shuttling. Together with transcriptomic analysis, our results reveal that cells maintain a cellular filter against decoupled bursts of transcription-factor activation but mount a stress response upon coordinated regulation, even in a subset of unstressed cells.

systems biology↗

bioRxiv2017

T-cell receptor sequencing of early stage breast cancer tumors identifies altered clonal structure of the T-cell repertoire

Tumor infiltrating T-cells play an important role in many cancers, and can improve prognosis and yield therapeutic targets. We characterized T-cells infiltrating both breast cancer tumors and the surrounding normal breast tissue to identify T-cells specific to each, as well as their abundance in peripheral blood. Using immune profiling of the T-cell beta chain repertoire in 16 patients with early stage breast cancer, we show that the clonal structure of the tumor is significantly different from adjacent breast tissue, with the tumor containing approximately 3-fold more T-cells, but with a lower fraction of unique sequences and higher clonality compared to normal breast. The clonal structure of T-cells in blood and normal breast is more similar than between blood and tumor and can be used to distinguish tumor from normal breast tissue in 14 of 16 patients. Many T-cells overlap between tissues from the same patient, including approximately 50% of T-cells between tumor and normal breast. Both solid tissues contain high-abundance "enriched" sequences that are absent or of low abundance in the other tissue. Many of these T-cells are either not detected or detected with very low frequency in the blood, suggesting the existence of separate compartments of T-cells in both tumor and normal breast. Enriched T-cell sequences are typically unique to each patient, but there is a subset of sequences that are shared between many different patients. We show that most of these are commonly generated sequences and thus unlikely to play an important role in the tumor microenvironment.

immunology↗

bioRxiv2017

Single-Cell RNAseq analysis of infiltrating neoplastic cells at the migrating front of human glioblastoma

Glioblastoma is the most common primary brain cancer in adults and is notoriously difficult to treat due to its diffuse nature. We performed single-cell RNAseq on 3589 cells in a cohort of four patients. We obtained cells from the tumor core as well as surrounding peripheral tissue. Our analysis revealed cellular variation in the tumors genome and transcriptome, We were able to identify infiltrating neoplastic cells in regions peripheral to the core lesions. Despite the existence of significant heterogeneity among neoplastic cells, we found that infiltrating GBM cells share a consistent gene signature between patients, suggesting a common mechanism of infiltration. Additionally, in investigating the immunological response to the tumors, we found transcriptionally distinct myeloid cell populations residing in the tumor core and the surrounding peritumoral space. Our data provide a detailed dissection of GBM cell types, revealing an abundance of novel information about tumor formation and migration.

cancer biology↗

bioRxiv2017

5-Hydroxymethylcytosine signatures in cell-free DNA provide information about tumor types and stages

5-Hydroxymethylcytosine (5hmC) is an important mammalian DNA epigenetic modification that has been linked to gene regulation and cancer pathogenesis. Here we explored the diagnostic potential of 5hmC in circulating cell-free DNA (cfDNA) using a sensitive chemical labeling-based low-input shotgun sequencing approach. We sequenced cell-free 5hmC from 49 patients of seven different cancer types and found distinct features that could be used to predict cancer types and stages with high accuracy. Specifically, we discovered that lung cancer leads to a progressive global loss of 5hmC in cfDNA, whereas hepatocellular carcinoma and pancreatic cancer lead to disease-specific changes in the cell-free hydroxymethylome. Our proof of principle results suggest that cell-free 5hmC signatures may potentially be used not only to identify cancer types but also to track tumor stage in some cancers.\n\nOne Sentence SummaryAnalyzing the epigenetic modification 5-hydroxymethylcysoine in circulating cell-free DNA reveals tumor tissue of origin and stages for cancer diagnostics.

genomics↗

bioRxiv2017

Classifying Drosophila Olfactory Projection Neuron Subtypes by Single-cell RNA Sequencing

How a neuronal cell type is defined and how this relates to its transcriptome are still open questions. The Drosophila olfactory projection neurons (PNs) are among the best-characterized neuronal types: Different PN classes target dendrites to distinct olfactory glomeruli and PNs of the same class exhibit indistinguishable anatomical and physiological properties. Using single-cell RNA-sequencing, we comprehensively characterized the transcriptomes of 40 PN classes and unequivocally identified transcriptomes for 6 classes. We found a new lineage-specific transcription factor that instructs PN dendrite targeting. Transcriptomes of closely-related PN classes exhibit the largest difference during circuit assembly, but become indistinguishable in adults, suggesting that neuronal subtype diversity peaks during development. Genes encoding transcription factors and cell-surface molecules are the most differentially expressed, indicating their central roles in specifying neuronal identity. Finally, we show that PNs use highly redundant combinatorial molecular codes to distinguish subtypes, enabling robust specification of cell identity and circuit assembly.

genomics↗

bioRxiv2017

Dynamics Of The Human Antibody Repertoire Following B-cell Depletion In Systemic Sclerosis

Systemic sclerosis with pulmonary arterial hypertension (SSc-PAH) is a debilitating and frequently lethal disease of unknown cause lacking effective treatment options. Lymphocyte anomalies and autoantibodies observed in systemic sclerosis have suggested an autoimmune character. Here we study the clonal structure of the B-cell repertoire in SSc-PAH using immunoglobulin heavy-chain sequencing before and after B-cell depletion. We found SSc-PAH to be associated with anomalies in B-cell development, namely altered VDJ rearrangement frequencies (reduced IGHV2-5 segment usage) and an increased somatic mutation-fixation probability in expanded B-cell lineages. SSc-PAH was also characterized by anomalies in B-cell homeostasis, namely an expanded IgD+ proportion with reduced mutation loads and an expanded proportion of highly antibody-secreting cells. Disease signatures pertaining to IGHV2-5 segment usage, IgD proportions and mutation loads were temporarily reversed after B-cell depletion. Analyzing the time course of B-cell depletion, we find that the kinetics of naive replenishment are predictable from baseline measurements alone, that release of plasma cells into the periphery can precede naive replenishment and that modes of B-cell receptor diversity are highly elastic. Our findings shed light on the humoral immune basis of SSc-PAH and provide insights into the effect of B-cell depletion on the antibody repertoire.\n\nAbbreviations

immunology↗

bioRxiv2017

The Human Cell Atlas

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body, by undertaking a Human Cell Atlas Project as an international collaborative effort. The aim would be to define all human cell types in terms of distinctive molecular profiles (e.g., gene expression) and connect this information with classical cellular descriptions (e.g., location and morphology). A comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, as well as provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas.

cell biology↗

bioRxiv2017

High fidelity hypothermic preservation of primary tissues in organ transplant preservative for single cell transcriptome analysis

BackgroundHigh-fidelity preservation strategies for primary tissues are in great demand in the single cell RNAseq community. A reliable method will greatly expand the scope of feasible collaborations and maximize the utilization of technical expertise. When choosing a method, standardizability is as important a factor to consider as fidelity due to the susceptibility of single-cell RNAseq analysis to technical noises. Existing approaches such as cryopreservation and chemical fixation are less than ideal for failing to satisfy either or both of these standards.\n\nResultsHere we propose a new strategy that leverages preservation schemes developed for organ transplantation. We evaluated the strategy by storing intact mouse kidneys in organ transplant preservative solution at hypothermic temperature for up to 4 days (6 hrs, 1, 2, 3, and 4 days), and comparing the quality of preserved and fresh samples using FACS and single cell RNAseq. We demonstrate that the strategy effectively maintained cell viability, transcriptome integrity, cell population heterogeneity, and transcriptome landscape stability for samples after up to 3 days of preservation. The strategy also facilitated the definition of the diverse spectrum of kidney resident immune cells, to our knowledge the first time at single cell resolution.\n\nConclusionsHypothermic storage of intact primary tissues in organ transplant preservative maintains the quality and stability of the transcriptome of cells for single cell RNAseq analysis. The strategy is readily generalizable to primary specimens from other tissue types for single cell RNAseq analysis.

genomics↗

bioRxiv2017

Microfluidic-based mini-metagenomics enables discovery of novel microbial lineages from complex environmental samples

Metagenomics and single-cell genomics have enabled the discovery of many new genomes from previously unknown branches of life. However, extracting novel genomes from complex mixtures of metagenomic data can still be challenging and in many respects represents an ill-posed problem which is generally approached with ad hoc methods. Here we present a microfluidic-based mini-metagenomic method which offers a statistically rigorous approach to extract novel microbial genomes from complex samples. In addition, by generating 96 sub-samples from each environmental sample, this method maintains high throughput, reduces sample complexity, and preserves single-cell resolution. We used this approach to analyze two hot spring samples from Yellowstone National Park and extracted 29 new genomes larger than 0.5 Mbps. These genomes represent novel lineages at different taxonomic levels, including three deeply branching lineages. Functional analysis revealed that these organisms utilize diverse pathways for energy metabolism. The resolution of this mini-metagenomic method enabled accurate quantification of genome abundance, even for genomes less than 1% in relative abundance. Our analyses also revealed a wide range of genome level single nucleotide polymorphism (SNP) distributions with nonsynonymous to synonymous ratio indicative of low to moderate environmental selection. The scale, resolution, and statistical power of microfluidic-based mini-metagenomic make it a powerful tool to dissect the genomic structure microbial communities while effectively preserving the fundamental unit of biology, the single cell.

microbiology↗

bioRxiv2017

Humans are colonized by many uncharacterized and highly divergent microbes

Blood circulates throughout the entire body and contains molecules drawn from virtually every tissue, including the microbes and viruses which colonize the body. Through massive shotgun sequencing of circulating cell-free DNA from the blood, we identified hundreds of new bacteria and viruses which represent previously unidentified members of the human microbiome. Analysing cumulative sequence data from 1,351 blood samples collected from 188 patients enabled us to assemble 7,190 contiguous regions (contigs) larger than 1 kbp, of which 3,761 are novel with little or no sequence homology in any existing databases. The vast majority of these novel contigs possess coding sequences, and we have validated their existence both by finding their presence in independent experiments and by performing direct PCR amplification. When their nearest neighbors are located in the tree of life, many of the organisms represent entirely novel taxa, showing that microbial diversity within the human body is substantially broader than previously appreciated.

microbiology↗

bioRxiv2017

Single cell transcriptome analysis of human pancreas reveals transcriptional signatures of aging and somatic mutation patterns.

As organisms age, cells accumulate genetic and epigenetic changes that eventually lead to impaired organ function or catastrophic transformation such as cancer. Since aging appears to be a stochastic process of increasing disorder1 cells in an organ will be individually affected in different ways - thus rendering bulk analyses of postmitotic adult tissues difficult to characterize. Here we directly measure the effects of aging in primary human tissue by performing single-cell transcriptome analysis of 2544 human pancreas cells from eight donors spanning six decades of life. We find that islet cells from older donors have increased levels of molecular disorder as measured both by noise in the transcriptome and by the number of cells which display inappropriate hormone expression, revealing a transcriptional instability associated with aging. By further analyzing the spectrum of somatic mutations in single cells, we found a specific age-dependent mutational signature characterized by C to A and C to G transversions. These mutations are indicators of oxidative stress and the signature is absent in single cells from human brain tissue or in a tumor cell line. We have used the single cell measurements of transcriptional noise and mutation level to identify molecular pathways correlated with these changes that could influence human disease. Our results demonstrate the feasibility of using single-cell RNA-seq data from primary cells to derive meaningful insights into the genetic processes that operate on aging human tissue and to determine molecular mechanisms coordinated with these processes.

genomics↗