Assaf Rotem

Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state.

Chromatin profiling provides a versatile means to investigate functional genomic elements and their regulation. However, current methods yield ensemble profiles that are insensitive to cell-to-cell variation. Here we combine microfluidics, DNA barcoding and sequencing to collect chromatin data at single-cell resolution. We demonstrate the utility of the technology by assaying thousands of individual cells and using the data to deconvolute a mixture of ES cells, fibroblasts and hematopoietic progenitors into high-quality chromatin state maps for each cell type. The data from each single cell are sparse, comprising on the order of 1,000 unique reads. However, by assaying thousands of ES cells, we identify a spectrum of subpopulations defined by differences in chromatin signatures of pluripotency and differentiation priming. We corroborate these findings by comparison to orthogonal single-cell gene expression data. Our method for single-cell analysis reveals aspects of epigenetic heterogeneity not captured by transcriptional analysis alone.

Synthesis of Monodisperse Microparticles from Non-Newtonian Polymer Solutions with Microfluidic Devices

Microfluidic devices can form emulsions that are highly uniform in size;[1–3] they can also form compound emulsions, in which each supradroplet contains exactly the same number of internal droplets, packed in exactly the same configuration.[4–6] Because the drops can be formed with a highly controlled structure and uniformity, they are useful as templates to synthesize monodisperse particles. In such a process, microfluidic devices are used to form droplets with the desired structure, which are then solidified to produce particles. This allows synthesis of particles with a variety of shapes, including Janus particles, nonspherical dimers, and core–shell capsules.[7–10] However, the fluid precursors must be compatible with the formation of drops in microfluidic devices: this precondition limits the applicability of this technique. For example, this circumstance requires fluids with a low viscosity, negligible viscoelastic response, and moderate interfacial tension. If even one of these constraints is not met, it is difficult to achieve drop formation in the stable dripping regime. Instead, jetting occurs, resulting in the production of polydisperse particles.[11] This represents a significant limitation, as the most useful materials for making particles typically have properties that do not meet these constraints. For example, lipid melts, due to their amphiphilic chemical properties, tend to be viscous and have low interfacial tension with oil or aqueous carrier phases. Solutions of long-chain polymers like poly(N-isopropylacrylamide) (pNIPAM)[12] or polyurethane–polybutadienediol (pU–pBDO),[13] form excellent particles, but tend to be viscous and have significant viscoelastic response at the shear rates needed for controlled drop formation.[1,11,14] As a consequence, these fluids, and many others like them, cannot be used to synthesize particles in microfluidic devices, greatly limiting the applicability of this technique. To overcome the limitations with such fluids, a more robust drop formation mechanism is needed.

DNA sequence analysis with droplet-based microfluidics

Droplet-based microfluidic techniques can form and process micrometer scale droplets at thousands per second. Each droplet can house an individual biochemical reaction, allowing millions of reactions to be performed in minutes with small amounts of total reagent. This versatile approach has been used for engineering enzymes, quantifying concentrations of DNA in solution, and screening protein crystallization conditions. Here, we use it to read the sequences of DNA molecules with a FRET-based assay. Using probes of different sequences, we interrogate a target DNA molecule for polymorphisms. With a larger probe set, additional polymorphisms can be interrogated as well as targets of arbitrary sequence.

High-Throughput Single-Cell Labeling (Hi- SCL) for RNA-Seq Using Drop-Based Microfluidics

The importance of single-cell level data is increasingly appreciated, and significant advances in this direction have been made in recent years. Common to these technologies is the need to physically segregate individual cells into containers, such as wells or chambers of a micro-fluidics chip. High-throughput Single-Cell Labeling (Hi-SCL) in drops is a novel method that uses drop-based libraries of oligonucleotide barcodes to index individual cells in a population. The use of drops as containers, and a microfluidics platform to manipulate them en-masse, yields a highly scalable methodological framework. Once tagged, labeled molecules from different cells may be mixed without losing the cell-of-origin information. Here we demonstrate an application of the method for generating RNA-sequencing data for multiple individual cells within a population. Barcoded oligonucleotides are used to prime cDNA synthesis within drops. Barcoded cDNAs are then combined and subjected to second generation sequencing. The data are deconvoluted based on the barcodes, yielding single-cell mRNA expression data. In a proof-of-concept set of experiments we show that this method yields data comparable to other existing methods, but with unique potential for assaying very large numbers of cells.

Rapid, targeted and culture-free viral infectivity assay in drop-based microfluidics

A key viral property is infectivity, and its accurate measurement is crucial for the understanding of viral evolution, disease and treatment. Currently viral infectivity is measured using plaque assays, which involve prolonged culturing of host cells, and whose measurement is unable to differentiate between specific strains and is prone to low number fluctuation. We developed a rapid, targeted and culture-free infectivity assay using high-throughput drop-based microfluidics. Single infectious viruses are incubated in a large number of picoliter drops with host cells for one viral replication cycle followed by in-drop gene-specific amplification to detect infection events. Using murine noroviruses (MNV) as a model system, we measure their infectivity and determine the efficacy of a neutralizing antibody for different variants of MNV. Our results are comparable to traditional plaque-based assays and plaque reduction neutralization tests. However, the fast, low-cost, highly accurate genomic-based assay promises to be a superior method for drug screening and isolation of resistant viral strains. Moreover our technique can be adapted to measuring the infectivity of other pathogens, such as bacteria and fungi.

Isolation and Analysis of Rare Norovirus Recombinants from Coinfected Mice Using Drop-Based Microfluidics

ABSTRACT Human noroviruses (HuNoVs) are positive-sense RNA viruses that can cause severe, highly infectious gastroenteritis. HuNoV outbreaks are frequently associated with recombination between circulating strains. Strain genotyping and phylogenetic analyses show that noroviruses often recombine in a highly conserved region near the junction of the viral polyprotein (open reading frame 1 [ORF1]) and capsid (ORF2) genes and occasionally within the RNA-dependent RNA polymerase (RdRP) gene. Although genotyping methods are useful for tracking changes in circulating viral populations, they report only the dominant recombinant strains and do not elucidate the frequency or range of recombination events. Furthermore, the relatively low frequency of recombination in RNA viruses has limited studies to cell culture or in vitro systems, which do not reflect the complexities and selective pressures present in an infected organism. Using two murine norovirus (MNV) strains to model coinfection, we developed a microfluidic platform to amplify, detect, and recover individual recombinants following in vitro and in vivo coinfection. One-step reverse transcriptase PCR (RT-PCR) was performed in picoliter drops with primers that identified the wild-type and recombinant progenies and scanned for recombination breakpoints at ∼1-kb intervals. We detected recombination between MNV strains at multiple loci spanning the viral protease, RdRP, and capsid ORFs and isolated individual recombinant RNA genomes that were present at a frequency of 1/300,000 or higher. This study is the first to examine norovirus recombination following coinfection of an animal and suggests that the exchange of RNA among viral genomes in an infected host occurs in multiple locations and is an important driver of genetic diversity. IMPORTANCE RNA viruses increase diversity and escape host immune barriers by genomic recombination. Studies using a number of viral systems indicate that recombination occurs via template switching by the virus-encoded RNA-dependent RNA polymerase (RdRP). However, factors that govern the frequency and positions of recombination in an infected organism remain largely unknown. This work leverages advances in the applied physics of drop-based microfluidics to isolate and sequence rare recombinants arising from the coinfection of mice with two distinct strains of murine norovirus. This study is the first to detect and analyze norovirus recombination in an animal model.

Artifact-Free Quantification and Sequencing of Rare Recombinant Viruses by Using Drop-Based Microfluidics.

Recombination is an important driver in the evolution of viruses and thus is key to understanding viral epidemics and improving strategies to prevent future outbreaks. Characterization of rare recombinant subpopulations remains technically challenging because of artifacts such as artificial recombinants, known as chimeras, and amplification bias. To overcome this, we have developed a high‐throughput microfluidic technique with a second verification step in order to amplify and sequence single recombinant viruses with high fidelity in picoliter drops. We obtained the first artifact‐free estimate of in vitro recombination rate between murine norovirus strains MNV‐1 and WU20 co‐infecting a cell (Prec=3.3×10−4±2×10−5) for a 1205 nt region. Our approach represents a time‐ and cost‐effective improvement over current methods, and can be adapted for genomic studies requiring artifact‐ and bias‐free selective amplification, such as microbial pathogens, or rare cancer cells.

A high-throughput drop microfluidic system for virus culture and analysis

High mutation rates and short replication times lead to rapid evolution in RNA viruses. New tools for high-throughput culture and analysis of viral phenotypes will enable more effective studies of viral evolutionary processes. A water-in-oil drop microfluidic system to study virus-cell interactions at the single event level on a massively parallel scale is described here. Murine norovirus (MNV-1) particles were co-encapsulated with individual RAW 264.7 cells in 65 pL aqueous drops formed by flow focusing in 50 μm microchannels. At low multiplicity of infection (MOI), viral titers increased greatly, reaching a maximum 18 h post-encapsulation. This system was employed to evaluate MNV-1 escape from a neutralizing monoclonal antibody (clone A6.2). Further, the system was validated as a means for testing escape from antibody neutralization using a series of viral point mutants. Finally, the replicative capacity of single viral particles in drops under antibody stress was tested. Under standard conditions, many RNA virus stocks harbor minority populations of genotypic and phenotypic variants, resulting in quasispecies. These data show that when single cells are encapsulated with single viral particles under antibody stress without competition from other virions, the number of resulting infectious particles is nearly equivalent to the number of viral genomes present. These findings suggest that lower fitness virions can infect cells successfully and replicate, indicating that the microfluidics system may serve as an effective tool for isolating mutants that escape evolutionary stressors.

Solving the Orientation Specific Constraints in Transcranial Magnetic Stimulation by Rotating Fields

Transcranial Magnetic Stimulation (TMS) is a promising technology for both neurology and psychiatry. Positive treatment outcome has been reported, for instance in double blind, multi-center studies on depression. Nonetheless, the application of TMS towards studying and treating brain disorders is still limited by inter-subject variability and lack of model systems accessible to TMS. The latter are required to obtain a deeper understanding of the biophysical foundations of TMS so that the stimulus protocol can be optimized for maximal brain response, while inter-subject variability hinders precise and reliable delivery of stimuli across subjects. Recent studies showed that both of these limitations are in part due to the angular sensitivity of TMS. Thus, a technique that would eradicate the need for precise angular orientation of the coil would improve both the inter-subject reliability of TMS and its effectiveness in model systems. We show here how rotation of the stimulating field relieves the angular sensitivity of TMS and provides improvements in both issues. Field rotation is attained by superposing the fields of two coils positioned orthogonal to each other and operated with a relative phase shift in time. Rotating field TMS (rfTMS) efficiently stimulates both cultured hippocampal networks and rat motor cortex, two neuronal systems that are notoriously difficult to excite magnetically. This opens the possibility of pharmacological and invasive TMS experiments in these model systems. Application of rfTMS to human subjects overcomes the orientation dependence of standard TMS. Thus, rfTMS yields optimal targeting of brain regions where correct orientation cannot be determined (e.g., via motor feedback) and will enable stimulation in brain regions where a preferred axonal orientation does not exist.

Whole‐Genome Sequencing of a Single Viral Species from a Highly Heterogeneous Sample

Metagenomic studies suggest that only a small fraction of the viruses that exist in nature have been identified and studied. Characterization of unknown viral genomes is hindered by the many genomes populating any virus sample. A new method is reported that integrates drop-based microfluidics and computational analysis to enable the purification of any single viral species from a complex mixed virus sample and the retrieval of complete genome sequences. By using this platform, the genome sequence of a 5243 bp dsDNA virus that was spiked into wastewater was retrieved with greater than 96% sequence coverage and more than 99.8% sequence identity. This method holds great potential for virus discovery since it allows enrichment and sequencing of previously undescribed viruses as well as known viruses.