Achieving better than 3 Å resolution by single particle cryo-EM at 200 keV, bioRxiv, 2017-05-26

AbstractTechnical and methodological advances in single-particle cryo-electron microscopy (cryo-EM) have expanded the technique into a resolution regime that was previously only attainable by X-ray crystallography. Although single-particle cryo-EM has proven to be a useful technique for determining the structures of biomedically relevant molecules at near-atomic resolution, nearly 98% of the structures resolved to better than 4 Å resolution have been determined using 300 keV transmission electron microscopes (TEMs). We demonstrate that it is possible to obtain cryo-EM reconstructions of macromolecular complexes at a range of sizes to better than 3 Å resolution using a 200 keV TEM. These structures are of sufficient quality to unambiguously assign amino acid rotameric conformations and identify ordered water molecules, features previously thought only to be resolvable using TEMs operating at 300 keV.

biorxiv biophysics 100-200-users 2017

scImpute Accurate And Robust Imputation For Single Cell RNA-Seq Data, bioRxiv, 2017-05-25

The emerging single cell RNA sequencing (scRNA-seq) technologies enable the investigation of transcriptomic landscapes at single-cell resolution. The analysis of scRNA-seq data is complicated by excess zero or near zero counts, the so-called dropouts due to the low amounts of mRNA sequenced within individual cells. Downstream analysis of scRNA-seq would be severely biased if the dropout events are not properly corrected. We introduce scImpute, a statistical method to accurately and robustly impute the dropout values in scRNA-seq data. ScImpute automatically identifies gene expression values affected by dropout events, and only perform imputation on these values without introducing new bias to the rest data. ScImpute also detects outlier or rare cells and excludes them from imputation. Evaluation based on both simulated and real scRNA-seq data on mouse embryos, mouse brain cells, human blood cells, and human embryonic stem cells suggests that scImpute is an effective tool to recover transcriptome dynamics masked by dropout events. scImpute is shown to correct false zero counts, enhance the clustering of cell populations and subpopulations, improve the accuracy of differential expression analysis, and aid the study of gene expression dynamics.

biorxiv bioinformatics 0-100-users 2017

A Complete Electron Microscopy Volume Of The Brain Of Adult Drosophila melanogaster, bioRxiv, 2017-05-23

Drosophila melanogaster has a rich repertoire of innate and learned behaviors. Its 100,000-neuron brain is a large but tractable target for comprehensive neural circuit mapping. Only electron microscopy (EM) enables complete, unbiased mapping of synaptic connectivity; however, the fly brain is too large for conventional EM. We developed a custom high-throughput EM platform and imaged the entire brain of an adult female fly. We validated the dataset by tracing brain-spanning circuitry involving the mushroom body (MB), intensively studied for its role in learning. Here we describe the complete set of olfactory inputs to the MB; find a new cell type providing driving input to Kenyon cells (the intrinsic MB neurons); identify neurons postsynaptic to Kenyon cell dendrites; and find that axonal arbors providing input to the MB calyx are more tightly clustered than previously indicated by light-level data. This freely available EM dataset will significantly accelerate Drosophila neuroscience.

biorxiv neuroscience 200-500-users 2017

Single-cell RNA-seq of rheumatoid arthritis synovial tissue using low cost microfluidic instrumentation, bioRxiv, 2017-05-23

AbstractDroplet-based single cell RNA-seq has emerged as a powerful technique for massively parallel cellular profiling. While these approaches offer the exciting promise to deconvolute cellular heterogeneity in diseased tissues, the lack of cost-effective, reliable, and user-friendly instrumentation has hindered widespread adoption of droplet microfluidic techniques. To address this, we have developed a microfluidic control instrument that can be easily assembled from 3D printed parts and commercially available components costing approximately $540. We adapted this instrument for massively parallel scRNA-seq and deployed it in a clinical environment to perform single cell transcriptome profiling of disaggregated synovial tissue from a rheumatoid arthritis patient. We sequenced 8,716 single cells from a synovectomy, revealing 16 transcriptomically distinct clusters. These encompass a comprehensive and unbiased characterization of the autoimmune infiltrate, including inflammatory T and NK subsets that contribute to disease biology. Additionally, we identified fibroblast subpopulations that are demarcated via THY1 (CD90) and CD55 expression. Further experiments confirm that these represent synovial fibroblasts residing within the synovial intimal lining and subintimal lining, respectively, each under the influence of differing microenvironments. We envision that this instrument will have broad utility in basic and clinical settings, enabling low-cost and routine application of microfluidic techniques, and in particular single-cell transcriptome profiling.

biorxiv genomics 100-200-users 2017

A comparison between single cell RNA sequencing and single molecule RNA FISH for rare cell analysis, bioRxiv, 2017-05-19

AbstractThe development of single cell RNA sequencing technologies has emerged as a powerful means of profiling the transcriptional behavior of single cells, leveraging the breadth of sequencing measurements to make inferences about cell type. However, there is still little understanding of how well these methods perform at measuring single cell variability for small sets of genes and what “transcriptome coverage” (e.g. genes detected per cell) is needed for accurate measurements. Here, we use single molecule RNA FISH measurements of 26 genes in thousands of melanoma cells to provide an independent reference dataset to assess the performance of the DropSeq and Fluidigm single cell RNA sequencing platforms. We quantified the Gini coefficient, a measure of rare-cell expression variability, and find that the correspondence between RNA FISH and single cell RNA sequencing for Gini, unlike for mean, increases markedly with per-cell library complexity up to a threshold of ∼2000 genes detected. A similar complexity threshold also allows for robust assignment of multi-genic cell states such as cell cycle phase. Our results provide guidelines for selecting sequencing depth and complexity thresholds for single cell RNA sequencing. More generally, our results suggest that if the number of genes whose expression levels are required to answer any given biological question is small, then greater transcriptome complexity per cell is likely more important than obtaining very large numbers of cells.

biorxiv genomics 0-100-users 2017

Cohesin loss eliminates all loop domains, leading to links among superenhancers and downregulation of nearby genes, bioRxiv, 2017-05-19

SUMMARYThe human genome folds to create thousands of intervals, called “contact domains,” that exhibit enhanced contact frequency within themselves. “Loop domains” form because of tethering between two loci - almost always bound by CTCF and cohesin – lying on the same chromosome. “Compartment domains” form when genomic intervals with similar histone marks co-segregate. Here, we explore the effects of degrading cohesin. All loop domains are eliminated, but neither compartment domains nor histone marks are affected. Loci in different compartments that had been in the same loop domain become more segregated. Loss of loop domains does not lead to widespread ectopic gene activation, but does affect a significant minority of active genes. In particular, cohesin loss causes superenhancers to co-localize, forming hundreds of links within and across chromosomes, and affecting the regulation of nearby genes. Cohesin restoration quickly reverses these effects, consistent with a model where loop extrusion is rapid.

biorxiv genomics 0-100-users 2017