SoupX removes ambient RNA contamination from droplet based single cell RNA sequencing data, bioRxiv, 2018-04-21
Droplet based single cell RNA sequence analyses assume all acquired RNAs are endogenous to cells. However, any cell free RNAs contained within the input solution are also captured by these assays. This sequencing of cell free RNA constitutes a background contamination that has the potential to confound the correct biological interpretation of single cell transcriptomic data. Here, we demonstrate that contamination from this soup of cell free RNAs is ubiquitous, experiment specific in its composition and magnitude, and can lead to erroneous biological conclusions. We present a method, SoupX, for quantifying the extent of the contamination and estimating background corrected, cell expression profiles that can be integrated with existing downstream analysis tools. We apply this method to two data-sets and show that the application of this method reduces batch effects, strengthens cell-specific quality control and improves biological interpretation.
biorxiv bioinformatics 100-200-users 2018Sub-2 Å Ewald Curvature Corrected Single-Particle Cryo-EM, bioRxiv, 2018-04-21
AbstractSingle-particle cryogenic electron microscopy (cryo-EM) provides a powerful methodology for structural biologists, but the resolutions typically attained with experimentally determined structures have lagged behind microscope capabilities. Here, we have exploited several technical solutions to improve resolution, including sub-Angstrom pixelation, per-particle CTF refinement, and most notably a correction for Ewald sphere curvature. The application of these methods on micrographs recorded on a base model Titan Krios enabled structure determination at ∼1.86-Å resolution of an adeno-associated virus serotype 2 variant (AAV2), an important gene-delivery vehicle.
biorxiv biophysics 100-200-users 2018Direct RNA Sequencing of the Complete Influenza A Virus Genome, bioRxiv, 2018-04-12
ABSTRACTFor the first time, a complete genome of an RNA virus has been sequenced in its original form. Previously, RNA was sequenced by the chemical degradation of radiolabelled RNA, a difficult method that produced only short sequences. Instead, RNA has usually been sequenced indirectly by copying it into cDNA, which is often amplified to dsDNA by PCR and subsequently analyzed using a variety of DNA sequencing methods. We designed an adapter to short highly conserved termini of the influenza virus genome to target the (-) sense RNA into a protein nanopore on the Oxford Nanopore MinION sequencing platform. Utilizing this method and total RNA extracted from the allantoic fluid of infected chicken eggs, we demonstrate successful sequencing of the complete influenza virus genome with 100% nucleotide coverage, 99% consensus identity, and 99% of reads mapped to influenza. By utilizing the same methodology we can redesign the adapter in order to expand the targets to include viral mRNA and (+) sense cRNA, which are essential to the viral life cycle. This has the potential to identify and quantify splice variants and base modifications, which are not practically measurable with current methods.
biorxiv genomics 100-200-users 2018Evaluation of UMAP as an alternative to t-SNE for single-cell data, bioRxiv, 2018-04-10
AbstractUniform Manifold Approximation and Projection (UMAP) is a recently-published non-linear dimensionality reduction technique. Another such algorithm, t-SNE, has been the default method for such task in the past years. Herein we comment on the usefulness of UMAP high-dimensional cytometry and single-cell RNA sequencing, notably highlighting faster runtime and consistency, meaningful organization of cell clusters and preservation of continuums in UMAP compared to t-SNE.
biorxiv bioinformatics 100-200-users 2018High-throughput mapping of long-range neuronal projection using in situ sequencing, bioRxiv, 2018-04-04
SummaryUnderstanding neural circuits requires deciphering interactions among myriad cell types defined by spatial organization, connectivity, gene expression, and other properties. Resolving these cell types requires both single neuron resolution and high throughput, a challenging combination with conventional methods. Here we introduce BARseq, a multiplexed method based on RNA barcoding for mapping projections of thousands of spatially resolved neurons in a single brain, and relating those projections to other properties such as gene or Cre expression. Mapping the projections to 11 areas of 3579 neurons in mouse auditory cortex using BARseq confirmed the laminar organization of the three top classes (IT, PT-like and CT) of projection neurons. In depth analysis uncovered a novel projection type restricted almost exclusively to transcriptionally-defined subtypes of IT neurons. By bridging anatomical and transcriptomic approaches at cellular resolution with high throughput, BARseq can potentially uncover the organizing principles underlying the structure and formation of neural circuits.
biorxiv neuroscience 100-200-users 2018Label-free prediction of three-dimensional fluorescence images from transmitted light microscopy, bioRxiv, 2018-03-27
Understanding living cells as integrated systems, a challenge central to modern biology, is complicated by limitations of available imaging methods. While fluorescence microscopy can resolve subcellular structure in living cells, it is expensive, slow, and damaging to cells. Here, we present a label-free method for predicting 3D fluorescence directly from transmitted light images and demonstrate that it can be used to generate multi-structure, integrated images.
biorxiv cell-biology 100-200-users 2018