Automated analysis of whole brain vasculature using machine learning, bioRxiv, 2019-04-19
SUMMARYTissue clearing methods enable imaging of intact biological specimens without sectioning. However, reliable and scalable analysis of such large imaging data in 3D remains a challenge. Towards this goal, we developed a deep learning-based framework to quantify and analyze the brain vasculature, named Vessel Segmentation & Analysis Pipeline (VesSAP). Our pipeline uses a fully convolutional network with a transfer learning approach for segmentation. We systematically analyzed vascular features of the whole brains including their length, bifurcation points and radius at the micrometer scale by registering them to the Allen mouse brain atlas. We reported the first evidence of secondary intracranial collateral vascularization in CD1-Elite mice and found reduced vascularization in the brainstem as compared to the cerebrum. VesSAP thus enables unbiased and scalable quantifications for the angioarchitecture of the cleared intact mouse brain and yields new biological insights related to the vascular brain function.GRAPHICAL ABSTRACT<jatsfig id=ufig1 position=float fig-type=figure orientation=portrait>Supporting material of VesSAP is available at <jatsext-link xmlnsxlink=httpwww.w3.org1999xlink ext-link-type=uri xlinkhref=httpDISCOtechnologies.orgVesSAP>httpDISCOtechnologies.orgVesSAP<jatsext-link><jatsgraphic xmlnsxlink=httpwww.w3.org1999xlink xlinkhref=613257_ufig1 position=float orientation=portrait >
biorxiv neuroscience 200-500-users 2019Shared morphological consequences of global warming in North American migratory birds, bioRxiv, 2019-04-19
ABSTRACTIncreasing temperatures associated with climate change are predicted to cause reductions in body size, a key determinant of animal physiology and ecology. Using a four-decade specimen series of 70,716 individuals of 52 North American migratory bird species, we demonstrate that increasing annual summer temperature over the 40-year period drove consistent reductions in body size across these diverse taxa. Concurrently, wing length – which impacts nearly all aspects of avian ecology and behavior – has consistently increased across taxa. Our findings suggest that warming-induced body size reduction is a general response to climate change, and reveal a similarly consistent shift in an ecologically-important dimension of body shape. We hypothesize that increasing wing length represents a compensatory adaptation to maintain migration as reductions in body size have increased the metabolic cost of flight. An improved understanding of warming-induced morphological changes, and their limits, are important for predicting biotic responses to global change.
biorxiv ecology 200-500-users 2019Droplet-based combinatorial indexing for massive scale single-cell epigenomics, bioRxiv, 2019-04-18
AbstractWhile recent technical advancements have facilitated the mapping of epigenomes at single-cell resolution, the throughput and quality of these methods have limited the widespread adoption of these technologies. Here, we describe a droplet microfluidics platform for single-cell assay for transposase accessible chromatin (scATAC-seq) for high-throughput single-cell profiling of chromatin accessibility. We use this approach for the unbiased discovery of cell types and regulatory elements within the mouse brain. Further, we extend the throughput of this approach by pairing combinatorial indexing with droplet microfluidics, enabling single-cell studies at a massive scale. With this approach, we measure chromatin accessibility across resting and stimulated human bone marrow derived cells to reveal changes in the cis- and trans- regulatory landscape across cell types and upon stimulation conditions at single-cell resolution. Altogether, we describe a total of 502,207 single-cell profiles, demonstrating the scalability and flexibility of this droplet-based platform.
biorxiv genomics 200-500-users 2019Massively parallel single-cell chromatin landscapes of human immune cell development and intratumoral T cell exhaustion, bioRxiv, 2019-04-18
AbstractUnderstanding complex tissues requires single-cell deconstruction of gene regulation with precision and scale. Here we present a massively parallel droplet-based platform for mapping transposase-accessible chromatin in tens of thousands of single cells per sample (scATAC-seq). We obtain and analyze chromatin profiles of over 200,000 single cells in two primary human systems. In blood, scATAC-seq allows marker-free identification of cell type-specific cis- and trans-regulatory elements, mapping of disease-associated enhancer activity, and reconstruction of trajectories of differentiation from progenitors to diverse and rare immune cell types. In basal cell carcinoma, scATAC-seq reveals regulatory landscapes of malignant, stromal, and immune cell types in the tumor microenvironment. Moreover, scATAC-seq of serial tumor biopsies before and after PD-1 blockade allows identification of chromatin regulators and differentiation trajectories of therapy-responsive intratumoral T cell subsets, revealing a shared regulatory program driving CD8+ T cell exhaustion and CD4+ T follicular helper cell development. We anticipate that droplet-based single-cell chromatin accessibility will provide a broadly applicable means of identifying regulatory factors and elements that underlie cell type and function.
biorxiv genomics 200-500-users 2019Developmentally regulated Shh expression is robust to TAD perturbations, bioRxiv, 2019-04-16
AbstractTopologically Associating Domains (TADs) have been proposed to both guide and constrain enhancer activity. Shh is located within a TAD known to contain all its enhancers. To investigate the importance of chromatin conformation and TAD integrity on developmental gene regulation, we have manipulated the Shh TAD – creating internal deletions, deleting CTCF sites including those at TAD boundaries, as well as larger deletions and inversions of TAD boundaries. Chromosome conformation capture and fluorescence in situ hybridisation assays were used the investigate changes in chromatin conformation that result from these manipulations. Our data suggest that the substantial alteration of TAD structure has no readily detectable effect on Shh expression patterns during development – except where enhancers are deleted - and results in no detectable phenotypes. Only in the case of a larger deletion of one TAD boundary could some ectopic influence of the Shh limb enhancer be detected on a gene - Mnx1 in the neighbouring TAD. Our data suggests that, contrary to expectations, the developmental regulation of Shh expression is remarkably robust to TAD perturbations.
biorxiv developmental-biology 200-500-users 2019Targeted Nanopore Sequencing with Cas9 for studies of methylation, structural variants, and mutations, bioRxiv, 2019-04-12
AbstractNanopore sequencing technology can rapidly and directly interrogate native DNA molecules. Often we are interested only in interrogating specific areas at high depth, but conventional enrichment methods have thus far proved unsuitable for long reads1. Existing strategies are currently limited by high input DNA requirements, low yield, short (<5kb) reads, time-intensive protocols, andor amplification or cloning (losing base modification information). In this paper, we describe a technique utilizing the ability of Cas9 to introduce cuts at specific locations and ligating nanopore sequencing adaptors directly to those sites, a method we term ‘nanopore Cas9 Targeted-Sequencing’ (nCATS).We have demonstrated this using an Oxford Nanopore MinION flow cell (Capacity >10Gb+) to generate a median 165X coverage at 10 genomic loci with a median length of 18kb, representing a several hundred-fold improvement over the 2-3X coverage achieved without enrichment. We performed a pilot run on the smaller Flongle flow cell (Capacity ~1Gb), generating a median coverage of 30X at 11 genomic loci with a median length of 18kb. Using panels of guide RNAs, we show that the high coverage data from this method enables us to (1) profile DNA methylation patterns at cancer driver genes, (2) detect structural variations at known hot spots, and (3) survey for the presence of single nucleotide mutations. Together, this provides a low-cost method that can be applied even in low resource settings to directly examine cellular DNA. This technique has extensive clinical applications for assessing medically relevant genes and has the versatility to be a rapid and comprehensive diagnostic tool. We demonstrate applications of this technique by examining the well-characterized GM12878 cell line as well as three breast cell lines (MCF-10A, MCF-7, MDA-MB-231) with varying tumorigenic potential as a model for cancer.ContributionsTG and WT constructed the study. TG performed the experiments. TG, IL, and FS analyzed the data. TG, JG, ER, RB and AH and developed the method. TG and WT wrote the paper
biorxiv genomics 200-500-users 2019