Profiling of pluripotency factors in individual stem cells and early embryos, bioRxiv, 2018-03-21
SUMMARYMajor cell fate decisions are governed by sequence-specific transcription factors (TFs) that act in small cell populations within developing embryos. To understand how TFs regulate cell fate it is important to identify their genomic binding sites in these populations. However, current methods cannot profile TFs genome-wide at or near the single cell level. Here we adapt the CUT&RUN method to profile chromatin proteins in low cell numbers, mapping TF-DNA interactions in single cells and individual pre-implantation embryos for the first time. Using this method, we demonstrate that the pluripotency TF NANOG is significantly more dependent on the SWISNF family ATPase BRG1 for association with its genomic targets in vivo than in cultured cells—a finding that could not have been made using traditional approaches. Ultra-low input CUT&RUN (uliCUT&RUN) enables interrogation of TF binding from low cell numbers, with broad applicability to rare cell populations of importance in development or disease.
biorxiv genomics 0-100-users 2018Recovering signals of ghost archaic introgression in African populations, bioRxiv, 2018-03-21
AbstractWhile introgression from Neanderthals and Denisovans has been well-documented in modern humans outside Africa, the contribution of archaic hominins to the genetic variation of present-day Africans remains poorly understood. Using 405 whole-genome sequences from four sub-Saharan African populations, we provide complementary lines of evidence for archaic introgression into these populations. Our analyses of site frequency spectra indicate that these populations derive 2-19% of their genetic ancestry from an archaic population that diverged prior to the split of Neanderthals and modern humans. Using a method that can identify segments of archaic ancestry without the need for reference archaic genomes, we built genome-wide maps of archaic ancestry in the Yoruba and the Mende populations that recover about 482 and 502 megabases of archaic sequence, respectively. Analyses of these maps reveal segments of archaic ancestry at high frequency in these populations that represent potential targets of adaptive introgression. Our results reveal the substantial contribution of archaic ancestry in shaping the gene pool of present-day African populations.One sentence summaryMultiple present-day African populations inherited genes from an unknown archaic population that diverged before modern humans and Neanderthals split.
biorxiv genomics 200-500-users 2018Exploring the Impact of Analysis Software on Task fMRI Results, bioRxiv, 2018-03-20
AbstractA wealth of analysis tools are available to fMRI researchers in order to extract patterns of task variation and, ultimately, understand cognitive function. However, this ‘methodological plurality’ comes with a drawback. While conceptually similar, two different analysis pipelines applied on the same dataset may not produce the same scientific results. Differences in methods, implementations across software packages, and even operating systems or software versions all contribute to this variability. Consequently, attention in the field has recently been directed to reproducibility and data sharing. Neuroimaging is currently experiencing a surge in initiatives to improve research practices and ensure that all conclusions inferred from an fMRI study are replicable.In this work, our goal is to understand how choice of software package impacts on analysis results. We use publically shared data from three published task fMRI neuroimaging studies, reanalyzing each study using the three main neuroimaging software packages, AFNI, FSL and SPM, using parametric and nonparametric inference. We obtain all information on how to process, analyze, and model each dataset from the publications. We make quantitative and qualitative comparisons between our replications to gauge the scale of variability in our results and assess the fundamental differences between each software package. While qualitatively we find broad similarities between packages, we also discover marked differences, such as Dice similarity coefficients ranging from 0.000 - 0.743 in comparisons of thresholded statistic maps between software. We discuss the challenges involved in trying to reanalyse the published studies, and highlight our own efforts to make this research reproducible.
biorxiv neuroscience 200-500-users 2018Third-generation in situ hybridization chain reaction multiplexed, quantitative, sensitive, versatile, robust, bioRxiv, 2018-03-20
ABSTRACTIn situ hybridization based on the mechanism of hybridization chain reaction (HCR) has addressed multi-decade challenges to imaging mRNA expression in diverse organisms, offering a unique combination of multiplexing, quantitation, sensitivity, resolution, and versatility. Here, with third-generation in situ HCR, we augment these capabilities using probes and amplifiers that combine to provide automatic background suppression throughout the protocol, ensuring that even if reagents bind non-specifically within the sample they will not generate amplified background. Automatic background suppression dramatically enhances performance and robustness, combining the benefits of higher signal-to-background with the convenience of using unoptimized probe sets for new targets and organisms. In situ HCR v3.0 enables multiplexed quantitative mRNA imaging with subcellular resolution in the anatomical context of whole-mount vertebrate embryos, multiplexed quantitative mRNA flow cytometry for high-throughput single-cell expression profiling, and multiplexed quantitative single-molecule mRNA imaging in thick autofluorescent samples.SUMMARYIn situ hybridization chain reaction (HCR) v3.0 exploits automatic background suppression to enable multiplexed quantitative mRNA imaging and flow cytometry with dramatically enhanced ease-of-use and performance.
biorxiv developmental-biology 0-100-users 2018All-optical electrophysiology reveals brain-state dependent changes in hippocampal subthreshold dynamics and excitability, bioRxiv, 2018-03-14
AbstractA technology to record membrane potential from multiple neurons, simultaneously, in behaving animals will have a transformative impact on neuroscience research1. Parallel recordings could reveal the subthreshold potentials and intercellular correlations that underlie network behavior2. Paired stimulation and recording can further reveal the input-output properties of individual cells or networks in the context of different brain states3. Genetically encoded voltage indicators are a promising tool for these purposes, but were so far limited to single-cell recordings with marginal signal to noise ratio (SNR) in vivo4-6. We developed improved near infrared voltage indicators, high speed microscopes and targeted gene expression schemes which enabled recordings of supra- and subthreshold voltage dynamics from multiple neurons simultaneously in mouse hippocampus, in vivo. The reporters revealed sub-cellular details of back-propagating action potentials, correlations in sub-threshold voltage between multiple cells, and changes in dynamics associated with transitions from resting to locomotion. In combination with optogenetic stimulation, the reporters revealed brain state-dependent changes in neuronal excitability, reflecting the interplay of excitatory and inhibitory synaptic inputs. These tools open the possibility for detailed explorations of network dynamics in the context of behavior.
biorxiv neuroscience 100-200-users 2018Widespread transcriptional scanning in the testis modulates gene evolution rates, bioRxiv, 2018-03-14
The testis expresses the largest number of genes of any mammalian organ, a finding that has long puzzled molecular biologists. Analyzing our single-cell transcriptomic maps of human and mouse spermatogenesis, we provide evidence that this widespread transcription serves to maintain DNA sequence integrity in the male germline by correcting DNA damage through 'transcriptional scanning'. Supporting this model, we find that genes expressed during spermatogenesis display lower mutation rates on the transcribed strand and have low diversity in the population. Moreover, this effect is fine-tuned by the level of gene expression during spermatogenesis. The unexpressed genes, which in our model do not benefit from transcriptional scanning, diverge faster over evolutionary time-scales and are enriched for sensory and immune-defense functions. Collectively, we propose that transcriptional scanning modulates germline mutation rates in a gene-specific manner, maintaining DNA sequence integrity for the bulk of genes but allowing for fast evolution in a specific subset.
biorxiv evolutionary-biology 200-500-users 2018