Coupled single-cell CRISPR screening and epigenomic profiling reveals causal gene regulatory networks, bioRxiv, 2018-09-17
SummaryHere we present Perturb-ATAC, a method which combines multiplexed CRISPR interference or knockout with genome-wide chromatin accessibility profiling in single cells, based on the simultaneous detection of CRISPR guide RNAs and open chromatin sites by assay of transposase-accessible chromatin with sequencing (ATAC-seq). We applied Perturb-ATAC to transcription factors (TFs), chromatin-modifying factors, and noncoding RNAs (ncRNAs) in ∼4,300 single cells, encompassing more than 63 unique genotype-phenotype relationships. Perturb-ATAC in human B lymphocytes uncovered regulators of chromatin accessibility, TF occupancy, and nucleosome positioning, and identified a hierarchical organization of TFs that govern B cell state, variation, and disease-associatedcis-regulatory elements. Perturb-ATAC in primary human epidermal cells revealed three sequential modules ofcis-elements that specify keratinocyte fate, orchestrated by the TFs JUNB, KLF4, ZNF750, CEBPA, and EHF. Combinatorial deletion of all pairs of these TFs uncovered their epistatic relationships and highlighted genomic co-localization as a basis for synergistic interactions. Thus, Perturb-ATAC is a powerful and general strategy to dissect gene regulatory networks in development and disease.Highlights<jatslist list-type=order><jatslist-item>A new method for simultaneous measurement of CRISPR perturbations and chromatin state in single cells.<jatslist-item><jatslist-item>Perturb-ATAC reveals regulatory factors that controlcis-element accessibility,trans-factor occupancy, and nucleosome positioning.<jatslist-item><jatslist-item>Perturb-ATAC reveals regulatory modules of coordinatedtrans-factor activity in B lymphoblasts.<jatslist-item><jatslist-item>Keratinocyte differentiation is orchestrated by synergistic activities of co-binding TFs oncis-elements.<jatslist-item>
biorxiv genomics 100-200-users 2018Optimization of T-DNA architecture for Cas9-mediated mutagenesis in Arabidopsis, bioRxiv, 2018-09-17
ABSTRACTBacterial CRISPR systems have been widely adopted to create operator-specified site-specific nucleases. Such nuclease action commonly results in loss-of-function alleles, facilitating functional analysis of genes and gene families We conducted a systematic comparison of components and T-DNA architectures for CRISPR-mediated gene editing in Arabidopsis, testing multiple promoters, terminators, sgRNA backbones and Cas9 alleles. We identified a T-DNA architecture that usually results in stable (i.e. homozygous) mutations in the first generation after transformation. Notably, the transcription of sgRNA and Cas9 in head-to-head divergent orientation usually resulted in highly active lines. Our Arabidopsis data may prove useful for optimization of CRISPR methods in other plants.
biorxiv plant-biology 100-200-users 2018A guide to performing Polygenic Risk Score analyses, bioRxiv, 2018-09-16
The application of polygenic risk scores (PRS) has become routine across genetic research. Among a range of applications, PRS are exploited to assess shared aetiology between phenotypes, to evaluate the predictive power of genetic data for use in clinical settings, and as part of experimental studies in which, for example, experiments are performed on individuals, or their biological samples (eg. tissues, cells), at the tails of the PRS distribution and contrasted. As GWAS sample sizes increase and PRS become more powerful, they are set to play a key role in personalised medicine. However, despite the growing application and importance of PRS, there are limited guidelines for performing PRS analyses, which can lead to inconsistency between studies and misinterpretation of results. Here we provide detailed guidelines for performing polygenic risk score analyses relevant to different methods for their calculation, outlining standard quality control steps and offering recommendations for best-practice. We also discuss different methods for the calculation of PRS, common misconceptions regarding the interpretation of results and future challenges.
biorxiv genomics 100-200-users 2018Evaluating the evidence for biotypes of depression attempted replication of Drysdale et.al. 2017, bioRxiv, 2018-09-16
AbstractBackgroundPsychiatric disorders are highly heterogeneous, defined based on symptoms with little connection to potential underlying biological mechanisms. A possible approach to dissect biological heterogeneity is to look for biologically meaningful subtypes. A recent study Drysdale et al. (2017) showed promising results along this line by simultaneously using resting state fMRI and clinical data and identified four distinct subtypes of depression with different clinical profiles and abnormal resting state fMRI connectivity. These subtypes were predictive of treatment response to transcranial magnetic stimulation therapy.ObjectiveHere, we attempted to replicate the procedure followed in the Drysdale et al. study and their findings in an independent dataset of a clinically more heterogeneous sample of 187 participants with depression and anxiety. We aimed to answer the following questions 1) Using the same procedure, can we find a statistically significant and reliable relationship between brain connectivity and clinical symptoms? 2) Is the observed relationship similar to the one found in the original study? 3) Can we identify distinct and reliable subtypes? 4) Do they have similar clinical profiles as the subtypes identified in the original study?MethodsWe followed the original procedure as closely as possible, including a canonical correlation analysis to find a low dimensional representation of clinically relevant resting state fMRI features, followed by hierarchical clustering to identify subtypes. We extended the original procedure using additional statistical tests, to test the statistical significance of the relationship between resting state fMRI and clinical data, and the existence of distinct subtypes. Furthermore, we examined the stability of the whole procedure using resampling.Results and ConclusionWe were not able to replicate the findings of the original study. Relationships between brain connectivity and clinical symptoms were not statistically significant and we also did not find clearly distinct subtypes of depression. We argue, that based on our rigorous approach and in-depth review of the original results, that the evidence for the existence of the distinct resting state connectivity based subtypes of depression is weak and should be interpreted with caution.
biorxiv neuroscience 100-200-users 2018Dating genomic variants and shared ancestry in population-scale sequencing data, bioRxiv, 2018-09-14
AbstractThe origin and fate of new mutations within species is the fundamental process underlying evolution. However, while previous efforts have been focused on characterizing the presence, frequency, and phenotypic impact of genetic variation, the evolutionary histories of most variants are largely unexplored. We have developed a non-parametric approach for estimating the date of origin of genetic variants that can be applied to large-scale genomic variation data sets. We demonstrate the accuracy and robustness of the approach through simulation and apply it to over 16 million single nucleotide poly-morphisms (SNPs) from two publicly available human genomic diversity resources. We characterize the differential relationship between variant frequency and age in different geographical regions and demonstrate the value of allele age in interpreting variants of known functional and selective importance. Finally, we use allele age estimates to power a rapid approach for inferring the genealogical history of a single genome or a group of individuals.
biorxiv genomics 100-200-users 2018Novel childhood experience suggests eccentricity drives organization of human visual cortex, bioRxiv, 2018-09-13
The functional organization of human high-level visual cortex, such as face and place-selective regions, is strikingly consistent across individuals. A fundamental, unanswered question in neuroscience is what dimensions of visual information constrain the development and topography of this shared brain organization? To answer this question, we scanned with fMRI a unique group of adults who, as children, engaged in extensive experience with a novel stimulus, Pokemon, that varied along critical dimensions (foveal bias, rectilinearity, size, animacy) from other ecological categories such as faces and places. We find that experienced adults not only demonstrate distinct and consistent distributed cortical responses to Pokemon, but their activations suggest that it is the experienced retinal eccentricity during childhood that predicts the locus of distributed responses to Pokemon in adulthood. These data advance our understanding about how childhood experience and functional constraints shape the functional organization of the human brain.
biorxiv neuroscience 100-200-users 2018