A Major Role for Common Genetic Variation in Anxiety Disorders, bioRxiv, 2017-10-17
AbstractAnxiety disorders are common, complex psychiatric disorders with twin heritabilities of 30-60%. We conducted a genome-wide association study of Lifetime Anxiety Disorder (n = 83 565) and an additional Current Anxiety Symptoms (n= 77 125) analysis. The liability scale common variant heritability estimate for Lifetime Anxiety Disorder was 26%, and for Current Anxiety Symptoms was 31%. Five novel genome-wide significant loci were identified including an intergenic region on chromosome 9 that has previously been associated with neuroticism, and a locus overlapping the BDNF receptor gene, NTRK2. Anxiety showed significant genetic correlations with depression and insomnia as well as coronary artery disease, mirroring findings from epidemiological studies. We conclude that common genetic variation accounts for a substantive proportion of the genetic architecture underlying anxiety.
biorxiv genetics 100-200-users 2017Amplification-free, CRISPR-Cas9 Targeted Enrichment and SMRT Sequencing of Repeat-Expansion Disease Causative Genomic Regions, bioRxiv, 2017-10-17
AbstractTargeted sequencing has proven to be an economical means of obtaining sequence information for one or more defined regions of a larger genome. However, most target enrichment methods require amplification. Some genomic regions, such as those with extreme GC content and repetitive sequences, are recalcitrant to faithful amplification. Yet, many human genetic disorders are caused by repeat expansions, including difficult to sequence tandem repeats.We have developed a novel, amplification-free enrichment technique that employs the CRISPR-Cas9 system for specific targeting multiple genomic loci. This method, in conjunction with long reads generated through Single Molecule, Real-Time (SMRT) sequencing and unbiased coverage, enables enrichment and sequencing of complex genomic regions that cannot be investigated with other technologies. Using human genomic DNA samples, we demonstrate successful targeting of causative loci for Huntington’s disease (HTT; CAG repeat), Fragile X syndrome (FMR1; CGG repeat), amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (C9orf72; GGGGCC repeat), and spinocerebellar ataxia type 10 (SCA10) (ATXN10; variable ATTCT repeat). The method, amenable to multiplexing across multiple genomic loci, uses an amplification-free approach that facilitates the isolation of hundreds of individual on-target molecules in a single SMRT Cell and accurate sequencing through long repeat stretches, regardless of extreme GC percent or sequence complexity content. Our novel targeted sequencing method opens new doors to genomic analyses independent of PCR amplification that will facilitate the study of repeat expansion disorders.
biorxiv genomics 0-100-users 2017Cluster Headache Comparing Clustering Tools for 10X Single Cell Sequencing Data, bioRxiv, 2017-10-17
AbstractThe commercially available 10X Genomics protocol to generate droplet-based single cell RNA-seq (scRNA-seq) data is enjoying growing popularity among researchers. Fundamental to the analysis of such scRNA-seq data is the ability to cluster similar or same cells into non-overlapping groups. Many competing methods have been proposed for this task, but there is currently little guidance with regards to which method offers most accuracy. Answering this question is complicated by the fact that 10X Genomics data lack cell labels that would allow a direct performance evaluation. Thus in this review, we focused on comparing clustering solutions of a dozen methods for three datasets on human peripheral mononuclear cells generated with the 10X Genomics technology. While clustering solutions appeared robust, we found that solutions produced by different methods have little in common with each other. They also failed to replicate cell type assignment generated with supervised labeling approaches. Furthermore, we demonstrate that all clustering methods tested clustered cells to a large degree according to the amount of genes coding for ribosomal protein genes in each cell.
biorxiv bioinformatics 100-200-users 2017Efficient generation of targeted large insertions in mouse embryos using 2C-HR-CRISPR, bioRxiv, 2017-10-17
Rapid and efficient generation of large fragment targeted knock-in mouse models is still a major hurdle in mouse genetics. Here we developed 2C-HR-CRISPR, a highly efficient gene editing method based on introducing CRISPR reagents into mouse embryos at the 2-cell stage, taking advantage of the likely increase in HR efficiency during the long G2 phase and open chromatin structure of the 2-cell embryo. With 2C-HR-CRISPR and a modified biotin-streptavidin approach to localize repair templates to target sites, we rapidly targeted 20 endogenous genes that are expressed in mouse blastocysts with fluorescent reporters and generated reporter mouse lines. We showcase the first live triple-color blastocyst with all three lineages differentially reported. Additionally, we demonstrated efficient double targeting, enabling rapid assessment of the auxin-inducible degradation system for probing protein function in mouse embryos. These methods open up exciting avenues for exploring cell fate decisions in the blastocyst and later stages of development. We also suggest that 2C-HR-CRISPR can be a better alternative to random transgenesis by ensuring transgene insertions at defined ‘safe harbor’ sites.
biorxiv genetics 100-200-users 2017Discrete attractor dynamics underlying selective persistent activity in frontal cortex, bioRxiv, 2017-10-16
AbstractShort-term memories link events separated in time, such as past sensation and future actions. Short-term memories are correlated with selective persistent activity, which can be maintained over seconds. In a delayed response task that requires short-term memory, neurons in mouse anterior lateral motor cortex (ALM) show persistent activity that instructs future actions. To elucidate the mechanisms underlying this persistent activity we combined intracellular and extracellular electrophysiology with optogenetic perturbations and network modeling. During the delay epoch, both membrane potential and population activity of ALM neurons funneled towards discrete endpoints related to specific movement directions. These endpoints were robust to transient shifts in ALM activity caused by optogenetic perturbations. Perturbations occasionally switched the population dynamics to the other endpoint, followed by incorrect actions. Our results are consistent with discrete attractor dynamics underlying short-term memory related to motor planning.
biorxiv neuroscience 0-100-users 2017Genomics in healthcare GA4GH looks to 2022, bioRxiv, 2017-10-16
AbstractThe Global Alliance for Genomics and Health (GA4GH), the standards-setting body in genomics for healthcare, aims to accelerate biomedical advancement globally. We describe the differences between healthcare- and research-driven genomics, discuss the implications of global, population-scale collections of human data for research, and outline mission-critical considerations in ethics, regulation, technology, data protection, and society. We present a crude model for estimating the rate of healthcare-funded genomes worldwide that accounts for the preparedness of each country for genomics, and infers a progression of cancer-related sequencing over time. We estimate that over 60 million patients will have their genome sequenced in a healthcare context by 2025. This represents a large technical challenge for healthcare systems, and a huge opportunity for research. We identify eight major practical, principled arguments to support the position that virtual cohorts of 100 million people or more would have tangible research benefits.
biorxiv genomics 100-200-users 2017