Biology subjects

O'Roak, B. J.

Publications and source records attributed to O'Roak, B. J..

8 recordsLinked to original sources

bioRxiv2024

Detailed phenotyping of Tbr1-2A-CreER knock-in mice demonstrates significant impacts on TBR1 protein levels and axon development

Spatiotemporal control of Cre-mediated recombination has been an invaluable tool for understanding key developmental processes. For example, knock-in of Cre into cell type marker gene loci drives Cre expression under endogenous promoter and enhancer sequences, greatly facilitating the study of diverse neuronal subtypes in the cerebral cortex. However, insertion of exogenous DNA into the genome can have unintended effects on local gene regulation or protein function that must be carefully considered. Here, we analyze a recently generated Tbr1-2A-CreER knock-in mouse line, where a 2A-CreER cassette was inserted in-frame just before the stop codon of the transcription factor gene Tbr1. Heterozygous TBR1 mutations in humans and mice are known to cause autism or autism-like behavioral phenotypes accompanied by structural brain malformations, most frequently a reduction of the anterior commissure. Thus, it is critical for modified versions of Tbr1 to exhibit true wild-type-like activity. We evaluated the Tbr1-2A-CreER allele for its potential impact on Tbr1 function and complementation to Tbr1 loss-of-function alleles. In mice with one copy of the Tbr1-2A-CreER allele, we identified reduction of TBR1 protein in early postnatal cortex along with thinning of the anterior commissure, suggesting hypersensitivity of this structure to TBR1 dosage. Comparing Tbr1-2A-CreER and Tbr1-null heterozygous and homozygous mice to Tbr1-null complementation crosses showed reductions of TBR1 dosage ranging from 28.4% to 95.9%. Using these combinatorial genotypes, we found that low levels of TBR1 protein ([~]16%) are sufficient to establish cortical layer positioning, while greater levels (>50%) are required for normal suppression of layer 5 identity. In total, these results strongly support the conclusion that Tbr1-2A-CreER is a hypomorphic allele. We advise caution when interpreting experiments using this allele, such as transcriptomic studies, considering the sensitivity of various corticogenic processes to TBR1 dosage and the association of heterozygous TBR1 mutations with complex neurodevelopmental disorders.

developmental biology↗

bioRxiv2022

Shared and distinct functional effects of patient-specific Tbr1 mutations on cortical development

Predicted loss-of-function and missense heterozygous de novo mutations of TBR1 are strongly associated with intellectual disability and autism. The functional effects of these heterogeneous mutations on cortical development and genotype-phenotype relationships have yet to be explored. We characterized mouse models carrying patient mutations A136PfsX80 and K228E, finding convergent and discordant phenotypes. The A136PfsX80 mutation is loss-of-function and allelic to the Tbr1 knockout. In contrast, K228E causes significant upregulation of TBR1. Heterozygosity of either mutation produces axon defects, including reduction of the anterior commissure, and CTIP2 downregulation in adult cortex. While mice lacking TBR1 show extensive cortical apoptosis and inverted layering, K228E homozygotes show normal apoptosis levels and a complex layering phenotype--suggesting partial, yet abnormal, function of the allele. The construct and face validity of these Tbr1 patient mutation mice suggests they will be valuable translational models for studying the function of this essential brain transcription factor.

neuroscience↗

bioRxiv2021

High-content single-cell combinatorial indexing

Single-cell genomics assays have emerged as a dominant platform for interrogating complex biological systems. Methods to capture various properties at the single-cell level typically suffer a tradeoff between cell count and information content, which is defined by the number of unique and usable reads acquired per cell. We and others have described workflows that utilize single-cell combinatorial indexing (sci)1, leveraging transposase-based library construction2 to assess a variety of genomic properties in high throughput; however, these techniques often produce sparse coverage for the property of interest. Here, we describe a novel adaptor-switching strategy, s3, capable of producing one-to-two order-of-magnitude improvements in usable reads obtained per cell for chromatin accessibility (s3-ATAC), whole genome sequencing (s3-WGS), and whole genome plus chromatin conformation (s3-GCC), while retaining the same high-throughput capabilities of predecessor sci technologies. We apply s3 to produce high-coverage single-cell ATAC-seq profiles of mouse brain and human cortex tissue; and whole genome and chromatin contact maps for two low-passage patient-derived cell lines from a primary pancreatic tumor.

genetics↗

bioRxiv2020

Cas12a-Capture: a novel, low-cost, and scalable method for targeted sequencing

Targeted sequencing remains a valuable technique for clinical and research applications. However, many existing technologies suffer from pervasive GC sequence content bias, high input DNA requirements, and high cost for custom panels. We have developed Cas12a-Capture, a low-cost and highly scalable method for targeted sequencing. The method utilizes preprogramed guide RNAs to direct CRISPR-Cas12a cleavage of double stranded DNA in vitro and then takes advantage of the resulting four to five nucleotide overhangs for selective ligation with a custom sequencing adapter. Addition of a second sequencing adapter and enrichment for ligation products generates a targeted sequence library. We first performed a pilot experiment with 7,176 guides targeting 3.5 megabases of DNA. Using these data, we modeled the sequence determinants of Cas12a-Capture efficiency, then designed an optimized set of 11,438 guides targeting 3.0 megabases. The optimized guide set achieves an average 64-fold enrichment of targeted regions with minimal GC bias. Cas12a-Capture variant calls had strong concordance with Illumina Platinum Genome calls, especially for SNVs, which could be improved by applying basic variant quality heuristics. We believe Cas12a-Capture has a wide variety of potential clinical and research applications and is amendable for selective enrichment for any double stranded DNA template or genome.

genomics↗

bioRxiv2019

Mutually exclusive autism mutations point to the circadian clock and PI3K signaling pathways

Mutual exclusivity analysis of genomic mutations has proven useful for detecting driver alterations in cancer patient cohorts. Here we demonstrate, for the first time, that this pattern is also present among de novo mutations in autism spectrum disorder. We analyzed three large whole genome sequencing studies and identified mutual exclusivity patterns within the most confident set of autism-related genes, as well as in the circadian clock and PI3K/AKT signaling pathways.

genetics↗

bioRxiv2019

Improved single-cell ATAC-seq reveals chromatin dynamics of in vitro corticogenesis

Development is a complex process that requires the precise modulation of regulatory gene networks controlled through dynamic changes in the epigenome. Single-cell-omic technologies provide an avenue for understanding the mechanisms of these processes by capturing the progression of epigenetic cell states during the course of cellular differentiation using in vitro or in vivo models1. However, current single-cell epigenomic methods are limited in the information garnered per individual cell, which in turn limits their ability to measure chromatin dynamics and state shifts. Single-cell combinatorial indexing (sci-) has been applied as a strategy for identifying single-cell-omic originating libraries and removes the necessity of single-cell, single-compartment chemistry2. Here, we report an improved sci-assay for transposase accessible chromatin by sequencing (ATAC-seq), which utilizes the small molecule inhibitor Pitstop 2 (scip-ATAC-seq)3. We demonstrate that these improvements, which theoretically could be applied to any in situ transposition method for single-cell library preparation, significantly increase the ability of transposase to enter the nucleus and generate highly complex single-cell libraries, without altering biological signal. We applied sci-ATAC-seq and scip-ATAC-seq to characterize the chromatin dynamics of developing forebrain-like organoids, an in vitro model of human corticogenesis4. Using these data, we characterized novel putative regulatory elements, compared the epigenome of the organoid model to human cortex data, generated a high-resolution pseudotemporal map of chromatin accessibility through differentiation, and measured epigenomic changes coinciding with a neurogenic fate decision point. Finally, we combined transcription factor motif accessibility with gene activity (GA) scores to directly observe the dynamics of complex regulatory programs that regulate neurogenesis through developmental pseudotime. Overall, scip-ATAC-seq increases information content per cell and bolsters the potential for future single-cell studies into complex developmental processes.

genomics↗

bioRxiv2017

Scalable and efficient single-cell DNA methylation sequencing by combinatorial indexing.

Here we present a novel method: single-cell combinatorial indexing for methylation analysis (sci-MET), which is the first highly scalable assay for whole genome methylation profiling of single cells. We use sci-MET to produce 2,697 total single-cell bisulfite sequencing libraries and achieve read alignment rates of 69 {+/-} 7%, comparable to those of bulk cell methods. As a proof of concept, we applied sci-MET to successfully deconvolve the cellular identity of a mixture of three human cell lines.

genomics↗

bioRxiv2016

Exonic somatic mutations contribute risk for autism spectrum disorder

Genetic risk factors for autism spectrum disorder (ASD) have yet to be fully elucidated. Somatic mosaic mutations (SMMs) have been implicated in several neurodevelopmental disorders and overgrowth syndromes. Here, we systematically evaluate SMMs by leveraging whole-exome sequencing (WES) data on a large family-based ASD cohort, the Simons Simplex Collection (SSC). We find evidence that ~10% of previously published de novo mutations are potentially SMMs. When using a custom somatic calling pipeline, we recalled all SSC WES data. We validated high and low confidence mutation predictions for a subset of families with single molecule molecular inversion probes. With these validation data, we iteratively developed a high confidence calling approach integrating logistic regression modeling and additional heuristics and applied it to the full cohort. Surprisingly, we found evidence of significant synonymous SMM burden in probands, with mutations more likely to be close to splicing sites. Overall, we observe no strong evidence of missense SMM burden. However, we do observe nominally significant signal for missense SMMs in those families without germline mutations, which strengthens specifically in genes intolerant to mutations. In contrast to missense germline mutations, missense SMMs show potential enrichment for chromatin modifiers. We observe 7-10% of parental mosaics are transmitted germline to a child as occult de novo mutations, which has important implications for recurrence risk for families and potential subclinical ASD features. Finally, we find SMMs in previously implicated high-confidence ASD risk genes, including CHD2, CTNNB1, KMT2C, SYNGAP1, and RELN, further suggesting that this class of mutations contribute to population risk.

genetics↗