Detection of de novo variants (DNVs) is critical for studies of disease-related variation and mutation rates. To accelerate DNV calling, we developed a graphics processing units-based workflow. We applied our workflow to whole-genome sequencing data from three parent-child sequenced cohorts including the Simons Simplex Collection (SSC), Simons Foundation Powering Autism Research (SPARK), and the 1000 Genomes Project (1000G) that were sequenced using DNA from blood, saliva, and lymphoblastoid cell lines (LCLs), respectively. The SSC and SPARK DNV callsets were within expectations for number of DNVs, percent at CpG sites, phasing to the paternal chromosome of origin, and average allele balance. However, the 1000G DNV callset was not within expectations and contained excessive DNVs that are likely cell line artifacts. Mutation signature analysis revealed 30% of 1000G DNV signatures matched B-cell lymphoma. Furthermore, we found variants in DNA repair genes and at Clinvar pathogenic or likely-pathogenic sites and significant excess of protein-coding DNVs in IGLL5; a gene known to be involved in B-cell lymphomas. Our study provides a new rapid DNV caller for the field and elucidates important implications of using sequencing data from LCLs for reference building and disease-related projects.

Background Previous research in autism and other neurodevelopmental disorders (NDDs) has indicated an important contribution of protein-coding (coding) de novo variants (DNVs) within specific genes. The role of de novo noncoding variation has been observable as a general increase in genetic burden but has yet to be resolved to individual functional elements. In this study, we assessed whole-genome sequencing data in 2671 families with autism (discovery cohort of 516 families, replication cohort of 2155 families). We focused on DNVs in enhancers with characterized in vivo activity in the brain and identified an excess of DNVs in an enhancer named hs737. Results We adapted the fitDNM statistical model to work in noncoding regions and tested enhancers for excess of DNVs in families with autism. We found only one enhancer (hs737) with nominal significance in the discovery (p = 0.0172), replication (p = 2.5 × 10−3), and combined dataset (p = 1.1 × 10−4). Each individual with a DNV in hs737 had shared phenotypes including being male, intact cognitive function, and hypotonia or motor delay. Our in vitro assessment of the DNVs showed they all reduce enhancer activity in a neuronal cell line. By epigenomic analyses, we found that hs737 is brain-specific and targets the transcription factor gene EBF3 in human fetal brain. EBF3 is genome-wide significant for coding DNVs in NDDs (missense p = 8.12 × 10−35, loss-of-function p = 2.26 × 10−13) and is widely expressed in the body. Through characterization of promoters bound by EBF3 in neuronal cells, we saw enrichment for binding to NDD genes (p = 7.43 × 10−6, OR = 1.87) involved in gene regulation. Individuals with coding DNVs have greater phenotypic severity (hypotonia, ataxia, and delayed development syndrome [HADDS]) in comparison to individuals with noncoding DNVs that have autism and hypotonia. Conclusions In this study, we identify DNVs in the hs737 enhancer in individuals with autism. Through multiple approaches, we find hs737 targets the gene EBF3 that is genome-wide significant in NDDs. By assessment of noncoding variation and the genes they affect, we are beginning to understand their impact on gene regulatory networks in NDDs.

Previous research in autism and other neurodevelopmental disorders (NDDs) has indicated an important contribution of de novo protein-coding variants within specific genes. The role of de novo noncoding variation has been observable as a general increase in genetic burden but has yet to be resolved to individual functional elements. In this study, we assessed whole-genome sequencing data in 2,671 families with autism, with a specific focus on de novo variation in enhancers with previously characterized in vivo activity. We identified three independent de novo mutations limited to individuals with autism in the enhancer hs737. These mutations result in similar phenotypic characteristics, affect enhancer activity in vitro, and preferentially occur in AAT motifs in the enhancer with predicted disruptions of transcription factor binding. We also find that hs737 is enriched for copy number variation in individuals with NDDs, is dosage sensitive in the human population, is brain-specific, and targets the NDD gene EBF3 that is genome-wide significant for protein coding de novo variants, demonstrating the importance of understanding all forms of variation in the genome.One Sentence SummaryWhole-genome sequencing in thousands of families reveals variants relevant to simplex autism in a brain enhancer of the well-established neurodevelopmental disorder gene EBF3.

Motivation An abundance of new reference genomes is becoming available through large-scale sequencing efforts. While the reference FASTA for each genome is available, there is currently no automated mechanism to query a specific sequence across all new reference genomes. Results We developed ACES (Analysis of Conservation with an Extensive list of Species) as a computational workflow to query specific sequences of interest (e.g., enhancers, promoters, exons) against reference genomes with an available reference FASTA. This automated workflow generates BLAST hits against each of the reference genomes, a multiple sequence alignment file, a graphical fragment assembly file, and a phylogenetic tree file. These data files can then be used by the researcher in several ways to provide key insights into conservation of the query sequence. Availability ACES is available at https://github.com/TNTurnerLab/ACES Supplementary information Supplementary Figure 1 is available online in Bioinformatics.