Using the linear references from the pangenome to discover missing autism variants

To better understand large-effect pathogenic variation associated with autism, we generated long-read sequencing (LRS) data to construct phased and near-complete genome assemblies (average contig N50 = 43 Mbp, QV = 56) for 189 individuals from 51 families with unsolved cases. We applied read- and assembly-based strategies to facilitate comprehensive characterization of de novo mutations, structural variants (SVs), and DNA methylation. Using LRS pangenome controls, we efficiently filtered >97% of common SVs exclusive to 87 offspring. We find no evidence of increased autosomal SV burden for probands when compared to unaffected siblings yet observe a suggestive trend toward an increased SV burden on the X chromosome among affected females. We establish a workflow to prioritize potential pathogenic variants by integrating autism risk genes and putative noncoding regulatory elements defined from ATAC-seq and CUT&Tag data from the developing cortex. In total, we identified three pathogenic variants in TBL1XR1, MECP2, and SYNGAP1, as well as nine candidate de novo and biallelic inherited homozygous SVs, most of which were missed by short-read sequencing. Our work highlights the potential of phased genomes to discover complex more pathogenic mutations and the power of the pangenome to restrict the focus on an increasingly smaller number of SVs for clinical evaluation.