Familial long-read sequencing increases yield of de novo mutations

• DOI: 10.1016/j.ajhg.2022.02.014
• Publication Date: 2022-03-14T16:18:27Z
• Authors: Michelle D. Noyes; William T. Harvey; David Porubsky; Arvis Sulovari; Ruiyang Li; Nicholas R. Rose; Peter A. Audano; Katherine M. Munson; Alexandra P. Lewis; Kendra Hoekzema; Tuomo Mantere; Tina A. Graves-Li...; Ashley D. Sanders; Sara Goodwin; Melissa Kramer; Younes Mokrab; Michael C. Zody; Alexander Hoischen; Jan O. Korbel; W. Richard McCombie; Evan E. Eichler
• Keywords: Radboudumc 4: lnfectious Diseases and Global Health RIMLS: Radboud Institute for Molecular Life Sciences; 0301 basic medicine; Nucleotides; Radboud University Medical Center; High-Throughput Nucleotide Sequencing; Genomics; Sequence Analysis, DNA; 03 medical and health sciences; Mutation; Humans; Female; Internal Medicine - Radboud University Medical Center; Software

Studies of de novo mutation (DNM) have typically excluded some of the most repetitive and complex regions of the genome because these regions cannot be unambiguously mapped with short-read sequencing data. To better understand the genome-wide pattern of DNM, we generated long-read sequence data from an autism parent-child quad with an affected female where no pathogenic variant had been discovered in short-read Illumina sequence data. We deeply sequenced all four individuals by using three sequencing platforms (Illumina, Oxford Nanopore, and Pacific Biosciences) and three complementary technologies (Strand-seq, optical mapping, and 10X Genomics). Using long-read sequencing, we initially discovered and validated 171 DNMs across two children-a 20% increase in the number of de novo single-nucleotide variants (SNVs) and indels when compared to short-read callsets. The number of DNMs further increased by 5% when considering a more complete human reference (T2T-CHM13) because of the recovery of events in regions absent from GRCh38 (e.g., three DNMs in heterochromatic satellites). In total, we validated 195 de novo germline mutations and 23 potential post-zygotic mosaic mutations across both children; the overall true substitution rate based on this integrated callset is at least 1.41 × 10-8 substitutions per nucleotide per generation. We also identified six de novo insertions and deletions in tandem repeats, two of which represent structural variants. We demonstrate that long-read sequencing and assembly, especially when combined with a more complete reference genome, increases the number of DNMs by >25% compared to previous studies, providing a more complete catalog of DNM compared to short-read data alone.