Long-read genome sequencing for the diagnosis of neurodevelopmental disorders

Abstract

<jats:title>Abstract</jats:title><jats:p>Exome and genome sequencing have proven to be effective tools for the diagnosis of neurodevelopmental disorders (NDDs), but large fractions of NDDs cannot be attributed to currently detectable genetic variation. This is likely, at least in part, a result of the fact that many genetic variants are difficult or impossible to detect through typical short-read sequencing approaches. Here, we describe a genomic analysis using Pacific Biosciences circular consensus sequencing (CCS) reads, which are both long (&gt;10 kb) and accurate (&gt;99% bp accuracy). We used CCS on six proband-parent trios with NDDs that were unexplained despite extensive testing, including genome sequencing with short reads. We identified variants and created <jats:italic>de novo</jats:italic> assemblies in each trio, with global metrics indicating these data sets are more accurate and comprehensive than those provided by short-read data. In one proband, we identified a likely pathogenic (LP), <jats:italic>de novo</jats:italic> L1-mediated insertion in <jats:italic>CDKL5</jats:italic> that results in duplication of exon 3, leading to a frameshift. In a second proband, we identified a <jats:italic>de novo</jats:italic> translocation affecting <jats:italic>DGKB</jats:italic> and <jats:italic>MLLT3</jats:italic>, which we show disrupts <jats:italic>MLLT3</jats:italic> transcript levels. We consider this a variant of uncertain significance (VUS). The breadth and quality of variant detection coupled to finding variants of clinical and research interest in two of six probands with unexplained NDDs strongly support the value of long-read genome sequencing for understanding rare disease.</jats:p>

Robust Detection of Somatic Mosaicism and Repeat Interruptions by Long-Read Targeted Sequencing in Myotonic Dystrophy Type 1

“…Here, we have analyzed, for the first time, two DM1 families carrying CTG repeats ranging from 170 to over 1000 CTG repeats using the Sequel II System. The Sequel II System generates longer reads, enabling higher CCS accuracy, and has higher throughputs than the Sequel and RSII Systems (data not shown and [ 18 , 42 , 43 ]). Here, the Sequel II System and bioinformatic tools give us the ability to simultaneously measure repeat numbers with high resolution, to resolve the complete sequence complex repeat expansions and to measure the degree of somatic mosaicism.…”

DeepTrio: Variant Calling in Families Using Deep Learning

“…In trio sequencing for rare disease, there is often greater importance in sequencing a child proband. To manage sequencing costs, studies often take the approach of sequencing the parents at a lower coverage than the child 36 . In order to evaluate performance in these scenarios, we performed downsampling of the parent samples while keeping the child coverage at 35x.…”

An Extensive Sequence Dataset of Gold-Standard Samples for Benchmarking and Development

“…We sequenced HG003, HG004, HG006, and HG007 with PacBio HiFi reads in order to contribute to Genome in a Bottle’s efforts to expand the confident regions. The high quality and mappability of PacBio HiFi data has recently proven instrumental in the first Telomere-to-Telomere human assembly[34] and undiagnosed rare disease[35]. Because we have sequence data for the same samples from PacBio HiFi and NovaSeq, we can quantify the differences in mappability and variant calling accuracy between the technologies.…”