Long-read trio sequencing of individuals with unsolved intellectual disability

Abstract

Long-read sequencing (LRS) has the potential to comprehensively identify all medically relevant genome variation, including variation commonly missed by short-read sequencing (SRS) approaches. To determine this potential, we performed LRS around 15×–40× genome coverage using the Pacific Biosciences Sequel I System for five trios. The respective probands were diagnosed with intellectual disability (ID) whose etiology remained unresolved after SRS exomes and genomes. Systematic assessment of LRS coverage showed that ~35 Mb of the human reference genome was only accessible by LRS and not SRS. Genome-wide structural variant (SV) calling yielded on average 28,292 SV calls per individual, totaling 12.9 Mb of sequence. Trio-based analyses which allowed to study segregation, showed concordance for up to 95% of these SV calls across the genome, and 80% of the LRS SV calls were not identified by SRS. De novo mutation analysis did not identify any de novo SVs, confirming that these are rare events. Because of high sequence coverage, we were also able to call single nucleotide substitutions. On average, we identified 3 million substitutions per genome, with a Mendelian inheritance concordance of up to 97%. Of these, ~100,000 were located in the ~35 Mb of the genome that was only captured by LRS. Moreover, these variants affected the coding sequence of 64 genes, including 32 known Mendelian disease genes. Our data show the potential added value of LRS compared to SRS for identifying medically relevant genome variation.

Long-read technologies identify a hidden inverted duplication in a family with choroideremia

“…Long-read sequencing technologies have been rapidly developing and seem to overcome the limitations of short reads in genetic research and molecular diagnoses of different human genetic diseases. 21 , 22 , 23 , 24 Their read length of several kilobases (1) simplifies the identification of SVs, 25 , 26 , 27 , 28 (2) simplifies the spanning of repeats and high GC-rich regions, 29 , 30 and (3) enables variant phasing. 24 , 31 , 32 Bionano optical genome mapping is a high-resolution cytogenetic technique and is based on ultra-high molecular weight (UHMW) DNA molecules that are fluorescently labeled at a 6-mer motif (CTTAAG).…”

MRSD: a novel quantitative approach for assessing suitability of RNA-seq in the clinical investigation of mis-splicing in Mendelian disease

“…This experimental RNAseq approach currently remains widespread (3,10,(13)(14)(15); however, our model may be readily applicable to RNA-seq generated using alternative methodologies, such as increased read length, with only minor variations in model performance ( Supplementary Figure 3). As other technologies, such as long-read (36)(37)(38), singlecell (39,40) and spatially resolved RNA-seq (41)(42)(43)(44), become more prevalent in a clinical setting, appropriate control datasets must be generated to develop corresponding MRSD models. Similarly, recent research has shown noticeable improvements to diagnostic yield for neuromuscular disorders by conducting RNAseq on in vitro myofibrils generated by a fibroblast-to-myofibril transdifferentiation protocol (45).…”

Diagnostic Analyses of Retinal Dystrophy Genes: Current Status and Perspective

“…Despite complete genome coverage in WGS, short-read technologies (Solexa/Illumina) may miss more complex structural rearrangements and even CNVs. Future perspective: Long-read WGS (based on PacBio sequencing, for example) is a promising approach to overcome these limitations [16,17].…”

“…Long-read NGS (e.g., PacBio) can conquer this problem. With contiguous reads of up to 60 kb [16], even distant variants can be attributed to the same or separate alleles (▶ Fig. 3).…”