Long-read sequencing for molecular diagnostics in constitutional genetic disorders

Abstract

Long-read sequencing (LRS) has been around for more than a decade, but widespread adoption of the technology has been slow due to the perceived high error rates and high sequencing cost. This is changing due to the recent advancements to produce highly accurate sequences and the reducing costs. LRS promises significant improvement over short read sequencing in four major areas: (1) better detection of structural variation (2) better resolution of highly repetitive or nonunique regions (3) accurate long-range haplotype phasing and (4) the detection of base modifications natively from the sequencing data. Several successful applications of LRS have demonstrated its ability to resolve molecular diagnoses where short-read sequencing fails to identify a cause. However, the argument for increased diagnostic yield from LRS remains to be validated. Larger cohort studies may be required to establish the realistic boundaries of LRS's clinical utility and analytical validity, as well as the development of standards for clinical applications. We discuss the limitations of the current standard of care, and contrast with the applications and advantages of two major LRS platforms, PacBio and Oxford Nanopore, for molecular diagnostics of constitutional disorders, and present a critical argument about the potential of LRS in diagnostic settings.

Keywords: Oxford Nanopore; PacBio; constitutional disorders; long-read sequencing; molecular diagnostics.

Figure 1.. Technologies used in comprehensive diagnostic test for nonsyndromic hearing loss, Audiome.

The comprehensive diagnostic test for Hearing Loss, Audiome, offered by the Genomic Diagnostics Lab at the Children’s Hospital of Philadelphia uses a tiered testing approach and a combination of six technologies for comprehensive variant detection (A,B,C,D,E,F). Tier 1 testing uses Sanger sequencing and fragment analysis focusing on GJB2 (DFNB1A; MIM# 220290) (A, B) and mitochondrial variants, with reflex to Tier 2 involving droplet-digital PCR (C) and long-range PCR followed by NGS (D) for STRC (MIM# 606440), a virtual exome “slice” for sequence and copy number variants (E) and exon-targeted custom-designed array CGH (F) for 132 genes.

Figure 2.. Comprehensive variant detection and phase resolution without parental samples using PacBio long-read sequencing.

Panel A shows the phased long-read sequencing (LRS) data for the patient who received an inconclusive result from the standard of care diagnostic testing for hearing loss (case vignette 1). Targeted exome analysis revealed two potentially pathogenic variants (SNV and a deletion) in the gene TRIOBP. The SNV and the deletion was 12 kb apart and the exome analysis was not able to resolve phase without additional sequencing of the parents. LRS detected these two variants and revealed these variants are in the same haplotype (cis) making it non-diagnostic. Panel B shows the phased LRS data for a patient who received an inconclusive result from the standard of care diagnostic testing for hearing loss (case vignette 2). Targeted long-range PCR followed by next-generation sequencing of STRC revealed a variant of uncertain significance in exon 8 and multiple likely pathogenic variants in exons 25 and 26. Variants identified in exons 25/26 of STRC are known to be present in the pseudogene STRCP1 and suggestive of a gene conversion event. Gene conversion events are challenging to resolve using short-reads due to the mappability issues in discriminating protein coding gene and its pseudogene. LRS was able to map reads unambiguously to the protein-coding gene, identified all the variants, and revealed that these variants were in different haplotypes (trans), confirming the gene conversion event to make this a diagnostic result.