Long read sequencing on its way to the routine diagnostics of genetic diseases

Abstract

The clinical application of technological progress in the identification of DNA alterations has always led to improvements of diagnostic yields in genetic medicine. At chromosome side, from cytogenetic techniques evaluating number and gross structural defects to genomic microarrays detecting cryptic copy number variants, and at molecular level, from Sanger method studying the nucleotide sequence of single genes to the high-throughput next-generation sequencing (NGS) technologies, resolution and sensitivity progressively increased expanding considerably the range of detectable DNA anomalies and alongside of Mendelian disorders with known genetic causes. However, particular genomic regions (i.e., repetitive and GC-rich sequences) are inefficiently analyzed by standard genetic tests, still relying on laborious, time-consuming and low-sensitive approaches (i.e., southern-blot for repeat expansion or long-PCR for genes with highly homologous pseudogenes), accounting for at least part of the patients with undiagnosed genetic disorders. Third generation sequencing, generating long reads with improved mappability, is more suitable for the detection of structural alterations and defects in hardly accessible genomic regions. Although recently implemented and not yet clinically available, long read sequencing (LRS) technologies have already shown their potential in genetic medicine research that might greatly impact on diagnostic yield and reporting times, through their translation to clinical settings. The main investigated LRS application concerns the identification of structural variants and repeat expansions, probably because techniques for their detection have not evolved as rapidly as those dedicated to single nucleotide variants (SNV) identification: gold standard analyses are karyotyping and microarrays for balanced and unbalanced chromosome rearrangements, respectively, and southern blot and repeat-primed PCR for the amplification and sizing of expanded alleles, impaired by limited resolution and sensitivity that have not been significantly improved by the advent of NGS. Nevertheless, more recently, with the increased accuracy provided by the latest product releases, LRS has been tested also for SNV detection, especially in genes with highly homologous pseudogenes and for haplotype reconstruction to assess the parental origin of alleles with de novo pathogenic variants. We provide a review of relevant recent scientific papers exploring LRS potential in the diagnosis of genetic diseases and its potential future applications in routine genetic testing.

Keywords: RNA sequencing; genetic diseases; long read sequencing; methylation; molecular diagnosis; single nucleotide variants; structural variants; tandem repeats.

FIGURE 1

Limitations of current diagnostic tests for tandem-repeat-related diseases. SRS: short read sequencing; RT-PCR: repeat primed-polymerase chain reaction; *its clinical application has been evaluated but technical issues prevented its transition to the diagnostic routine.

FIGURE 2

Suggested diagnostic workflows that include long read sequencing. (A) diagnostic workflow to be adopted in case the patient presents with a complex phenotype, not suggestive for a specific genetic condition. Long read sequencing can help define copy number variants of uncertain significance, a targeted approach can be useful to study the second allele of a recessive gene when a monoallelic variant is identified and, finally, a whole genome approach can be an additional diagnostic tool in case neither single nucleotide variants nor copy number variants are detected with other techniques. (B) diagnostic workflow to be adopted in case the patient presents with clinical features that are suggestive for a specific genetic condition. Targeted long read sequencing can help diagnose mendelian diseases involving difficult-to-sequence genes and tandem repeat-related disorders. aCGH: array-comparative genomic hybridization; AD: autosomal dominant; CNV: copy number variant; LRS: long read sequencing; MLPA: multiplex ligation-dependent probe amplification; MS-MLPA: methylation-specific multiplex ligation-dependent probe amplification; SRS: short read sequencing; SNV: single nucleotide variant; SV: structural variant; TR: tandem repeat.