Scalable detection of technically challenging variants through modified next‐generation sequencing

Abstract

Background: Some clinically important genetic variants are not easily evaluated with next-generation sequencing (NGS) methods due to technical challenges arising from high-similarity copies (e.g., PMS2, SMN1/SMN2, GBA1, HBA1/HBA2, CYP21A2), repetitive short sequences (e.g., ARX polyalanine repeats, FMR1 AGG interruptions in CGG repeats, CFTR poly-T/TG repeats), and other complexities (e.g., MSH2 Boland inversions). Methods:We customized our NGS processes to detect the technically challenging variants mentioned above with adaptations including target enrichment and bioinformatic masking of similar sequences. Adaptations were validated with samples of known genotypes.Results: Our adaptations provided high-sensitivity and high-specificity detection for most of the variants and provided a high-sensitivity primary assay to be followed with orthogonal disambiguation for the others. The sensitivity of the NGS adaptations was 100% for all of the technically challenging variants. Specificity was 100% for those in PMS2, GBA1, SMN1/SMN2, and HBA1/HBA2, and for the MSH2 Boland inversion; 97.8%-100% for CYP21A2 variants; and 85.7% for ARX polyalanine repeats.Conclusions: NGS assays can detect technically challenging variants when chemistries and bioinformatics are jointly refined. The adaptations described support a scalable, cost-effective path to identifying all clinically relevant variants within a single sample.

The spectrum of movement disorders in young children with ARX‐related epilepsy‐dyskinesia syndrome

“…However, in the context of ARX ‐related disorder, panel and exome sequencing may fail to provide the diagnosis because the most common type of ARX disease‐causing variants, polyalanine tract expansions, can be missed. This is due to multiple mechanisms such as suboptimal polymerase processivity and fidelity at repetitive sequences, 11 as was the case in Patient #2 and #3. WGS and single‐gene testing for ARX include a dedicated evaluation for the polyalanine tract expansion and should be considered alongside other genetic investigations for male infants with epilepsy‐dyskinesia syndromes, male infants with epileptic spasms in the setting of agenesis of the corpus callosum with or without ambiguous genitalia and in the association between hand dystonia and epilepsy in infancy or early childhood, which is considered highly suggestive of ARX ‐related disorder and should prompt specific genetic testing 12,13 .…”

Machine learning in onco-pharmacogenomics: a path to precision medicine with many challenges

“…Moreover, NGS has only recently been introduced into clinical practice to assess diagnosis and prognosis and to evaluate therapeutic strategies, but it is still a niche and there is room for improvement. In addition, sequencing of some parts of the genome remains challenging, e.g., highly polygenic regions, pseudogenes, triplet expansions, low complexity regions, short repetitive sequences, regions of high-similarity, and complex structural rearrangements ( Treangen and Salzberg, 2012 ; Rojahn et al, 2022 ). It is estimated that approximately 14% of clinically relevant genetic tests are located in these genomic regions ( Lincoln et al, 2021 ), and correct variant identification can be difficult.…”

“…On the one hand, short tandem repeats and complex structural variants known to play a role in the pathogenesis of certain diseases can now be sequenced using a targeted approach with long reads sequencing technology, as longer reads are expected to generate appropriate sequence length that overlaps better during assembly ( Stevanovski et al, 2022 ). On the other hand, these technical difficulties can be at least partially overcome by adapting NGS analysis workflows accordingly ( Rojahn et al, 2022 ). However, detecting variants in these regions remains challenging and difficult to validate.…”