Clinical Long-Read Sequencing: A Game-Changer in Pediatric Genetic Disease Diagnosis

Highlight

– Clinical long-read sequencing (LRS) significantly improves diagnostic rates (P = .004) over standard-of-care (SOC) genetic testing in pediatric patients.
– LRS reduces time to diagnosis substantially, averaging 27 days versus 62 days for SOC and enables earlier reporting of negative results.
– The integrated detection capabilities of LRS capture complex structural variants, repeat expansions, and methylation abnormalities that are often missed in traditional tests.
– By consolidating multiple SOC genetic tests, LRS simplifies and expedites the diagnostic workflow significantly in clinical practice.

Study Background and Disease Burden

Genetic diseases represent a substantial burden for pediatric populations, often presenting with heterogenous symptoms that require comprehensive molecular diagnostics to enable precise diagnosis and management. Traditional genetic testing involves multiple sequential tests such as exome sequencing (ES), genome sequencing (GS), chromosomal microarray (CMA), karyotyping, fluorescence in situ hybridization (FISH), and targeted panels, which prolong time to diagnosis and increase costs. While GS has shown modest incremental gains (4%-8%) over ES alone, the overall diagnostic improvement (<1%) compared to combinations of standard tests remains marginal. Unexplained cases often persist after standard testing, in part due to difficulties in detecting certain genetic variants including repeat expansions, methylation changes, and complex structural rearrangements. Recent advances in long-read sequencing (LRS) technology provide a promising approach to simultaneously interrogate these variant types in a single assay, potentially transforming genetic diagnostics. The study by Thiffault et al. aimed to evaluate clinical LRS as a first-line diagnostic test in pediatric patients to assess its impact on diagnostic yield, turnaround time, and the ability to consolidate standard-of-care approaches.

Study Design

This controlled diagnostic accuracy study enrolled 235 pediatric patients aged 0 to 18 years who underwent clinical high-fidelity (HiFi) LRS, compared against 513 age- and phenotype-matched controls who received standard-of-care (SOC) genetic testing during inpatient evaluation. Standard tests in the control cohort included expedited ES/GS, karyotyping, FISH, CMA, and targeted panels per clinical indication. The study followed the Standards for Reporting of Diagnostic Accuracy (STARD) guidelines and excluded variants of uncertain significance, incidental or carrier findings, and pathogenic variants not directly relevant to the presenting clinical symptoms. Outcome measures included diagnostic yield (diagnoses explaining primary clinical features), turnaround time from test order to report, number of tests required per diagnosis, and variant detection profiles. Statistical analysis employed χ² tests for diagnostic rates and t tests for comparisons of turnaround time.

Key Findings

The LRS cohort demonstrated a significantly higher diagnostic yield compared to the SOC group (P = .004). Specifically, among 235 patients tested with LRS, 87 diagnostic findings were reported, while the 513 controls yielded fewer diagnoses in relation to the tested cohort size. The mean turnaround time from test ordering to diagnostic report was significantly reduced with LRS (27 days) versus SOC (62 days) (P = .048). Even for patients with negative results, report issuance was faster with LRS (29 days) compared to SOC (91 days) (P < .001).

One pivotal driver of LRS’s diagnostic advantage was its ability to detect complex structural variants (SVs), copy number variants, repeat expansions, and methylation abnormalities in an integrated fashion. Notably, 16 of 87 (18.3%) diagnostic variants in the LRS group were identified through such comprehensive variant detection, including rare repeat expansion disorders and epigenetic changes like GNAS methylation abnormalities—features not readily captured by standard clinical tests. While 81.6% of diagnostic genotypes detected by LRS would have been detectable by ES, GS, or CMA, the single-test approach of LRS eliminated the need for multiple sequential testing (6.1 tests on average in SOC versus 2.7 in LRS cases).

Figure 2. Long-Read Sequencing (LRS) Compared to Standard Genetic Testing Among Matched Pediatric Cases.

The SOC group’s diagnostic delays largely stemmed from the requirement of multiple genetic test orders: 57 SOC cases required multiple tests to reach diagnosis, incurring turnaround times exceeding four months. In terms of test-efficiency ratios, SOC cases averaged 1.6 tests per diagnostic finding compared to nearly one test per diagnosis in LRS cases, highlighting LRS’s potential to streamline and shorten the diagnostic odyssey.

Technical challenges specific to LRS included occasional sequencing yield deficits necessitating reloading and limitations in tertiary bioinformatics analysis tools. However, these did not substantially diminish LRS performance benefits in clinical settings.

Expert Commentary

The study by Thiffault et al. substantiates LRS as a transformative first-line genomic assay in pediatric clinical genetics, offering a unified platform that overcomes the inherent limitations of current sequential SOC testing. Importantly, the ability of LRS to detect a spectrum of variant classes—including repeat expansions and epigenetic modifications—which often require specialized and seldom concurrently ordered tests, provides unique clinical value.

Experts appreciate that while framework interpretations of LRS findings utilize variant types identified by conventional assays, clinical practice tends to selectively employ testing based on clinical suspicion, frequently leaving repeat expansions or methylation defects undetected due to atypical presentations. LRS mitigates this by capturing diverse variant types in a single assay irrespective of preliminary clinical assumptions.

The reduced diagnostic turnaround times witnessed offer vital clinical impacts, especially in pediatric populations where early diagnosis can inform management, prognostication, and genetic counseling. While challenges remain in analytic workflows and bioinformatics maturation, this study sets a benchmark for clinical integration of LRS technologies.

Future iterations of LRS applied in clinical settings are poised to expand pathogenic variant discovery, particularly within noncoding genomic regions currently poorly interrogated by short-read or targeted sequencing.

Conclusion

Clinical high-fidelity long-read sequencing represents a significant advance in the genetic diagnosis of pediatric diseases. By consolidating multiple standard genetic tests into a single comprehensive assay, LRS improves diagnostic yield, shortens turnaround times, and enhances detection of clinically relevant structural, epigenetic, and expansion variants under-recognized by traditional testing. Its integration into clinical pathways promises to streamline genetic diagnostic workflows, mitigate delays, and improve patient care. Continued refinement in sequencing yield and bioinformatics analysis will further strengthen its clinical utility. Overall, LRS heralds a new era in clinical genomics, offering profound implications for timely, accurate diagnosis of complex genetic disorders in children.

References

Thiffault I, Farrow E, Barrett C, Scott M, Ross A, Means JC, Cheung WA, Johnson AF, Koseva B, McLennan R, Grundberg E, Bi C, Schwendinger-Schreck C, Yoo B, Johnston JJ, Del Viso F, Paolillo V, Herriges J, Zhang L, Gibson M, Cohen ASA, Alaimo J, Saunders CJ, Pastinen T. Clinical Long-Read Sequencing Test for Genetic Disease Diagnosis. JAMA Pediatr. 2025 Sep 22:e253320. doi: 10.1001/jamapediatrics.2025.3320 IF: 18.0 Q1 B1. Epub ahead of print. PMID: 40982260 IF: 18.0 Q1 B1; PMCID: PMC12455484 IF: 18.0 Q1 B1.