Clinical Evaluation of Long-Read Sequencing for Telomere Length Assessment in Human Blood and Lung Tissues

Telomere length (TL) measurement is increasingly used in the evaluation of pulmonary fibrosis and suspected telomere biology disorders, with flow cytometry-fluorescence in situ hybridisation (Flow-FISH) representing the current clinical reference standard. However, Flow-FISH provides a population-averaged relative TL, has a high analytical variability and is largely restricted to PBMC-based measurements. We evaluated a long-read sequencing-based assay, Telo-Seq, for clinical TL assessment in an Australian cohort. In this observational study, TL was measured by both Flow-FISH and Telo-Seq in PBMC from 156 healthy controls and a small cohort of 24 patients undergoing clinical evaluation for interstitial lung disease, using the same blood processing and assay workflows. In an exploratory subset of samples, TL was also assessed in whole lung lavage fluid and explanted lung tissue. In the assessment of PBMC, Telo-Seq correlated with Flow-FISH (R² ≈ 0.77) and showed lower inter-assay variability (CV ~1.4% vs. ~4.6%). Classification relative to assay-specific age-adjusted 10th-percentile thresholds was largely concordant between Flow-FISH and Telo-Seq, although discordant cases occurred in both directions near clinically relevant thresholds. Long-read sequencing provides a reproducible approach to TL assessment and supports further evaluation in disease-relevant tissues.