Incremental Clinical Value of Single-Molecule Nanopore Sequencing in Thalassemia Testing: A Prospective, Double-blind, Multicenter Study
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Background
Thalassemia is one of the most common monogenic disorders worldwide. Current screening strategies combining hematological testing with molecular assays still carry a risk of missed diagnoses and suboptimal efficiency, particularly for complex structural variants and rare mutations.
Methods
In this prospective, double-blind, multicenter cohort study of 3,842 participants (3,362 pregnant women and 480 male partners), we conducted a head-to-head comparison to systematically evaluate the incremental clinical value and detection performance of single-molecule nanopore sequencing in thalassemia (SMITH) against conventional hematological testing and next-generation sequencing (NGS).
Findings
The overall concordance rate between NGS and SMITH was 98.6% (3789/3842). The discrepant cases (n=53) were directly attributed to the superior detection capabilities of SMITH, which successfully identified complex structural rearrangements—including 45 α-globin gene triplications and four HKαα alleles—that were missed by NGS. Furthermore, SMITH accurately detected four rare variants (αα c.134_135insT /αα, αα c.-22(C>T) /αα, β N /β c.316-290delinsAGGGCAATAATTT , and β 3.5 kb deletion /β N ) and resolved ten trans and three cis configurations within the globin gene alleles. Clinically, these technical advantages translated to a 9.3% (5/54) increase in the detection rate of high-risk prenatal couples, effectively preventing one birth affected by moderate-to-severe thalassemia. Additionally, SMITH corrected a diagnostic discrepancy in one case (HKαα vs. -α 3.7 ), sparing the couple from an unnecessary invasive procedure.
Interpretation
Our findings demonstrate that SMITH provides a powerful platform for resolving globin gene rearrangements, detecting rare variants, and enabling direct haplotype phasing. By effectively eliminating diagnostic blind spots, SMITH is expected to become an optimal method for thalassemia prevention programs.
Funding
This study was supported by the Chinese National Natural Science Foundation Projects 81760037 and 82271894.
Research in context
Evidence before this study
Next-generation sequencing (NGS) has substantially advanced carrier screening for thalassemia. However, its analytical blind spots—particularly in highly homologous regions, complex structural variants (SVs), and haplotype phasing—directly compromise the accuracy of genetic counseling and prenatal diagnosis.
Long-read sequencing holds promise to address these gaps by generating reads that span entire gene clusters. Prior to this study, research on long-read sequencing for thalassemia screening had been extensive. To systematically evaluate its clinical value, we searched the literature published over the past decade. However, most previous studies compared long-read sequencing with conventional PCR-based methods, which do not allow assessment of its incremental value relative to current NGS-based screening pathways. To date, no large-scale, prospective, head-to-head study has compared NGS with single-molecule nanopore sequencing in a real-world prenatal screening setting. Therefore, the incremental clinical value of long-read sequencing in this context remains to be systematically quantified.
Added value of this study
In this prospective, double-blind, multicenter cohort study of 3,842 participants, we performed a head-to-head comparison between standard NGS and our novel Single-Molecule nanopore sequencing In THalassemia (SMITH) in a real-world prenatal screening setting. To our knowledge, this is the first large-scale, prospective study to directly compare long-read sequencing with standard NGS and to link technical performance with clinical outcomes. SMITH increased the detection rate of high-risk couples by 9.3%, prevented one moderate-to-severe thalassemia birth, and corrected a diagnostic misclassification that spared a couple from unnecessary invasive amniocentesis. These findings provide detailed, clinically actionable insights not previously reported.
Implications of all the available evidence
These findings establish SMITH as a major advance in thalassemia prevention. By eliminating key diagnostic blind spots—complex structural variants, rare mutations, and unresolved allelic phases—SMITH improves the accuracy of genetic counseling and prenatal diagnosis. The reduction in missed high-risk couples and preventable severe thalassemia births has direct implications for public health policy, particularly in high-prevalence regions. Future work should prioritise cost-effectiveness analyses and large-scale implementation studies to guide the integration of SMITH into routine population-based screening programmes.
