Rapid Detection of Hemoglobinopathy Variants Using One-Step Library Preparation and Nanopore Sequencing

Xu Yang; Shang Yi; Xingyu Wei; Guiyun Zheng; Long Wu; Li Zhang; Guojun Ouyang; Sheng He; Xuexi Yang; Fang Yang

doi:10.1093/clinchem/hvag072

Rapid Detection of Hemoglobinopathy Variants Using One-Step Library Preparation and Nanopore Sequencing

Clin Chem. 2026 Jun 23:hvag072. doi: 10.1093/clinchem/hvag072. Online ahead of print.

Authors

Xu Yang^{1

2}, Shang Yi³, Xingyu Wei⁴, Guiyun Zheng⁴, Long Wu², Li Zhang², Guojun Ouyang², Sheng He³, Xuexi Yang¹, Fang Yang⁴

Affiliations

¹ Department of Institute of Antibody Engineering, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, China.
² Guangzhou Darui Biotechnology Co., Ltd, Guangzhou, China.
³ Department of Guangxi Clinical Medical Research Center for Birth Defects, Guangxi Key Laboratory of Reproductive Health and Birth Defects Prevention and Control, Guangxi Zhuang Autonomous Region Women and Children Care Hospital, Nanning, China.
⁴ Fetal Medicine and Prenatal Diagnosis Department, Zhujiang Hospital, Southern Medical University, Guangzhou, China.

PMID: 42335171
DOI: 10.1093/clinchem/hvag072

Abstract

Background: Third-generation sequencing (TGS) enables comprehensive detection of rare hemoglobin (Hb) variants due to its long-read capability. However, library preparation is often labor-intensive and time-consuming. This study developed a novel one-step library preparation (OSLP) method for TGS. By integrating barcode sequences into target-specific primers, the approach enables immediate barcoding on amplification, streamlining identification of hemoglobinopathy variants.

Methods: A retrospective set of DNA samples (n = 455; 394 adult, 61 prenatal) underwent OSLP targeting of the HBA2/1, HBG2/1, HBD, and HBB genes on the Qitan Nanopore platform for variant and structural variation detection. Samples were divided into validation and application cohorts based on prior testing with multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing alongside Gap-polymerase chain reaction (GAP-PCR)/reverse dot blot (RDB).

Results: In the validation cohort, OSLP-TGS showed 100% concordance with routine methods (MLPA, Sanger, GAP-PCR, RDB), including 294 samples for α-globin genotyping [8 structural variants (SVs), 17 single nucleotide variants (SNVs)/insertions-deletions (indels)], 138 for β-globin genotyping (4 SVs, 22 SNVs/indels), and 61 prenatal samples for combined genotyping. The method directly spanned breakpoints and determined cis/trans configurations. In the application cohort, OSLP-TGS-Nanopore identified additional variants not covered by routine genotyping: 9 extra α-globin SNVs in 16/100 samples and 8 extra β-globin SNVs in 33/256 samples. Notably, 3 samples with the -SEA deletion harbored co-inherited novel variants, explaining their disproportionately low Hb A2 levels.

Conclusions: The OSLP-TGS-Nanopore assay provides an efficient, comprehensive solution for detecting rare hemoglobinopathy variants, with a framework extendable to population screening for diverse genetic disorders.

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Abstract

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