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

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Title: Rapid Detection of Hemoglobinopathy Variants Using One-Step Library Preparation and Nanopore Sequencing.
Authors: Yang X; Department of Institute of Antibody Engineering, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, China.; Guangzhou Darui Biotechnology Co., Ltd, Guangzhou, China., Yi S; 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., Wei X; Fetal Medicine and Prenatal Diagnosis Department, Zhujiang Hospital, Southern Medical University, Guangzhou, China., Zheng G; Fetal Medicine and Prenatal Diagnosis Department, Zhujiang Hospital, Southern Medical University, Guangzhou, China., Wu L; Guangzhou Darui Biotechnology Co., Ltd, Guangzhou, China., Zhang L; Guangzhou Darui Biotechnology Co., Ltd, Guangzhou, China., Ouyang G; Guangzhou Darui Biotechnology Co., Ltd, Guangzhou, China., He S; 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., Yang X; Department of Institute of Antibody Engineering, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, China., Yang F; Fetal Medicine and Prenatal Diagnosis Department, Zhujiang Hospital, Southern Medical University, Guangzhou, China.
Source: Clinical chemistry [Clin Chem] 2026 Oct 05; Vol. 72 (10), pp. 1079-1092.
Publication Type: Journal Article
Language: English
Journal Info: Publisher: Oxford University Press Country of Publication: England NLM ID: 9421549 Publication Model: Print Cited Medium: Internet ISSN: 1530-8561 (Electronic) Linking ISSN: 00099147 NLM ISO Abbreviation: Clin Chem Subsets: MEDLINE
Imprint Name(s): Publication: 2020- : Oxford : Oxford University Press
Original Publication: Baltimore, Md. : P.B. Hoeber, [c1955-
MeSH Terms: Hemoglobinopathies*/genetics , Hemoglobinopathies*/diagnosis , Nanopore Sequencing*/methods , Gene Library*, beta-Globins/genetics ; Humans ; Retrospective Studies ; Genetic Variation
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.
(© Association for Diagnostics & Laboratory Medicine 2026. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)
Grant Information: 2023A1515110676 Guangdong Basic and Applied Basic Research Foundation; 2024B03J1036 Science and Technology Program of Guangzhou
Substance Nomenclature: 0 (beta-Globins)
Entry Date(s): Date Created: 20260623 Date Completed: 20261005 Latest Revision: 20261005
Update Code: 20261005
DOI: 10.1093/clinchem/hvag072
PMID: 42335171
Database: MEDLINE
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  Data: Rapid Detection of Hemoglobinopathy Variants Using One-Step Library Preparation and Nanopore Sequencing.
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  Data: <searchLink fieldCode="AU" term="%22Yang+X%22">Yang X</searchLink>; Department of Institute of Antibody Engineering, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, China.; Guangzhou Darui Biotechnology Co., Ltd, Guangzhou, China.<br /><searchLink fieldCode="AU" term="%22Yi+S%22">Yi S</searchLink>; 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.<br /><searchLink fieldCode="AU" term="%22Wei+X%22">Wei X</searchLink>; Fetal Medicine and Prenatal Diagnosis Department, Zhujiang Hospital, Southern Medical University, Guangzhou, China.<br /><searchLink fieldCode="AU" term="%22Zheng+G%22">Zheng G</searchLink>; Fetal Medicine and Prenatal Diagnosis Department, Zhujiang Hospital, Southern Medical University, Guangzhou, China.<br /><searchLink fieldCode="AU" term="%22Wu+L%22">Wu L</searchLink>; Guangzhou Darui Biotechnology Co., Ltd, Guangzhou, China.<br /><searchLink fieldCode="AU" term="%22Zhang+L%22">Zhang L</searchLink>; Guangzhou Darui Biotechnology Co., Ltd, Guangzhou, China.<br /><searchLink fieldCode="AU" term="%22Ouyang+G%22">Ouyang G</searchLink>; Guangzhou Darui Biotechnology Co., Ltd, Guangzhou, China.<br /><searchLink fieldCode="AU" term="%22He+S%22">He S</searchLink>; 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.<br /><searchLink fieldCode="AU" term="%22Yang+X%22">Yang X</searchLink>; Department of Institute of Antibody Engineering, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, China.<br /><searchLink fieldCode="AU" term="%22Yang+F%22">Yang F</searchLink>; Fetal Medicine and Prenatal Diagnosis Department, Zhujiang Hospital, Southern Medical University, Guangzhou, China.
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  Data: <searchLink fieldCode="JN" term="%229421549%22">Clinical chemistry</searchLink> [Clin Chem] 2026 Oct 05; Vol. 72 (10), pp. 1079-1092.
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  Data: <i>Publisher: </i><searchLink fieldCode="PB" term="%22Oxford+University+Press%22">Oxford University Press </searchLink><i>Country of Publication: </i>England <i>NLM ID: </i>9421549 <i>Publication Model: </i>Print <i>Cited Medium: </i>Internet <i>ISSN: </i>1530-8561 (Electronic) <i>Linking ISSN: </i><searchLink fieldCode="IS" term="%2200099147%22">00099147 </searchLink><i>NLM ISO Abbreviation: </i>Clin Chem <i>Subsets: </i>MEDLINE
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  Data: <i>Publication</i>: 2020- : Oxford : Oxford University Press<br /><i>Original Publication</i>: Baltimore, Md. : P.B. Hoeber, [c1955-
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  Data: <searchLink fieldCode="MM" term="%22Hemoglobinopathies%22">Hemoglobinopathies*</searchLink>/<searchLink fieldCode="MM" term="%22Hemoglobinopathies+genetics%22">genetics</searchLink> <br /><searchLink fieldCode="MM" term="%22Hemoglobinopathies%22">Hemoglobinopathies*</searchLink>/<searchLink fieldCode="MM" term="%22Hemoglobinopathies+diagnosis%22">diagnosis</searchLink> <br /><searchLink fieldCode="MM" term="%22Nanopore+Sequencing%22">Nanopore Sequencing*</searchLink>/<searchLink fieldCode="MM" term="%22Nanopore+Sequencing+methods%22">methods</searchLink> <br /><searchLink fieldCode="MM" term="%22Gene+Library%22">Gene Library*</searchLink><br /><searchLink fieldCode="MH" term="%22beta-Globins%22">beta-Globins</searchLink>/<searchLink fieldCode="MH" term="%22beta-Globins+genetics%22">genetics</searchLink> ; <searchLink fieldCode="MH" term="%22Humans%22">Humans</searchLink> ; <searchLink fieldCode="MH" term="%22Retrospective+Studies%22">Retrospective Studies</searchLink> ; <searchLink fieldCode="MH" term="%22Genetic+Variation%22">Genetic Variation</searchLink>
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  Label: Abstract
  Group: Ab
  Data: 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.<br />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).<br />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.<br />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.<br /> (© Association for Diagnostics & Laboratory Medicine 2026. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)
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  Data: 2023A1515110676 Guangdong Basic and Applied Basic Research Foundation; 2024B03J1036 Science and Technology Program of Guangzhou
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