Application practice of the long read single-molecule sequencing technology in clinical genetics_West China Medical Journal

Authors：

REN Jun ^1,2 , GAO Meng ^1,2 , PENG Cuiting ^1,2 ,  LIU Shanling ^1,2

1. Department of Medical Genetics / Center for Prenatal Diagnosis, West China Second University Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. China;
2. Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, Chengdu, Sichuan 610041, P. R. China;

Corresponding author：

LIU Shanling, Email: sunny630@126.com

Keywords：

Long read single-molecule sequencing technology; monogenic disorder; chromosomal structural abnormality; preimplantation genetic testing for monogenic disorder

DOI：

10.7507/1002-0179.202405023

Video：

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In recent years, the application of the long read single-molecule sequencing technology in clinical genetics testing has been increasingly widespread. Due to its sequencing process not requiring amplification and its characteristic of single-molecule sequencing and long reads, it has unique advantages compared to traditional molecular genetics testing such as Sanger sequencing, next-generation sequencing and chromosomal microarray. In this article, its application in the detection of chromosomal structural abnormalities, monogenic diseases, and preimplantation genetic testing for monogenic diseases were discussed. Along with a comparative analysis of the advantages and disadvantages of the long read single-molecule sequencing technology in these areas compared to traditional molecular genetics testing techniques. This article preliminarily explores the application prospects and value of the long read single-molecule sequencing technology in clinical genetics testing.

Citation： REN Jun, GAO Meng, PENG Cuiting, LIU Shanling. Application practice of the long read single-molecule sequencing technology in clinical genetics. West China Medical Journal, 2025, 40(1): 92-96. doi: 10.7507/1002-0179.202405023 Copy

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9.	Madan K. Balanced complex chromosome rearrangements: reproductive aspects. A review. Am J Med Genet A, 2012, 158A(4): 947-963.
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11.	Chau MHK, Li Y, Dai P, et al. Investigation of the genetic etiology in male infertility with apparently balanced chromosomal structural rearrangements by genome sequencing. Asian J Androl, 2022, 24(3): 248-254.
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14.	Zeng X, Lin D, Liang D, et al. Gene sequencing and result analysis of balanced translocation carriers by third-generation gene sequencing technology. Sci Rep, 2023, 13(1): 7004.
15.	Goodwin S, McPherson JD, McCombie WR. Coming of age: ten years of next-generation sequencing technologies. Nat Rev Genet, 2016, 17(6): 333-351.
16.	Hassan S, Bahar R, Johan MF, et al. Next-generation sequencing (NGS) and third- generation sequencing (TGS) for the diagnosis of thalassemia. Diagnostics (Basel), 2023, 13(3): 373.
17.	Liang Q, Gu W, Chen P, et al. A more universal approach to comprehensive analysis of thalassemia alleles (CATSA). J Mol Diagn, 2021, 23(9): 1195-1204.
18.	Liang Q, He J, Li Q, et al. Evaluating the clinical utility of a long-read sequencing- based approach in prenatal diagnosis of thalassemia. Clin Chem, 2023, 69(3): 239-250.
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22.	中华医学会医学遗传学分会遗传病临床实践指南撰写组. α-地中海贫血的临床实践指南. 中华医学遗传学杂志, 2020, 37(3): 235-242.
23.	Liu Y, Chen M, Liu J, et al. Comprehensive analysis of congenital adrenal hyperplasia using long-read sequencing. Clin Chem, 2022, 68(7): 927-939.
24.	Liu Y, Li D, Yu D, et al. Comprehensive analysis of hemophilia A (CAHEA): towards full characterization of the F8 gene variants by long-read sequencing. Thromb Haemost, 2023, 123(12): 1151-1164.
25.	Liang Q, Liu Y, Liu Y, et al. Comprehensive analysis of fragile X syndrome: full characterization of the FMR1 locus by long-read sequencing. Clin Chem, 2022, 68(12): 1529-1540.
26.	Yao F, Hao N, Li D, et al. Long-read sequencing enables comprehensive molecular genetic diagnosis of fabry disease. Hum Genomics, 2024, 18(1): 133.
27.	Tsuiko O, El Ayeb Y, Jatsenko T, et al. Preclinical workup using long-read amplicon sequencing provides families with de novo pathogenic variants access to universal preimplantation genetic testing. Hum Reprod, 2023, 38(3): 511-519.
28.	Capuano I, Buonanno P, Riccio E, et al. Therapeutic advances in ADPKD: the future awaits. J Nephrol, 2022, 35(2): 397-415.
29.	Reiterová J, Tesař V. Autosomal dominant polycystic kidney disease: from pathophysiology of cystogenesis to advances in the treatment. Int J Mol Sci, 2022, 23(6): 3317.
30.	Peng C, Chen H, Ren J, et al. A long-read sequencing and SNP haplotype-based novel preimplantation genetic testing method for female ADPKD patient with de novo PKD1 mutation. BMC Genomics, 2023, 24(1): 521.
31.	Mariya T, Shichiri Y, Sugimoto T, et al. Clinical application of long-read nanopore sequencing in a preimplantation genetic testing pre-clinical workup to identify the junction for complex Xq chromosome rearrangement-related disease. Prenat Diagn, 2023, 43(3): 304-313.
32.	Pei Z, Deng K, Lei C, et al. Identifying balanced chromosomal translocations in human embryos by Oxford nanopore sequencing and breakpoints region analysis. Front Genet, 2022, 12: 810900.

1. Logsdon GA, Vollger MR, Eichler EE. Long-read human genome sequencing and its applications. Nat Rev Genet, 2020, 21(10): 597-614.
2. Xiao T, Zhou W. The third generation sequencing: the advanced approach to genetic diseases. Transl Pediatr, 2020, 9(2): 163-173.
3. Ling X, Wang C, Li L, et al. Third-generation sequencing for genetic disease. Clin Chim Acta, 2023, 551: 117624.
4. Eid J, Fehr A, Gray J, et al. Real-time DNA sequencing from single polymerase molecules. Science, 2009, 323(5910): 133-138.
5. Clarke J, Wu HC, Jayasinghe L, et al. Continuous base identification for single-molecule nanopore DNA sequencing. Nat Nanotechnol, 2009, 4(4): 265-270.
6. Flowers NJ, Burgess T, Giouzeppos O, et al. Genome-wide noninvasive prenatal screening for carriers of balanced reciprocal translocations. Genet Med, 2020, 22(12): 1944-1955.
7. Dong Z, Wang H, Chen H, et al. Identification of balanced chromosomal rearrangements previously unknown among participants in the 1000 genomes project: implications for interpretation of structural variation in genomes and the future of clinical cytogenetics. Genet Med, 2018, 20(7): 697-707.
8. Alfarawati S, Fragouli E, Colls P, et al. First births after preimplantation genetic diagnosis of structural chromosome abnormalities using comparative genomic hybridization and microarray analysis. Hum Reprod, 2011, 26(6): 1560-1574.
9. Madan K. Balanced complex chromosome rearrangements: reproductive aspects. A review. Am J Med Genet A, 2012, 158A(4): 947-963.
10. Pellestor F, Anahory T, Lefort G, et al. Complex chromosomal rearrangements: origin and meiotic behavior. Hum Reprod Update, 2011, 17(4): 476-494.
11. Chau MHK, Li Y, Dai P, et al. Investigation of the genetic etiology in male infertility with apparently balanced chromosomal structural rearrangements by genome sequencing. Asian J Androl, 2022, 24(3): 248-254.
12. Zhang S, Pei Z, Lei C, et al. Detection of cryptic balanced chromosomal rearrangements using high-resolution optical genome mapping. J Med Genet, 2023, 60(3): 274-284.
13. Madjunkova S, Sundaravadanam Y, Antes R, et al. Detection of structural rearrangements in embryos. N Engl J Med, 2020, 382(25): 2472-2474.
14. Zeng X, Lin D, Liang D, et al. Gene sequencing and result analysis of balanced translocation carriers by third-generation gene sequencing technology. Sci Rep, 2023, 13(1): 7004.
15. Goodwin S, McPherson JD, McCombie WR. Coming of age: ten years of next-generation sequencing technologies. Nat Rev Genet, 2016, 17(6): 333-351.
16. Hassan S, Bahar R, Johan MF, et al. Next-generation sequencing (NGS) and third- generation sequencing (TGS) for the diagnosis of thalassemia. Diagnostics (Basel), 2023, 13(3): 373.
17. Liang Q, Gu W, Chen P, et al. A more universal approach to comprehensive analysis of thalassemia alleles (CATSA). J Mol Diagn, 2021, 23(9): 1195-1204.
18. Liang Q, He J, Li Q, et al. Evaluating the clinical utility of a long-read sequencing- based approach in prenatal diagnosis of thalassemia. Clin Chem, 2023, 69(3): 239-250.
19. Peng C, Zhang H, Ren J, et al. Analysis of rare thalassemia genetic variants based on third-generation sequencing. Sci Rep, 2022, 12(1): 9907.
20. Zhang Q, Fan X, Xu M, et al. Hb H disease caused by multiple mutations in the polyadenylation signal site and - -^SEA/αα. Hemoglobin, 2017, 41(3): 189-192.
21. Charoenwijitkul T, Singha K, Fucharoen G, et al. Molecular characteristics of α⁺-thalassemia (3.7 kb deletion) in Southeast Asia: molecular subtypes, haplotypic heterogeneity, multiple founder effects and laboratory diagnostics. Clin Biochem, 2019, 71: 31-37.
22. 中华医学会医学遗传学分会遗传病临床实践指南撰写组. α-地中海贫血的临床实践指南. 中华医学遗传学杂志, 2020, 37(3): 235-242.
23. Liu Y, Chen M, Liu J, et al. Comprehensive analysis of congenital adrenal hyperplasia using long-read sequencing. Clin Chem, 2022, 68(7): 927-939.
24. Liu Y, Li D, Yu D, et al. Comprehensive analysis of hemophilia A (CAHEA): towards full characterization of the F8 gene variants by long-read sequencing. Thromb Haemost, 2023, 123(12): 1151-1164.
25. Liang Q, Liu Y, Liu Y, et al. Comprehensive analysis of fragile X syndrome: full characterization of the FMR1 locus by long-read sequencing. Clin Chem, 2022, 68(12): 1529-1540.
26. Yao F, Hao N, Li D, et al. Long-read sequencing enables comprehensive molecular genetic diagnosis of fabry disease. Hum Genomics, 2024, 18(1): 133.
27. Tsuiko O, El Ayeb Y, Jatsenko T, et al. Preclinical workup using long-read amplicon sequencing provides families with de novo pathogenic variants access to universal preimplantation genetic testing. Hum Reprod, 2023, 38(3): 511-519.
28. Capuano I, Buonanno P, Riccio E, et al. Therapeutic advances in ADPKD: the future awaits. J Nephrol, 2022, 35(2): 397-415.
29. Reiterová J, Tesař V. Autosomal dominant polycystic kidney disease: from pathophysiology of cystogenesis to advances in the treatment. Int J Mol Sci, 2022, 23(6): 3317.
30. Peng C, Chen H, Ren J, et al. A long-read sequencing and SNP haplotype-based novel preimplantation genetic testing method for female ADPKD patient with de novo PKD1 mutation. BMC Genomics, 2023, 24(1): 521.
31. Mariya T, Shichiri Y, Sugimoto T, et al. Clinical application of long-read nanopore sequencing in a preimplantation genetic testing pre-clinical workup to identify the junction for complex Xq chromosome rearrangement-related disease. Prenat Diagn, 2023, 43(3): 304-313.
32. Pei Z, Deng K, Lei C, et al. Identifying balanced chromosomal translocations in human embryos by Oxford nanopore sequencing and breakpoints region analysis. Front Genet, 2022, 12: 810900.

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