第三代测序技术在血型基因多态性研究的应用

doi:10.3969/j.issn.1671-2587.2025.05.018

临床输血与检验 ›› 2025, Vol. 27 ›› Issue (5): 701-711.DOI: 10.3969/j.issn.1671-2587.2025.05.018

第三代测序技术在血型基因多态性研究的应用

梁丽欣¹, 梁结玲¹, 魏亚明^2,3,4, 苑召虎^2,3,4

¹肇庆市第一人民医院输血科,广东肇庆 526000;
²广州市第一人民医院输血医学科,广东广州 510180;
³广东省精准输血技术工程研究中心,广东广州 510180;
⁴广州医科大学输血与血液病研究所,广东广州 510180

收稿日期:2025-09-05 修回日期:2025-09-12 出版日期:2025-10-20 发布日期:2025-10-11
通讯作者: 苑召虎,主要从事输血免疫学方面研究,（E-mail）eyyuanzhaohu@scut.edu.cn。共同通信作者：魏亚明,主要从事输血免疫学方面研究,（E-mail）eywym@scut.edu.cn。
作者简介:梁丽欣,主要从事输血医学方面研究,（E-mail）419084196@qq.com。

The Application of Third-generation Sequencing Technology in the Study of Blood Group Gene Polymorphism

LIANG Lixin¹, LIANG Jieling¹, WEI Yaming^2,3,4, YUAN Zhaohu^2,3,4

¹Department of Blood Transfusion Zhaoqing First People's Hospital, Zhaoqing, Guangdong 526000;
²Department of Blood Transfusion Guangzhou First People's Hospital, Guangdong 510180;
³Guangdong Engineering Research Center for Precision Blood Transfusion Technology, Guangdong 510180;
⁴Institute of Blood Transfusion and Hematology, Guangzhou Medical University, Guangzhou, Guangdong 510180

Received:2025-09-05 Revised:2025-09-12 Online:2025-10-20 Published:2025-10-11

摘要/Abstract

摘要： 血型基因多态性在遗传学研究、临床输血和移植免疫等领域中具有重要意义,血型基因多态性决定了血型抗原多态性。近年来,精准医学基因检测行业领域里的研究热点——第三代测序（third-generation sequencing, TGS）技术,即单分子测序技术,为整个基因领域打开了一扇全新的大门。基于TGS技术的血型基因研究成果相继发表,持续拓展着该技术在血型多态性分析中的应用边界。本综述梳理TGS技术在血型基因多态性研究的最新进展及其在临床输血医学中的应用。

关键词: 第三代测序, 血型基因, 单分子实时测序, 牛津纳米孔测序

Abstract: Blood group gene polymorphism holds significant importance in the fields of genetic research, clinical blood transfusion, and transplant immunology, as it determines the polymorphism of blood group antigens. In recent years, third-generation sequencing (TGS) technology, also known as single-molecule sequencing, has emerged as a research hotspot in the precision medicine genetic testing industry, opening a new door for the entire field of genetics. Research findings on blood group genes based on TGS technology have been successively published, continuously expanding the application boundaries of this technology in blood group polymorphism analysis. This review summarizes the latest advancements in TGS technology for studying blood group gene polymorphism and its applications in clinical transfusion medicine.

Key words: Third-generation sequencing, Blood group genes, Single molecule real-time, Oxford nanopore sequencing

中图分类号:

R394.3

梁丽欣, 梁结玲, 魏亚明, 苑召虎. 第三代测序技术在血型基因多态性研究的应用[J]. 临床输血与检验, 2025, 27(5): 701-711.

LIANG Lixin, LIANG Jieling, WEI Yaming, YUAN Zhaohu. The Application of Third-generation Sequencing Technology in the Study of Blood Group Gene Polymorphism[J]. JOURNAL OF CLINICAL TRANSFUSION AND LABORATORY MEDICINE, 2025, 27(5): 701-711.

参考文献

[1] 赵桐茂. 血型分子分型技术的进展和应用[J]. 临床输血与检验,2017,19(5):530-536.
[2] 李晓健,黄象艳. 红细胞血型分子诊断技术的研究进展[J]. 国际输血及血液学杂志,2021,44(3):271-276.
[3] Table of blood group systems. International Society of Blood Transfusion Working Party [EB/OL]. (2025, 5). https://www.isbtweb.org/resource/tableofbloodgroupsystems.html.
[4] LI H Y,GUO K.Blood group testing[J]. Front Med,2022, 9:827619.
[5] SIMTONG P,JUMNAINSONG A,LEELAYUWAT C.Genetic profile of RHCE,Kell,Duffy,Kidd,Diego and MNS hybrid glycophorins blood groups in ethnic northeastern Thais: Alleles,genotypes and risk of alloimmunisation[J]. Transfus Med,2024,34(4):287-297.
[6] 李小飞,马春娅,蔡晓红,等. 红细胞血型抗原拓展匹配适用范围中国专家共识[J]. 临床输血与检验,2024,26(3): 289-298.
[7] 梁伟,周建霖,杨亮,等. PCR-SSP技术在红细胞ABO血型快速基因分型中的应用研究[J]. 中华全科医学,2015, 13(8):1323-1327.
[8] CROSSLEY B M,BAI J F,GLASER A,et al.Guidelines for Sanger sequencing and molecular assay monitoring[J]. J Vet Diagn Invest,2020,32(6):767-775.
[9] 刘婉彤,童梅,林福玉,等. 分子诊断技术的临床应用进展[J]. 生物技术通讯,2020,31(2):240-250.
[10] HE Y M,HONG X Z,ZHANG J J,et al.Analysis of the genomic sequence of ABO allele using next-generation sequencing method[J]. Front Immunol,2022,13:814263.
[11] BRONNER I F,DAWSON E,PARK N,et al.Evaluation of controls, quality control assays, and protocol optimisations for PacBio HiFi sequencing on diverse and challenging samples[J]. Front Genet,2025,15:1505839.
[12] SALAVA H,DEÁK T,CZEPE C,et al. Sample and library preparation for PacBio long-read sequencing in grapevine[J]. Methods Mol Biol,2024,2787:183-197.
[13] 李红英,覃再隆. 单分子测序技术的应用与研究进展[J]. 广西医学,2024,46(12):1935-1940.
[14] QUAIL M A,SMITH M,COUPLAND P,et al.A tale of three next generation sequencing platforms: comparison of Ion Torrent,Pacific Biosciences and Illumina MiSeq sequencers[J]. BMC Genomics,2012,13:341.
[15] RHOADS A,AU K F.PacBio sequencing and its applications[J]. Genomics Proteomics Bioinformatics, 2015,13(5):278-289.
[16] CHANG X Y,HAN W T,CHEN X M,et al.Re-annotation improved large-scale assembly of the reef-building coral Acropora intermedia[J]. Sci Data,2025,12(1):1504.
[17] YUDA A,NAKAMURA T,MOMOSE S,et al.A comprehensive approach for identifying filaggrin mutations and copy number variants by long-read sequencing[J]. Genomics,2025,117(4):111055.
[18] NARDONE G G,ANDRIOLETTI V,SANTIN A,et al.A hitchhiker guide to structural variant calling: a comprehensive benchmark through different sequencing technologies[J]. Biomedicines,2025,13(8):1949.
[19] SMRT® sequencing—Delivering highly accurate long reads to drive discovery in life science. PacBio—Sequence with confidence. [EB/OL]. (2021, 12). https://www.pacb.com/wp-content/uploads/SMRT-Sequencing-Brochure-Delivering-highly-accurate-long-reads-to-drive-discovery-in-life-science.pdf.
[20] DEAMER D,AKESON M,BRANTON D.Three decades of nanopore sequencing[J]. Nat Biotechnol,2016,34(5): 518-524.
[21] YU G C,XU X D,SHEN Y Q,et al.Diagnostic accuracy of nanopore sequencing for the rapid diagnosis of pulmonary tuberculosis: A protocol for a systematic review and meta-analysis[J]. PLoS One,2024,19(6): e0304162.
[22] WANG Y H,ZHAO Y,BOLLAS A,et al.Nanopore sequencing technology,bioinformatics and applications[J]. Nat Biotechnol,2021,39(11):1348-1365.
[23] GERSHOW M,GOLOVCHENKO J A.Recapturing and trapping single molecules with a solid-state nanopore[J]. Nat Nanotechnol,2007,2(12):775-779.
[24] TUNCEL G,BALCı M C,AKAN G,et al.An Oxford nanopore technologies-based sequencing assay for molecular diagnosis of phenylketonuria and variant frequencies in a Turkish cohort[J]. Int J Genomics,2025, 2025:5552662.
[25] WARREN R L,BIROL I.Streaming long-read sequence alignments for HLA predictions using HLAminer[J]. Curr Protoc,2025,5(3):e70124.
[26] WU H C,CHIU Y T,WU I C,et al.Streamlining whole genome sequencing for clinical diagnostics with ONT technology[J]. Sci Rep,2025,15(1):6270.
[27] ZHANG T,XING W Q,WANG A M,et al.Comparison of long-read methods for sequencing and assembly of lepidopteran pest genomes[J]. Int J Mol Sci,2022,24(1). DOI:10.3390/ijms24010649.
[28] MOZHEIKO E,YI H,LU A Z,et al.Pre-assembly NGS correction of ONT reads achieves HiFi-level assembly quality[J]. Genome,2025,68:1-9.
[29] LIU Y,TAHA H B,ZHANG Q Y,et al.NANOME: A Nextflow pipeline for haplotype-aware allele-specific consensus DNA methylation detection by nanopore long-read sequencing[J]. bioRxiv,2025. DOI:10.1101/2025.06.29.662079.
[30] ABDOLLAHZADEH E,MORTAZAVI A.Dogme: A nextflow pipeline for reprocessing nanopore RNA and DNA modifications[J]. bioRxiv,2025. DOI:10.1101/2025.06.04.657941.
[31] TOUNSI W A,LENIS V P,TAMMI S M,et al.Rh blood group D antigen genotyping using a portable nanopore-based sequencing device: proof of principle[J]. Clin Chem,2022,68(9):1196-1201.
[32] ZHANG Z,AN H H,VEGE S,et al.Accurate long-read sequencing allows assembly of the duplicated RHD and RHCE genes harboring variants relevant to blood transfusion[J]. Am J Hum Genet,2022,109(1):180-191.
[33] PAYNE A,HOLMES N,RAKYAN V,et al.BulkVis: a graphical viewer for Oxford nanopore bulk FAST5 files[J]. Bioinformatics,2019,35(13):2193-2198.
[34] GUEUNING M,THUN G A,WITTIG M,et al.Haplotype sequence collection of ABO blood group alleles by long-read sequencing reveals putative A1-diagnostic variants[J]. Blood Adv,2023,7(6):878-892.
[35] 刘静怡,崔丹荔,王芳,等. 重庆地区RhD变异型无偿献血者的基因多态性及其表型研究[J]. 中国输血杂志,2024, 37(8):879-885.
[36] XU H,SU X M,ZUO Q Q,et al.The research of a large-scale analysis platform for MNS blood group identification based on long-read sequencing[J]. Transfus Med Rev,2024,38(4):150836.
[37] 唐炳娣,蔡仲仁,邓泳诗,等. 1例RHD-CE(3-7)-D基因重组与RHCE变异型患者的血清学与分子生物学分析[J]. 分子诊断与治疗杂志,2024,16(6):1183-1186.
[38] LI M H,WANG L P,LI A J,et al.Integrated analyses reveal unexpected complex inversion and recombination in RH genes[J]. Blood Adv,2024,8(12):3154-3165.
[39] WANG Z,CHU Y S,XIAO Y L,et al.Detecting serologically difficult ABO blood groups using single-molecule real-time sequencing technology[J]. Vox Sang,2024,119(10):1096-1105.
[40] THUN G A,GUEUNING M,SIGURDARDOTTIR S,et al.Novel regulatory variant in ABO intronic RUNX1 binding site inducing a(3) phenotype[J]. Vox Sang,2024,119(4):377-382.
[41] 李艳,周丽玲,马肇泽,等. 25例B亚型/AB亚型的ABO基因序列分析[J]. 临床输血与检验,2024,26(6):787-791.
[42] KOMINATO Y,TSUCHIYA T,HATA N,et al.Transcription of human ABO histo-blood group genes is dependent upon binding of transcription factor CBF/NF-Y to minisatellite sequence[J]. J Biol Chem,1997, 272(41):25890-25898.
[43] KIM T Y,YU H B,SEO J Y,et al.Molecular basis of weak A subgroups in the Korean population: Identification of three novel subgroup-causing variants in the ABO regulatory regions[J]. Transfusion,2022,62(2):286-291.
[44] SANO R,NAKAJIMA T,TAKAHASHI K,et al.Expression of ABO blood-group genes is dependent upon an erythroid cell-specific regulatory element that is deleted in persons with the B(m) phenotype[J]. Blood, 2012,119(22):5301-5310.
[45] SHAO L N,XIA Y X,YANG Y C,et al.PacBio third-generation sequencing reveals an ABO gene promoter mutation,c.-35_-18del,leading to weakened B antigen expression[J]. Ann Lab Med,2024,44(6):614-616.
[46] HONG X Z,YING Y L,ZHANG J J,et al.Six splice site variations,three of them novel,in the ABO gene occurring in nine individuals with ABO subtypes[J]. J Transl Med,2021,19(1):470.
[47] KONG Y K,WANG L,KONG C Q,et al.A novel c.29-3C>G variant on the B allele forms the Bel phenotype[J]. Transfusion,2024,64(7):E28-E29.
[48] SHAO L N,SONG W Q,ZHOU L,et al.Characterization of a novel AEL allele harboring a c.28+5G>A mutation on the ABO*A2.01 background: a study utilizing PacBio third-generation sequencing and functional assays[J]. Front Immunol,2024,15:1396426.
[49] HE Y L,YU L,ZHANG J W,et al.Serological and molecular characterization of novel ABO variants including an interesting B(A) subgroup[J]. Transfusion, 2024,64(12):2364-2370.
[50] FICHOU Y,BERLIVET I,RICHARD G,et al.Defining blood group gene reference alleles by long-read sequencing: proof of concept in the ACKR1 gene encoding the Duffy antigens[J]. Transfus Med Hemother,2020, 47(1):23-32.
[51] LANE W J,GLEADALL N S,AESCHLIMANN J,et al.Multiple GYPB gene deletions associated with the U- phenotype in those of African ancestry[J]. Transfusion, 2020,60(6):1294-1307.
[52] YE L Y,WANG C,LI A J,et al.Targeted next-generation sequencing and long-read HiFi sequencing provide novel insights into clinically significant KLF1 variants[J]. BMC Genomics,2024,25(1):230.
[53] RAUD L,KA C,GOURLAOUEN I,et al.Functional analysis of novel RHD variants: splicing disruption is likely to be a common mechanism of variant D phenotype[J]. Transfusion,2019,59(4):1367-1375.
[54] 罗亚林. “亚洲型”DEL红细胞表达完整D抗原的分子基础研究[D]. 广州:南方医科大学,2024.
[55] 梁延连,苏宇清,吴凡,等. RhD mRNA剪接体实时荧光定量方法的建立[J]. 中国输血杂志,2017,30(10):1126-1128.
[56] 官笑羽,纪欣,李玲,等. 基于三代测序技术的RHD、RHCE基因mRNA研究[J]. 中国输血杂志,2023,36(2): 115-120.
[57] 李慧,冯晨晨,陈青. ABO血型基因分型方法的进展与应用[J]. 中国实验血液学杂志,2022,30(2):622-626.
[58] JOHNSEN J M.Using red blood cell genomics in transfusion medicine[J]. Hematology Am Soc Hematol Educ Program,2015,2015:168-176.
[59] SANDLER S G,HORN T,KELLER J,et al.A model for integrating molecular-based testing in transfusion services[J]. Blood Transfus,2016,14(6):566-572.
[60] FLEGEL W A,GOTTSCHALL J L,DENOMME G A. Implementing mass-scale red cell genotyping at a blood center[J]. Transfusion,2015,55(11):2610-2615; quiz2609.
[61] QING Y,ZOU H M,LIU B J,et al.A novel frameshift mutation in RHAG leads to Rh(null) phenotype in a Chinese individual[J]. Transfusion,2024,64(5):789-792.
[62] SHAO L N,YANG Y C,XIA Y X,et al.Novel missense mutation c.797T>C (p.Met266Thr) gives rise to the rare B(A) phenotype in a Chinese family[J]. Vox Sang,2024, 119(4):383-387.
[63] 李剑平,李娇,李晓丰. 红细胞血型的免疫学检测与基因检测的现状和展望[J]. 诊断学理论与实践,2015,14(6):491-493.
[64] 朱发明,毛伟,张志欣. 血小板配合性输注的分析和展望[J]. 中国输血杂志,2022,35(11):1097-1100.
[65] ZHAO P Z,LYU Q L,XU Y,et al.A novel approach to simultaneous genotyping of human platelet antigen systems and human leucocyte antigen class Ⅰ loci using PacBio long-read sequencing[J]. Vox Sang,2025,120(1): 63-70.
[66] JACKSON M E,BAKER J M.Hemolytic disease of the fetus and newborn: historical and current state[J]. Clin Lab Med,2021,41(1):133-151.
[67] CLARKE G,BODNAR M,LOZANO M,et al.Vox Sanguinis International forum on the selection and preparation of blood components for intrauterine transfusion[J]. Vox Sang,2020,115(8):e18-e38.
[68] 姚文娟,高珺,穆启明,等. 亚洲型DEL母亲产生抗-c致新生儿溶血病的母子输血救治[J]. 中国输血杂志,2024, 37(3):352-356.
[69] 卢霞,潘英芳,赵爱平,等. RhD阴性患者输注“亚洲型”DEL型红细胞短期内引起高效价抗-D的同种免疫研究[J]. 临床输血与检验,2023,25(6):835-839.
[70] JI Y L,LUO Y L,WEN J Z,et al.Patients with Asian-type DEL can safely be transfused with RhD-positive blood[J]. Blood,2023,141(17):2141-2150.
[71] KOELEWIJN J M,VRIJKOTTE T G M,DE HAAS M,et al. Risk factors for the presence of non-Rhesus D red blood cell antibodies in pregnancy[J]. BJOG,2009, 116(5):655-664.
[72] 储玉霜,肖彦琳,望喆,等. 单分子实时测序在HLA基因分型中的应用初探[J]. 临床输血与检验,2024,26(3):359-365.
[73] WESTHOFF C M,FLOCH A.Blood group genotype matching for transfusion[J]. Br J Haematol,2025,206(1): 18-32.
[74] VOLLGER M R,LOGSDON G A,AUDANO P A,et al.Improved assembly and variant detection of a haploid human genome using single-molecule,high-fidelity long reads[J]. Ann Hum Genet,2020,84(2):125-140.
[75] LOGSDON G A,VOLLGER M R,EICHLER E E.Long-read human genome sequencing and its applications[J]. Nat Rev Genet,2020,21(10):597-614.
[76] LI C H,CHNG K R,BOEY E J H,et al. INC-Seq: accurate single molecule reads using nanopore sequencing[J]. Gigascience,2016,5(1):34.
[77] WILSON B D,EISENSTEIN M,SOH H T.High-fidelity nanopore sequencing of ultra-short DNA targets[J]. Anal Chem,2019,91(10):6783-6789.
[78] 朱孟琳,何燕琴,谢水莲,等. 纳米孔基因测序技术在HLA基因分型中的应用[J]. 赣南医学院学报,2024,44(1): 99-104.
[79] VEGE S,FLOCH A,BURGOS A,et al.Two novel variants that disrupt the RHCE start Codon with varying effect on RhCE antigen expression[J]. Transfusion, 2023,63(4):E12-E14.
[80] WANG C B,TANG Y C,ZHANG P P,et al.Detection and phenotype analysis of a novel Ael blood group allele[J]. Vox Sang,2024,119(1):74-78.
[81] OU G Y,WEI Y L,LI M,et al.Identification of a novel A allele with a c.421T>C variant on the ABO*A1.02 allele in a Chinese individual[J]. Transfusion,2024, 64(6):E23-E25.
[82] SHAO L,MA L,XIAO J Y,et al.PacBio third-generation sequencing detects a new variant,c.27delC,in exon 1 of the ABO gene resulting in a weak B phenotype[J]. Transfus Med,2025,35(1):103-105.
[83] 杨贺才,关茵,马晓莉,等. 新等位基因c.175_176insGA致B el亚型的家系分析[J]. 中华检验医学杂志,2024,47(10): 1206-1211.
[84] 望喆. 基于三代测序技术在ABO疑难血型鉴定中的应用[D]. 合肥:安徽医科大学,2024.
[85] 梁爽,吴凡,梁延连,等. ABO基因c.917T>C变异对B糖基转移酶表达及功能的影响[J]. 中国实验血液学杂志, 2025,33(1):269-275.
[86] SHAO L N,YANG Y C,XIA Y X,et al.Identification of a novel O allele with c.777C>G on an ABO*O.01.02 background[J]. Transfusion,2024,64(4):E11-E12.
[87] SHAO L N,XIA Y X,LI C X,et al.Identification of a novel DEL allele with c.411G>C on the RHD*01EL.01 allele background[J]. Transfusion,2024,64(6):E26-E27.
[88] CI X,FAN X X,LV P,et al.Identification of a novel RHCE*Ce (829G>A) allele associated with absence of C and e antigens expression[J]. Transfus Med,2024, 34(5):445-449.
[89] SHAO L N,YANG Y C,LI C X,et al.Identification of a novel FUT1 allele with c.425G > A on the FUT1*01 allele background[J]. Indian J Hematol Blood Transfus, 2025,41(2):393-397.

第三代测序技术在血型基因多态性研究的应用

The Application of Third-generation Sequencing Technology in the Study of Blood Group Gene Polymorphism

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 4

编辑推荐

Metrics

本文评价

关于本刊

作者中心

在线期刊

访问统计

联系我们

[1]	任道菊, 杨金华, 李小薇, 肖军, 李翠莹. 应用孕妇外周血cff-DNA检测胎儿RHD血型基因的方法学研究^*[J]. 临床输血与检验, 2025, 27(2): 163-168.
[2]	储玉霜, 肖彦琳, 望喆, 冯青青, 李亭亭, 卞茂红. 单分子实时测序在HLA基因分型中的应用初探^*[J]. 临床输血与检验, 2024, 26(3): 359-365.
[3]	赵桐茂. 血型分子分型技术的进展和应用[J]. 临床输血与检验, 2017, 19(5): 530-536.
[4]	宋艳艳, 周雪莹, 欧阳旭, 徐敏, 于笑难. B（A）02亚型分子生物学鉴定1例[J]. 临床输血与检验, 2016, 18(4): 338-341.