Advances and Applications of Nanoscale Nanopore Sequencing Technology

doi:10.3870/j.issn.1672-8009.2026.04.015

Abstract

Abstract: As an innovative high-throughput sequencing technology,nanopore sequencing has become a vital tool in genomics research and clinical diagnostics due to its unique advantages of real-time detection,long-read capability,and PCR-free amplification.In recent years,significant advancements in sequencing speed and data accuracy have driven progress in genome sequencing,transcriptome analysis,and multi-omics integration studies.However,nanopore sequencing still faces challenges such as signal noise,complex data processing,and accuracy improvement.This article systematically reviews the fundamental principles and technological evolution of nanopore sequencing,with a focus on its latest advancements in genome sequencing,transcriptome analysis,and clinical applications.In addition,by exploring current technical bottlenecks and future development directions,this article aims to provide comprehensive theoretical support and practical guidance for researchers,thereby promoting the widespread application of nanopore sequencing technology in life sciences and medicine.

Key words: nanopore sequencing, high-throughput sequencing, genomics, transcriptome, clinical diagnosis, technological progress

CLC Number:

Q819

WU Haiming, ZHAN Weiwu, LIU Jiacui. Advances and Applications of Nanoscale Nanopore Sequencing Technology[J]. Journal of Medical Molecular Biology, 2026, 23(4): 471-476.

References

[1] LANG J,SUN J,YANG Z,et al.Nano2 NGS-Muta:a framework for converting nanopore sequencing data to NGS-liked sequencing data for hotspot mutation detection[J].NAR Genom Bioinform,2022,4(2):lqac033.
[2] XIE S,LEUNG A W,ZHENG Z,et al.Applications and potentials of nanopore sequencing in the(epi)genome and(epi)transcriptome era[J].Innovation(Camb),2021,2(4):100153.
[3] ZHU Z.SqueezeCall:nanopore basecalling using a Squeezeformer network[J].GigaByte,2025,2025:gigabyte148.
[4] YEH Y M,LU Y C.MSRCall:a multi-scale deep neural network to basecall Oxford Nanopore sequences[J].Bioinformatics,2022,38(16):3877-3884.
[5] LIAO Y,GONG J,WANG X,et al.Applying nanopore sequencing in the etiological diagnosis of bloodstream infection[J].Front Microbiol,2025,16:1554965.
[6] RIAZ N,LEUNG P,BARTON K,et al.Adaptation of Oxford Nanopore technology for hepatitis C whole genome sequencing and identification of within-host viral variants[J].BMC Genomics,2021,22(1):148.
[7] SHARMA A,BANSAL S,MOORE M D,et al.Exploring the frontiers of nanopore sequencing in food safety and food microbiology[J].Annu Rev Food Sci Technol,2025,16(1):219-244.
[8] Chen Z,He X.Application of third-generation sequencing in cancer research[J].Medical Review,2021,1(2):150-171.
[9] SUN K,LIU Y,ZHOU X,et al.Nanopore sequencing technology and its application in plant virus diagnostics[J].Front Microbiol,2022,13:939666.
[10] WANG Y,ZHAO Y,BOLLAS A,et al.Nanopore sequencing technology,bioinformatics and applications[J].Nat Biotechnol,2021,39(11):1348-1365.
[11] BERKOVICH A K,PYSHKINA O A,ZORINA A A,et al.Direct determination of the structure of single biopolymer molecules using nanopore sequencing[J].Biochemistry,2024,89(Suppl 1):S234-S248.
[12] MAKAŁOWSKI W,SHABARDINA V.Bioinformatics of nanopore sequencing[J].J Hum Genet,2020,65(1):61-67.
[13] MACKENZIE M,ARGYROPOULOS C.An introduction to nanopore sequencing:Past,present,and future considerations[J].Micromachines,2023,14(2):459.
[14] NAPIERALSKI A,NOWAK R.Basecalling using joint raw and event nanopore data sequence-to-sequence processing[J].Sensors(Basel),2022,22(6):2275.
[15] RUSK N.More accurate nanopore sequencing[J].Nat Methods,2019,16(6):460.
[16] DUMSCHOTT K,SCHMIDT M H,CHAWLA H S,et al.Oxford Nanopore sequencing:New opportunities for plant genomics[J].J Exp Bot,2020,71(18):5313-5322.
[17] MOSS E L,MAGHINI D G,BHATT A S.Complete,closed bacterial genomes from microbiomes using nanopore sequencing[J].Nat Biotechnol,2020,38(6):701-707.
[18] RAMIREZ P,SUN W,DEHKORDI S K,et al.Long-read sequencing reveals genomic and epigenomic variation in the dark genome of human Alzheimer’s disease[J].Alzheimer’s & Dementia,2025,21(11):e70852.
[19] QIAO G,XU P,GUO T,et al.Genome-wide detection of structural variation in some sheep breeds using whole-genome long-read sequencing data[J].Zeitschrift für Tierzuchtung und Züchtungsbiologie,2024,141(4):403-414.
[20] LAMB H J,NGUYEN L T,COPLEY J P,et al.Imputation strategies for genomic prediction using nanopore sequencing[J].BMC Biol,2023,21(1):286.
[21] SUZUKI M,HASHIMOTO Y,HIRABAYASHI A,et al.Genomic epidemiological analysis of antimicrobial-resistant bacteria with nanopore sequencing[M]//Arakawa K.Nanopore Sequencing:Methods and Protocols.New York:Springer,2023:227-246.
[22] BOWDEN R,DAVIES R W,HEGER A,et al.Sequencing of human genomes with nanopore technology[J].Nat Commun,2019,10(1):1869.
[23] XIONG J,CHEN K K,XIE N B,et al.Bisulfite-free and single-base resolution detection of epigenetic DNA modification of 5-methylcytosine by methyltransferase-directed labeling with APOBEC3 A deamination sequencing[J].Anal Chem,2022,94(44):15489-15498.
[24] WANG Z X,CHEN F,HE B D,et al.Direct sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution unravels their distinct roles in Alzheimer’s disease[J].Adv Sci(Weinh),2025,12(38):e07843.
[25] MITTAL S,JENA M K,PATHAK B.Machine learning-assisted direct RNA sequencing with epigenetic RNA modification detection via quantum tunneling[J].Anal Chem,2024,96(28):11516-11524.
[26] ZHANG J,XIE S,XU J,et al.Cancer biomarkers discovery of methylation modification with direct high-throughput nanopore sequencing[J].Front Genet,2021,12:672804.
[27] CAI X,YAO Y.Epigenetic modifications of immune cells in rheumatoid arthritis[J].Ann Med,2025,57(1):2533432.
[28] WHITACRE L K,WILDHABER M L,JOHNSON G S,et al.Exploring genetic variation and population structure in a threatened species,Noturus placidus,with whole-genome sequence data[J].G3(Bethesda),2022,12(4):jkac046.
[29] CHOI J Y,LYE Z N,GROEN S C,et al.Nanopore sequencing-based genome assembly and evolutionary genomics of circum-basmati rice[J].Genome Biol,2020,21(1):21.
[30] XIANG X,GUO Y,ZHAO L,et al.Whole genome sequencing and structural variations provide insights into the body size traits of Hu sheep[J].Sci Data,2025,12(1):1373.
[31] MAXWELL L M,CLARK J D,WALSH J,et al.Ecological characteristics explain neutral genetic variation of three coastal sparrow species[J].Mol Ecol,2024,33(8):e17316.
[32] OGDEN R,VASILJEVIC N,PROST S.Nanopore sequencing in non-human forensic genetics[J].Emerg Top Life Sci,2021,5(3):465-473.
[33] FUJIMOTO A,WONG J H,YOSHII Y,et al.Whole-genome sequencing with long reads reveals complex structure and origin of structural variation in human genetic variations and somatic mutations in cancer[J].Genome Med,2021,13(1):65.
[34] WORKMAN R E,TANG A D,TANG P S,et al.Nanopore native RNA sequencing of a human poly(A)transcriptome[J].Nat Methods,2019,16(12):1297-1305.
[35] PHILPOTT M,WATSON J,THAKURTA A,et al.Nanopore sequencing of single-cell transcriptomes with scCOLOR-seq[J].Nat Biotechnol,2021,39(12):1517-1520.
[36] LI J,GUAN D,HALSTEAD M M,et al.Transcriptome annotation of 17 porcine tissues using nanopore sequencing technology[J].Anim Genet,2023,54(1):35-44.
[37] HU X F,JIN M J,GONG Z X,et al.Full-length transcriptome profile of Apis cerana revealed by nanopore sequencing[J].Int J Mol Sci,2024,25(19):10833.
[38] MOCK A,BRAUN M,SCHOLL C,et al.Transcriptome profiling for precision cancer medicine using shallow nanopore cDNA sequencing[J].Sci Rep,2023,13(1):2378.
[39] ZHANG L L,ZHANG C,PENG J P.Application of nanopore sequencing technology in the clinical diagnosis of infectious diseases[J].Biomed Environ Sci,2022,35(5):381-392.
[40] RENF,MA J,DANG L,et al.Potential of nanopore sequencing for tuberculosis diagnosis and drug resistance detection[J].BMC Infect Dis,2024,24(1):1469.
[41] 贾晓芳,张小楠,凌云,等。纳米孔测序技术在重症新冠肺炎患者继发感染诊断中的应用[J].浙江大学学报(医学版),2021,50(6):748-754.
JIA X,ZHANG X,LING Y,et al.Application of nanopore sequencing in diagnosis of secondary infections in patients with severe COVID-19[J].J Zhejiang Univ Med Sci,2021,50(6):748-754.
[42] CHENJ,XU F.Application of nanopore sequencing in the diagnosis and treatment of pulmonary infections[J].Mol Diagn Ther,2023,27(6):685-701.
[43] KUMBURU HH,SHAYO M,VAN ZWETSLAAR M,et al.Nanopore sequencing technology for clinical diagnosis of infectious diseases where laboratory capacity is meager:A case report[J].Heliyon,2023,9(7):e17439.
[44] SUX,LIN Q,LIU B,et al.The promising role of nanopore sequencing in cancer diagnostics and treatment[J].Cell Insight,2025,4(2):100229.
[45] YUG,FANG L,SHEN Y,et al.Targeted nanopore sequencing using clinical specimens for the rapid diagnosis of extrapulmonary tuberculosis[J].BMC Infect Dis,2024,24(1):710.
[46] DIXONK,SHEN Y,O’NEILL K,et al.Defining the heterogeneity of unbalanced structural variation underlying breast cancer susceptibility by nanopore genome sequencing[J].Eur J Hum Genet,2023,31(5):602-606.
[47] KATSMAN E,ORLANSKI S,MARTIGNANO F,et al.Detecting cell-of-origin and cancer-specific methylation features of cell-free DNA from Nanopore sequencing[J].Genome Biol,2022,23(1):158.