Comprehensive recombinant adeno-associated virus characterization is essential for establishing the knowledge base required to ensure clinical safety and efficacy, yet current long-read methods suffer from library preparation biases that obscure genome integrity. We present AviNP-seq, a blindspot-free nanopore sequencing framework utilizing one-end-sufficient ligation and Cas9–ribonucleoprotein (RNP) linearization to minimize terminal selection. Applied to a 1.5–6.5 kb panel, AviNP-seq delineates a sharp packaging cliff at 5.0–5.2 kb and reveals that sequence structure modulates integrity by 2–5× at fixed lengths. It unmasks covalent head-to-tail tandems in sub-3 kb vectors, detecting them with significantly higher sensitivity than PacBio HiFi. The Cas9–RNP step boosts ligation yield ∼7-fold, providing an unbiased assessment of genome integrity (≥95% inverted terminal repeat [ITR]-to-ITR). In addition, the assay quantifies plasmid impurities down to 0.05% with linear response. By integrating integrity mapping, tandem detection, and impurity profiling into a rapid (<36 h), low-input workflow, AviNP-seq provides a robust analytical tool to guide vector design and de-risk early-stage process development.
WangD, TaiPWL, GaoG. Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov2019;18(5):358–378; doi: 10.1038/s41573-019-0012-9
2.
HighKA, RoncaroloMG. Gene therapy. N Engl J Med2019;381(5):455–464; doi: 10.1056/NEJMra1706910
3.
WangJH, GesslerDJ, ZhanW, et al.Adeno-associated virus as a delivery vector for gene therapy of human diseases. Signal Transduct Target Ther2024;9(1):78; doi: 10.1038/s41392-024-01780-w
4.
ByrneBJ, FlaniganKM, MatesanzSE, et al.Current clinical applications of AAV-mediated gene therapy. Mol Ther2025;33(6):2479–2516; doi: 10.1016/j.ymthe.2025.04.045
5.
XieJ, MaoQ, TaiPWL, et al.Short DNA hairpins compromise recombinant adeno-associated virus genome homogeneity. Mol Ther2017;25(6):1363–1374; doi: 10.1016/j.ymthe.2017.03.028
6.
KapranovP, ChenL, DederichD, et al.Native molecular state of adeno-associated viral vectors revealed by single-molecule sequencing. Hum Gene Ther2012;23(1):46–55; doi: 10.1089/hum.2011.160
7.
DongB, NakaiH, XiaoW. Characterization of genome integrity for oversized recombinant AAV vector. Mol Ther2010;18(1):87–92; doi: 10.1038/mt.2009.258
8.
KosakaM, SajikiAF, FujitaK, et al.Evaluation of the loading capacity and patterns of packaged DNA in AAV genomes of different sizes using long-read sequencing. Mol Ther Methods Clin Dev2025;33(2):101474; doi: 10.1016/j.omtm.2025.101474
9.
TranNT, LecomteE, SaleunS, et al.Human and insect cell-produced recombinant adeno-associated viruses show differences in genome heterogeneity. Hum Gene Ther2022;33(7–8):371–388; doi: 10.1089/hum.2022.050
10.
ZhangJ, GuoP, YuX, et al.Subgenomic particles in rAAV vectors result from DNA lesion/break and non-homologous end joining of vector genomes. Mol Ther Nucleic Acids2022;29:852–861; doi: 10.1016/j.omtn.2022.08.027
11.
ShmidtAA, EgorovaTV. PCR-Based analytical methods for quantification and quality control of recombinant adeno-associated viral vector preparations. Pharmaceuticals (Basel)2021;15(1):23; doi: 10.3390/ph15010023
12.
WangY, MenonN, ShenS, et al.A qPCR method for AAV genome titer with ddPCR-Level of accuracy and precision. Mol Ther Methods Clin Dev2020;19:341–346; doi: 10.1016/j.omtm.2020.09.017
13.
LockM, AlviraMR, ChenSJ, et al.Absolute determination of single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR. Hum Gene Ther Methods2014;25(2):115–125; doi: 10.1089/hgtb.2013.131
14.
DobnikD, KogovsekP, JakominT, et al.Accurate quantification and characterization of adeno-associated viral vectors. Front Microbiol2019;10:1570; doi: 10.3389/fmicb.2019.01570
15.
McCartyDM, FuH, MonahanPE, et al.Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo. Gene Ther2003;10(26):2112–2118; doi: 10.1038/sj.gt.3302134
16.
LecomteE, TournaireB, CogneB, et al.Advanced characterization of DNA molecules in rAAV vector preparations by single-stranded virus next-generation sequencing. Mol Ther Nucleic Acids2015;4(10):e260; doi: 10.1038/mtna.2015.32
17.
Penaud-BudlooM, LecomteE, Guy-DucheA, et al.Accurate identification and quantification of DNA species by next-generation sequencing in adeno-associated viral vectors produced in insect cells. Hum Gene Ther Methods2017;28(3):148–162; doi: 10.1089/hgtb.2016.185
18.
LecomteE, SaleunS, BolteauM, et al.The SSV-Seq 2.0 PCR-Free method improves the sequencing of adeno-associated viral vector genomes containing GC-Rich regions and homopolymers. Biotechnol J2021;16(1):e2000016; doi: 10.1002/biot.202000016
19.
OziolorEM, KumpfSW, QianJ, et al.Comparing molecular and computational approaches for detecting viral integration of AAV gene therapy constructs. Mol Ther Methods Clin Dev2023;29:395–405; doi: 10.1016/j.omtm.2023.04.009
20.
RadukicMT, BrandtD, HaakM, et al.Nanopore sequencing of native adeno-associated virus (AAV) single-stranded DNA using a transposase-based rapid protocol. NAR Genom Bioinform2020;2(4):lqaa074; doi: 10.1093/nargab/lqaa074
21.
TaiPWL, XieJ, FongK, et al.Adeno-associated virus genome population sequencing achieves full vector genome resolution and reveals human-vector chimeras. Mol Ther Methods Clin Dev2018;9:130–141; doi: 10.1016/j.omtm.2018.02.002
22.
GilpatrickT, LeeI, GrahamJE, et al.Targeted nanopore sequencing with Cas9-guided adapter ligation. Nat Biotechnol2020;38(4):433–438; doi: 10.1038/s41587-020-0407-5
23.
NamkungS, TranNT, ManokaranS, et al.Direct ITR-to-ITR nanopore sequencing of AAV vector genomes. Hum Gene Ther2022;33(21–22):1187–1196; doi: 10.1089/hum.2022.143
24.
ZhangJ, YuX, ChrzanowskiM, et al.Thorough molecular configuration analysis of noncanonical AAV genomes in AAV vector preparations. Mol Ther Methods Clin Dev2024;32(1):101215; doi: 10.1016/j.omtm.2024.101215
25.
TranNT, HeinerC, WeberK, et al.AAV-Genome population sequencing of vectors packaging CRISPR components reveals design-influenced heterogeneity. Mol Ther Methods Clin Dev2020;18:639–651; doi: 10.1016/j.omtm.2020.07.007
26.
TraversKJ, ChinCS, RankDR, et al.A flexible and efficient template format for circular consensus sequencing and SNP detection. Nucleic Acids Res2010;38(15):e159; doi: 10.1093/nar/gkq543
27.
GargS. Towards routine chromosome-scale haplotype-resolved reconstruction in cancer genomics. Nat Commun2023;14(1):1358; doi: 10.1038/s41467-023-36689-5
28.
Dunker-SeidlerF, BreunigK, HaubnerM, et al.Recombinant AAV batch profiling by nanopore sequencing elucidates product-related DNA impurities and vector genome length distribution. Mol Ther Methods Clin Dev2025;33(1):101417; doi: 10.1016/j.omtm.2025.101417
29.
ZhangJP, LiXL, LiGH, et al.Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage. Genome Biol2017;18(1):35; doi: 10.1186/s13059-017-1164-8
30.
LiG, TianS, SunX, et al.Leveraging CRISPR-Cas9 for accurate detection of AAV-Neutralizing antibodies: The AAV-HDR method. Hum Gene Ther2024;35(13–14):490–505; doi: 10.1089/hum.2023.129
31.
KohlbrennerE, WeberT. Production and characterization of vectors based on the cardiotropic AAV serotype 9. Methods Mol Biol2017;1521:91–107; doi: 10.1007/978-1-4939-6588-5_6
32.
FuYW, DaiXY, WangWT, et al.Dynamics and competition of CRISPR-Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing. Nucleic Acids Res2021;49(2):969–985; doi: 10.1093/nar/gkaa1251
33.
WenW, QuanZJ, LiSA, et al.Effective control of large deletions after double-strand breaks by homology-directed repair and dsODN insertion. Genome Biol2021;22(1):236; doi: 10.1186/s13059-021-02462-4
34.
JainM, KorenS, MigaKH, et al.Nanopore sequencing and assembly of a human genome with ultra-long reads. Nat Biotechnol2018;36(4):338–345; doi: 10.1038/nbt.4060
35.
SereikaM, KirkegaardRH, KarstSM, et al.Oxford Nanopore R10.4 long-read sequencing enables the generation of near-finished bacterial genomes from pure cultures and metagenomes without short-read or reference polishing. Nat Methods2022;19(7):823–826; doi: 10.1038/s41592-022-01539-7
36.
QuanZJ, LiSA, YangZX, et al.GREPore-seq: A robust workflow to detect changes after Gene editing through long-range PCR and nanopore sequencing. Genomics Proteomics Bioinformatics2023;21(6):1221–1236; doi: 10.1016/j.gpb.2022.06.002
WuZ, YangH, ColosiP. Effect of genome size on AAV vector packaging. Mol Ther2010;18(1):80–86; doi: 10.1038/mt.2009.255
39.
LaiY, YueY, DuanD. Evidence for the failure of adeno-associated virus serotype 5 to package a viral genome > or = 8.2 kb. Mol Ther2010;18(1):75–79; doi: 10.1038/mt.2009.256
40.
DongJY, FanPD, FrizzellRA. Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. Hum Gene Ther1996;7(17):2101–2112; doi: 10.1089/hum.1996.7.17-2101
41.
Ben-AssaN, CoyneMJ, FomenkovA, et al.Analysis of a phase-variable restriction modification system of the human gut symbiont Bacteroides fragilis. Nucleic Acids Res2020;48(19):11040–11053; doi: 10.1093/nar/gkaa824
42.
DuckworthAT, BilottiK, PotapovV, et al.Profiling DNA ligase substrate specificity with a pacific biosciences single-molecule real-time sequencing assay. Curr Protoc2023;3(3):e690; doi: 10.1002/cpz1.690
43.
Gonzalez-SandovalA, PekrunK, TsujiS, et al.The AAV capsid can influence the epigenetic marking of rAAV delivered episomal genomes in a species dependent manner. Nat Commun2023;14(1):2448; doi: 10.1038/s41467-023-38106-3
44.
MaurerAC, WeitzmanMD. Adeno-Associated virus genome interactions important for vector production and transduction. Hum Gene Ther2020;31(9–10):499–511; doi: 10.1089/hum.2020.069
45.
ZhengW, JiangL, LeiQ, et al.Development and validation of quantitative real-time PCR for the detection of residual CHO host cell DNA and optimization of sample pretreatment method in biopharmaceutical products. Biol Proced Online2019;21(1):17; doi: 10.1186/s12575-019-0105-1
WrightJF. Manufacturing and characterizing AAV-based vectors for use in clinical studies. Gene Ther2008;15(11):840–848; doi: 10.1038/gt.2008.65
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
