Nanopore sequensing: theory and practice

Highlights
A comparative study was conducted on the suitability of the domestic "Nanoporus" platform and its foreign counterpart, MinION, for solving the tasks of the NBC Protection Corps.
The comparable effectiveness of the "Nanoporus" platform was demonstrated after applying post-processing algorithms.
Key advantages of the "Nanoporus" platform are its cost-effectiveness and capability for autonomous operation.
Relevance. The study is determined by the need to develop domestic technologies for the rapid identification of pathogenic biological agents (PBA) in field conditions by the NBC Protection Corps.
Purpose of the study is a comprehensive review of the theoretical foundations and practical applications of nanopore sequencing technology, as well as the prospects for its implementation in the NBC Protection Corps of the Russian Armed Forces.
Materials and Methods. The quality of genome assemblies from the "Nanoporus" (Nanoporus, Russia) and MinION (Oxford Nanopore Technologies, UK) sequencing systems was compared. The reference strain E. coli XL1-Blue (Evrogen, Russia) was used as the biological model. Experimental nanopore sequencing was performed, including DNA extraction, library preparation, and subsequent bioinformatic data processing using the Flye and Medaka tools. Genome assembly completeness and accuracy were analyzed based on N50 and Q-score metrics.
Results. It was shown that the domestic "Nanoporus" platform provides high genome assembly quality, comparable to the results obtained with the MinION system, but at significantly lower operational costs. The platform is compact and suitable for autonomous operation in field conditions.
Conclusion. The "Nanoporus" platform is promising for integration into the mobile laboratories of the NBC Protection Corps of the Russian Armed Forces and can be used for solving PBA identification tasks.

1. Wang Y, Zhao Y, Bollas A, Wang Y, Au KF. Nanopore sequencing technology, bioinformatics and applications. Nature Biotechnology. 2021;39(11):1348–65. https://doi.org/10.1038/s41587-021-01108-x

2. Peng M, Davis ML, Bentz ML. Short-read and long-read whole genome sequencing for SARS-CoV-2 variants identification. Viruses. 2025;17(4):584. https://doi.org/10.3390/v17040584

3. Brown A, Patel R, Chen S. Cost-effective genomic solutions: the impact of nanopore sequencing in lowresource settings. BMC Genomics. 2022;23(1):100. https://doi.org/10.1186/s12864-022-09001-9

4. Amarasinghe SL, Su S, Dong X, Zappia L, Ritchie ME, Gouil Q. Opportunities and challenges in long-read sequencing data analysis. Genome Biology. 2020;21(1):30. https://doi.org/10.1186/s13059-020-1935-5

5. Logsdon GA, Vollger MR, Eichler EE. Long-read human genome sequencing and its applications. Nature Reviews Genetics. 2020;21(10):597–614. https://doi.org/10.1038/s41576-020-0236-x

6. Pugh J. The Current State of Nanopore Sequencing. Methods in Molecular Biology. 2023;2632:3–14. https://doi.org/10.1007/978-1-0716-2996-3_1

7. Deamer D, Akeson M, Branton D. Three decades of nanopore sequencing. Nature Biotechnology. 2016;34:518–24. https://doi.org/10.1038/nbt.3423

8. Jain M, Olsen HE, Paten B, Akeson M. The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community. Genome Biology. 2016;17:239. https://doi.org/10.1186/s13059-016-1103-0

9. Quick J, Loman NJ, Duraffour S, Simpson JT, Severi E, Cowley L, et al. Real-time, portable genome sequencing for Ebola surveillance. Nature. 2016;530(7589):228–32. https://doi.org/10.1038/nature16996

10. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988;239(4839):487–91. https://doi.org/10.1126/science.2448875

11. Taudt A, Colomé-Tatché M, Johannes F. Genetic sources of population epigenomic variation. Nature Reviews Genetics. 2016;17(6):319–32. https://doi.org/10.1038/nrg.2016.45

12. Li H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. 2018;34(18):3094–100. https://doi.org/10.1093/bioinformatics/bty191

13. De Coster W, Hert DS, Schultz DT, Cruts M, Van BC. NanoPack: visualizing and processing long-read sequencing data. Bioinformatics. 2018;34(15):2666–9. https://doi.org/10.1093/bioinformatics/bty149

14. Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. Canu: scalable and accurate longread assembly via adaptive k-mer weighting. Genome Research. 2017;27(5):722–36. https://doi.org/10.1101/gr.215087.116

15. Ruan J, Li H. Fast and accurate long-read assembly with wtdbg2. Nature Methods. 2020;17:155–8. https://doi.org/10.1038/s41592-019-0669-3

16. Sedlazeck FJ, Rescheneder P, Smolka M, Fang H, Nattestad M, Haeseler A, et al. Accurate detection of complex structural variations using single-molecule sequencing. Nature Methods. 2018;15:461–8. https://doi.org/10.1038/s41592-018-0001-7

17. Тham CY, Tirado-Magallanes R, Goh Y, Fullwood MJ, Koh BTH, Wang W, et al. NanoVar: accurate characterization of patients' genomic structural variants using low-depth nanopore sequencing. Genome Biology. 2020;21:56. https://doi.org/10.1186/s13059-020-01968-7

18. Shafin K, Pesout T, Chang P-C, Nattestad M, Kolesnikov A, Goel S, et al. Haplotype-aware variant calling with PEPPER-Margin-DeepVariant enables high accuracy in nanopore long-reads. Nature Methods. 2021;18:1322–32. https://doi.org/10.1038/s41592-021-01299-w

19. Simpson JT, Workman RE, Zuzarte PC, David M, Dursi LJ, Timp W. Detecting DNA cytosine methylation using nanopore sequencing. Nature Methods. 2017;14:407–10. https://doi.org/10.1038/nmeth.4184

20. Kim D, Song L, Breitwieser FP, Salzberg SL. Centrifuge: rapid and sensitive classification of metagenomic sequences. Genome Research. 2016;26:1721–9. https://doi.org/10.1101/gr.210641.116

21. Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G, et al. Integrative genomics viewer. Nature Biotechnology. 2011;29(1):24–6. https://doi.org/10.1038/nbt.1754

22. Wick RR, Schultz MB, Zobel J, Holt KE. Bandage: interactive visualization of de novo genome assemblies. Bioinformatics. 2015;31(20):3350–2. https://doi.org/10.1093/bioinformatics/btv383

23. Nicholls SM, Quick JC, Tang S, Loman NJ. Ultra-deep, long-read nanopore sequencing of mock microbial community standards. GigaScience. 2019;8(5):giz043. https://doi.org/10.1093/gigascience/giz043

24. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, et al. Initial sequencing and analysis of the human genome. Nature. 2001;409(6822):860–921. https://doi.org/10.1038/35057062

25. Mardis ER. Next-generation DNA sequencing methods. Annual Review of Genomics and Human Genetics. 2008;9:387–402. https://doi.org/10.1146/annurev.genom.9.081307.164359

26. Goodwin S, McPherson JD, McCombie WR. Coming of age: Ten years of next-generation sequencing technologies. Nature Reviews Genetics. 2016;17(6):333–51. https://doi.org/10.1038/nrg.2016.49