# Unlocking the full potential of nanopore sequencing: tips, tricks, and advanced data analysis techniques

**Authors:** Daria Meyer, Winfried Goettsch, Jannes Spangenberg, Bettina Stieber, Sebastian Krautwurst, Martin Hölzer, Christian Brandt, Jörg Linde, Christian Höner zu Siederdissen, Akash Srivastava, Milena Zarkovic, Damian Wollny, Manja Marz

PMC · DOI: 10.1093/nar/gkag023 · Nucleic Acids Research · 2026-02-02

## TL;DR

This paper explores how to improve nanopore sequencing by offering tips and advanced data analysis techniques to maximize its potential.

## Contribution

The paper provides comprehensive guidelines and statistically supported insights to address challenges in nanopore sequencing.

## Key findings

- Nanopore sequencing allows real-time analysis of long nucleic acid strands in portable devices.
- The paper identifies persistent challenges in protocols and technology that hinder optimal results.
- It offers interdisciplinary approaches to enhance information gain from nanopore sequencing.

## Abstract

Nucleic acid sequencing is the process of identifying the sequence of DNA or RNA, with DNA used for genomes and RNA for transcriptomes. Deciphering this information has the potential to greatly advance our understanding of genomic features and cellular functions. In comparison to other available sequencing methods, nanopore sequencing stands out due to its unique advantages of processing long nucleic acid strands in real time, within a small portable device, enabling the rapid analysis of samples in diverse settings. Evolving over the past decade, nanopore sequencing remains in a state of ongoing development and refinement, resulting in persistent challenges in protocols and technology. This article employs an interdisciplinary approach, evaluating experimental and computational methods to address critical gaps in our understanding in order to maximize the information gain from this advancing technology. Here, we present both overview and analysis of all aspects of nanopore sequencing by providing statistically supported insights. Thus, we aim to provide fresh perspectives on nanopore sequencing and give comprehensive guidelines for the diverse challenges that frequently impede optimal experimental outcomes.

Graphical Abstract

## Full-text entities

- **Genes:** ENO2 (phosphopyruvate hydratase ENO2) [NCBI Gene 856579]
- **Diseases:** ONT (MESH:C000719218), COVID-19 (MESH:D000086382)
- **Chemicals:** platinum (MESH:D010984), cytosine (MESH:D003596), 5mC (-), silica (MESH:D012822), deuterium (MESH:D003903), 5-hydroxymethylcytosine (MESH:C011865), chloroform (MESH:D002725), poly(A) (MESH:D011061), 5-methylcytosine (MESH:D044503), N6-methyladenine (MESH:C005955), phenol (MESH:D019800), 4mC (MESH:C000612305), adenine (MESH:D000225), nucleotide (MESH:D009711), DCS (MESH:D003523)
- **Species:** Bacillus subtilis (species) [taxon 1423], Bacteria Latreille et al. 1825 (Bacteria stick insect, genus) [taxon 629395], Lambdavirus (genus) [taxon 186765], Escherichia coli (E. coli, species) [taxon 562], Mus musculus (house mouse, species) [taxon 10090], Gammacoronavirus (genus) [taxon 694013], Severe acute respiratory syndrome coronavirus 2 (no rank) [taxon 2697049], Saccharomyces cerevisiae (baker's yeast, species) [taxon 4932], Homo sapiens (human, species) [taxon 9606], Plum pox virus (no rank) [taxon 12211]
- **Cell lines:** R9 — Rattus norvegicus (Rat), Transformed cell line (CVCL_4282), R10 — Mus musculus (Mouse), Transformed cell line (CVCL_0155)

## Full text

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## Figures

11 figures with captions in the complete paper: https://tomesphere.com/paper/PMC12862388/full.md

## References

106 references — full list in the complete paper: https://tomesphere.com/paper/PMC12862388/full.md

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Source: https://tomesphere.com/paper/PMC12862388