Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Readfish enables targeted nanopore sequencing of gigabase-sized genomes


Nanopore sequencers can be used to selectively sequence certain DNA molecules in a pool by reversing the voltage across individual nanopores to reject specific sequences, enabling enrichment and depletion to address biological questions. Previously, we achieved this using dynamic time warping to map the signal to a reference genome, but the method required substantial computational resources and did not scale to gigabase-sized references. Here we overcome this limitation by using graphical processing unit (GPU) base-calling. We show enrichment of specific chromosomes from the human genome and of low-abundance organisms in mixed populations without a priori knowledge of sample composition. Finally, we enrich targeted panels comprising 25,600 exons from 10,000 human genes and 717 genes implicated in cancer, identifying PMLRARA fusions in the NB4 cell line in <15 h sequencing. These methods can be used to efficiently screen any target panel of genes without specialized sample preparation using any computer and a suitable GPU. Our toolkit, readfish, is available at

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Human-genome-scale selective sequencing.
Fig. 2: Adaptive sequencing enriching for the least abundant genome and ensuring uniform 40× coverage.
Fig. 3: Adaptive sequencing enriching for the least abundant genome with centrifuge read classification and ensuring uniform 50× coverage.
Fig. 4: Half-exome panel targeted sequencing.
Fig. 5: COSMIC panel targeted sequencing.
Fig. 6: COSMIC panel targeted sequencing of NB4.

Similar content being viewed by others

Data availability

All reads generated in the course of this study are available from the ENA under project ID PRJEB36644.

Code availability

Our code is available open source at See also “readfish code availability” above.


  1. Loose, M., Malla, S. & Stout, M. Real-time selective sequencing using nanopore technology. Nat. Methods 13, 751–754 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Masutani, B. & Morishita, S. A framework and an algorithm to detect low-abundance DNA by a handy sequencer and a palm-sized computer. Bioinformatics 35, 584–592 (2019).

    Article  CAS  PubMed  Google Scholar 

  3. Kovaka, S., Fan, Y., Ni, B., Timp, W. & Schatz, M. C. Targeted nanopore sequencing by real-time mapping of raw electrical signal with UNCALLED. Nat. Biotechnol. (2020).

  4. Edwards, H. S. et al. Real-time selective sequencing with RUBRIC: Read Until with Basecall and Reference-Informed Criteria. Sci. Rep. 9, 11475 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Rang, F. J., Kloosterman, W. P. & de Ridder, J. From squiggle to basepair: computational approaches for improving nanopore sequencing read accuracy. Genome Biol. 19, 90 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Li, H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34, 3094–3100 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Kim, D., Song, L., Breitwieser, F. P. & Salzberg, S. L. Centrifuge: rapid and sensitive classification of metagenomic sequences. Genome Res. 26, 1721–1729 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Tate, J. G. et al. COSMIC: the Catalogue of Somatic Mutations in Cancer. Nucleic Acids Res. 47, D941–D947 (2019).

    Article  CAS  PubMed  Google Scholar 

  9. Mozziconacci, M.-J. et al. Molecular cytogenetics of the acute promyelocytic leukemia-derived cell line NB4 and of four all-trans retinoic acid–resistant subclones. Genes Chromosomes Cancer 35, 261–270 (2002).

    Article  CAS  PubMed  Google Scholar 

  10. Jain, M. et al. Nanopore sequencing and assembly of a human genome with ultra-long reads. Nat. Biotechnol. 36, 338–345 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Charalampous, T. et al. Nanopore metagenomics enables rapid clinical diagnosis of bacterial lower respiratory infection. Nat. Biotechnol. 37, 783–792 (2019).

    Article  CAS  PubMed  Google Scholar 

  12. Marotz, C. A. et al. Improving saliva shotgun metagenomics by chemical host DNA depletion. Microbiome 6, 42 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Nicholls, S. M., Quick, J. C., Tang, S. & Loman, N. J. Ultra-deep, long-read nanopore sequencing of mock microbial community standards. Gigascience 8, giz043 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  14. Kolmogorov, M., Yuan, J., Lin, Y. & Pevzner, P. A. Assembly of long, error-prone reads using repeat graphs. Nat. Biotechnol. 37, 540–546 (2019).

    Article  CAS  PubMed  Google Scholar 

  15. Kozarewa, I., Armisen, J., Gardner, A. F., Slatko, B. E. & Hendrickson, C. L. Overview of target enrichment strategies. Curr. Protoc. Mol. Biol. 112, 7.21.1–7.21.23 (2015).

    Article  PubMed  Google Scholar 

  16. Gnirke, A. et al. Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nat. Biotechnol. 27, 182–189 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Gilpatrick, T. et al. Targeted nanopore sequencing with Cas9-guided adapter ligation. Nat. Biotechnol. 38, 433–438 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Loose, M. Finding the needle: targeted nanopore sequencing and CRISPR-Cas9. CRISPR J. 1, 265–267 (2018).

    Article  PubMed  Google Scholar 

  19. Cunningham, F. et al. Ensembl 2019. Nucleic Acids Res. 47, D745–D751 (2019).

    Article  CAS  PubMed  Google Scholar 

  20. Heller, D. & Vingron, M. SVIM: structural variant identification using mapped long reads. Bioinformatics 35, 2907–2915 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Sedlazeck, F. J. et al. Accurate detection of complex structural variations using single-molecule sequencing. Nat. Meth. 15, 461–468 (2018).

    Article  CAS  Google Scholar 

  22. Beyter, D., Ingimundardottir, H. & Eggertsson, H. P. Long read sequencing of 1,817 Icelanders provides insight into the role of structural variants in human disease. Preprint at bioRxiv (2019).

  23. Pedersen, B. S. & Quinlan, A. R. Mosdepth: quick coverage calculation for genomes and exomes. Bioinformatics 34, 867–868 (2018).

    Article  CAS  PubMed  Google Scholar 

  24. Zook, J. M. et al. An open resource for accurately benchmarking small variant and reference calls. Nat. Biotechnol. 37, 561–566 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Jeffares, D. C. et al. Transient structural variations have strong effects on quantitative traits and reproductive isolation in fission yeast. Nat. Commun. 8, 14061 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Nattestad, M., Aboukhalil, R., Chin, C.-S. & Schatz, M. C. Ribbon: intuitive visualization for complex genomic variation. Bioinformatics (2020).

  27. Pruitt, K. D. & Maglott, D. R. RefSeq and LocusLink: NCBI gene-centered resources. Nucleic Acids Res. 29, 137–140 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references


We thank J. Quick, J. Tyson, J. Simpson and N. Loman for helpful comments and (mainly) criticisms and E. Birney, N. Goldman and A. Senf for helpful insights and discussion on these approaches. We thank M. Hubank and L. Gallagher for access to materials and reagents as well as general boundless enthusiasm. We thank M. Jain for assisting in manipulating data. We also thank S. Reid, C. Wright, C. Seymour, J. Pugh and G. Pimm from ONT for advice on MinKNOW and Guppy operations as well as extensive troubleshooting. This work was supported by the Biotechnology and Biological Sciences Research Council (grant numbers BB/N017099/1, R.M. and M.L.; BB/M020061/1, M.L.; and BB/M008770/1, 1949454 A.P.), the Wellcome Trust (grant number 204843/Z/16/Z, N.H. and M.L.) and the Defence Science and Technology Laboratory (grant number DSTLX-1000138444, R.M. and M.L.).

Author information

Authors and Affiliations



M.L. and A.P. conceived the study. A.P., N.H. and M.L. acquired data. T.C. and R.M. designed and implemented metagenomics applications. A.P., B.J.D. and M.L. analyzed and interpreted data. All authors discussed the results and contributed to the final manuscript.

Corresponding author

Correspondence to Matthew Loose.

Ethics declarations

Competing interests

M.L. was a member of the MinION access program and has received free flow cells and sequencing reagents in the past. M.L. has received reimbursement for travel, accommodation and conference fees to speak at events organized by ONT.

Additional information

Peer review information Nature Biotechnology thanks Jan Korbel and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–19, Tables 1–5, Note 1 and Data 1 description.

Reporting Summary

Supplementary Data 1

COSMIC panel coordinates for selective sequencing and the mean coverage across each run.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Payne, A., Holmes, N., Clarke, T. et al. Readfish enables targeted nanopore sequencing of gigabase-sized genomes. Nat Biotechnol 39, 442–450 (2021).

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research