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Readfish enables targeted nanopore sequencing of gigabase-sized genomes

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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

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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.

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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.


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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.).

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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.

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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.

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

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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.

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

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