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...

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Bibliographic Details
Published inNature biotechnology Vol. 39; no. 4; pp. 442 - 450
Main Authors Payne, Alexander, Holmes, Nadine, Clarke, Thomas, Munro, Rory, Debebe, Bisrat J, Loose, Matthew
Format Journal Article
LanguageEnglish
Published United States Nature Publishing Group 01.04.2021
Subjects
Analysis
Cell Line, Tumor
Chromosomes
Computational Biology - methods
Computer applications
Computer programs
Deoxyribonucleic acid
Depletion
DNA
DNA sequencing
Enrichment
Exons
Gene sequencing
Genes
Genome Size
Genome, Human
Genomes
Graphics processing units
High-Throughput Nucleotide Sequencing
Human genome
Humans
Nanopore Sequencing - instrumentation
Nanotechnology
Neoplasms - genetics
Nucleotide sequence
Nucleotide sequencing
Oncogene Proteins, Fusion - genetics
Porosity
Sample preparation
Science
Sequence Analysis, DNA
Software
Technology application
United Kingdom
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Summary: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 PML-RARA 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 https://www.github.com/looselab/readfish .
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ISSN:1087-0156
1546-1696
DOI:10.1038/s41587-020-00746-x