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Targeted Nanopore Sequencing with Cas9 for studies of methylation, structural variants, and mutations

Timothy Gilpatrick, Isac Lee, James E. Graham, Etienne Raimondeau, Rebecca Bowen, Andrew Heron, View ORCID ProfileFritz J Sedlazeck, View ORCID ProfileWinston Timp
doi: https://doi.org/10.1101/604173
Timothy Gilpatrick
1Department of Biomedical Engineering, Johns Hopkins University (Baltimore, USA)
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Isac Lee
1Department of Biomedical Engineering, Johns Hopkins University (Baltimore, USA)
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James E. Graham
2Oxford Nanopore Technologies (Oxford, UK)
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Etienne Raimondeau
2Oxford Nanopore Technologies (Oxford, UK)
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Rebecca Bowen
2Oxford Nanopore Technologies (Oxford, UK)
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Andrew Heron
2Oxford Nanopore Technologies (Oxford, UK)
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Fritz J Sedlazeck
3Human Genome Sequencing Center, Baylor College of Medicine (Houston, USA)
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  • ORCID record for Fritz J Sedlazeck
Winston Timp
1Department of Biomedical Engineering, Johns Hopkins University (Baltimore, USA)
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  • ORCID record for Winston Timp
  • For correspondence: wtimp@jhu.edu
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Abstract

Nanopore sequencing technology can rapidly and directly interrogate native DNA molecules. Often we are interested only in interrogating specific areas at high depth, but conventional enrichment methods have thus far proved unsuitable for long reads1. Existing strategies are currently limited by high input DNA requirements, low yield, short (<5kb) reads, time-intensive protocols, and/or amplification or cloning (losing base modification information). In this paper, we describe a technique utilizing the ability of Cas9 to introduce cuts at specific locations and ligating nanopore sequencing adaptors directly to those sites, a method we term ‘nanopore Cas9 Targeted-Sequencing’ (nCATS).

We have demonstrated this using an Oxford Nanopore MinION flow cell (Capacity >10Gb+) to generate a median 165X coverage at 10 genomic loci with a median length of 18kb, representing a several hundred-fold improvement over the 2-3X coverage achieved without enrichment. We performed a pilot run on the smaller Flongle flow cell (Capacity ~1Gb), generating a median coverage of 30X at 11 genomic loci with a median length of 18kb. Using panels of guide RNAs, we show that the high coverage data from this method enables us to (1) profile DNA methylation patterns at cancer driver genes, (2) detect structural variations at known hot spots, and (3) survey for the presence of single nucleotide mutations. Together, this provides a low-cost method that can be applied even in low resource settings to directly examine cellular DNA. This technique has extensive clinical applications for assessing medically relevant genes and has the versatility to be a rapid and comprehensive diagnostic tool. We demonstrate applications of this technique by examining the well-characterized GM12878 cell line as well as three breast cell lines (MCF-10A, MCF-7, MDA-MB-231) with varying tumorigenic potential as a model for cancer.

Contributions TG and WT constructed the study. TG performed the experiments. TG, IL, and FS analyzed the data. TG, JG, ER, RB and AH and developed the method. TG and WT wrote the paper

Footnotes

  • We have recalled the data with the latest basecaller and added a flongle sequencing run. We also had other small textural changes and added minor analyses

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted June 04, 2019.
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Targeted Nanopore Sequencing with Cas9 for studies of methylation, structural variants, and mutations
Timothy Gilpatrick, Isac Lee, James E. Graham, Etienne Raimondeau, Rebecca Bowen, Andrew Heron, Fritz J Sedlazeck, Winston Timp
bioRxiv 604173; doi: https://doi.org/10.1101/604173
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Targeted Nanopore Sequencing with Cas9 for studies of methylation, structural variants, and mutations
Timothy Gilpatrick, Isac Lee, James E. Graham, Etienne Raimondeau, Rebecca Bowen, Andrew Heron, Fritz J Sedlazeck, Winston Timp
bioRxiv 604173; doi: https://doi.org/10.1101/604173

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