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Depletion of Abundant Sequences by Hybridization (DASH): Using Cas9 to remove unwanted high-abundance species in sequencing libraries and molecular counting applications

W Gu, View ORCID ProfileED Crawford, View ORCID ProfileBD O’Donovan, View ORCID ProfileMR Wilson, View ORCID ProfileED Chow, View ORCID ProfileH Retallack, View ORCID ProfileJL DeRisi
doi: https://doi.org/10.1101/031708
W Gu
1Departments of Pathology and Laboratory Medicine, University of California, San Francisco
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ED Crawford
2Department of Biochemistry and Biophysics, University of California, San Francisco; Howard Hughes Medical Institute
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BD O’Donovan
3Integrative Program in Quantitative Biology, Bioinformatics, University of California San Francisco
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MR Wilson
4Department of Neurology, University of California, San Francisco
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ED Chow
5Center for Advanced Technology, Department of Biochemistry and Biophysics, University of California, San Francisco
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H Retallack
6Medical Scientist Training Program, Biomedical Sciences Graduate Program, University of California, San Francisco
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JL DeRisi
2Department of Biochemistry and Biophysics, University of California, San Francisco; Howard Hughes Medical Institute
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Abstract

* Wei Gu and Emily Crawford contributed equally to this work

Background: With widespread adoption of next-generation sequencing (NGS) technologies, the need has arisen for a broadly applicable method to remove unwanted high-abundance species prior to sequencing. We introduce DASH (Depletion of Abundant Sequences by Hybridization), a facile technique for targeted depletion of undesired sequences.

Results: Sequencing libraries are DASHed with recombinant Cas9 protein complexed with a library of single guide RNAs (sgRNAs) programmed to target unwanted species for cleavage, thus preventing them from consuming sequencing space. We demonstrate up to 99% reduction of mitochondrial ribosomal RNA (rRNA) in HeLa cells, and enrichment of pathogen sequences up to 4-fold in metagenomic samples from patients with infectious diseases. Similarly, we demonstrate the utility of DASH in the context of cancer diagnostics by significantly increasing the detectable fraction of KRAS mutant sequences over the predominant wild-type allele.

Conclusion: This simple single-tube method is reprogrammable for virtually any sample type to increase sequencing yield without additional cost.

Footnotes

  • Email addresses:

    WG: microfluidics{at}gmail.com

    EDC1 (Crawford): Emily.Crawford{at}ucsf.edu

    BDO: Brian.Odonovan{at}ucsf.edu

    MRW: Michael.Wilson{at}ucsf.edu

    EDC2 (Chow): Eric.Chow{at}ucsf.edu

    HR: Hanna.Retallack{at}ucsf.edu

    JLD: Joe{at}derisilab.ucsf.edu

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-NC-ND 4.0 International license.
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Posted November 20, 2015.
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Depletion of Abundant Sequences by Hybridization (DASH): Using Cas9 to remove unwanted high-abundance species in sequencing libraries and molecular counting applications
W Gu, ED Crawford, BD O’Donovan, MR Wilson, ED Chow, H Retallack, JL DeRisi
bioRxiv 031708; doi: https://doi.org/10.1101/031708
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Depletion of Abundant Sequences by Hybridization (DASH): Using Cas9 to remove unwanted high-abundance species in sequencing libraries and molecular counting applications
W Gu, ED Crawford, BD O’Donovan, MR Wilson, ED Chow, H Retallack, JL DeRisi
bioRxiv 031708; doi: https://doi.org/10.1101/031708

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