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Title: Dual-barcoded shotgun expression library sequencing for high-throughput characterization of functional traits in bacteria

Abstract

A major challenge in genomics is the knowledge gap between sequence and its encoded function. Gain-of-function methods based on gene overexpression are attractive avenues for phenotype-based functional screens, but are not easily applied in high-throughput across many experimental conditions. Here, we present Dual Barcoded Shotgun Expression Library Sequencing (Dub-seq), a method that uses random DNA barcodes to greatly increase experimental throughput. As a demonstration of this approach, we construct a Dub-seq library with Escherichia coli genomic DNA, performed 155 genome-wide fitness assays in 52 experimental conditions, and identified overexpression phenotypes for 813 genes. We show that Dub-seq data is reproducible, accurately recapitulates known biology, and identifies hundreds of novel gain-of-function phenotypes for E. coli genes, a subset of which we verified with assays of individual strains. Dub-seq provides complementary information to loss-of-function approaches and will facilitate rapid and systematic functional characterization of microbial genomes.

Authors:
ORCiD logo [1];  [1];  [1];  [1];  [1]; ORCiD logo [2];  [3]; ORCiD logo [3]
  1. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  2. Univ. of California, Berkeley, CA (United States)
  3. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1508060
Alternate Identifier(s):
OSTI ID: 1581084
Grant/Contract Number:  
AC02-05CH11231; SC0008812
Resource Type:
Accepted Manuscript
Journal Name:
Nature Communications
Additional Journal Information:
Journal Volume: 10; Journal Issue: 1; Journal ID: ISSN 2041-1723
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Mutalik, Vivek K., Novichkov, Pavel S., Price, Morgan N., Owens, Trenton K., Callaghan, Mark, Carim, Sean, Deutschbauer, Adam M., and Arkin, Adam P. Dual-barcoded shotgun expression library sequencing for high-throughput characterization of functional traits in bacteria. United States: N. p., 2019. Web. doi:10.1038/s41467-018-08177-8.
Mutalik, Vivek K., Novichkov, Pavel S., Price, Morgan N., Owens, Trenton K., Callaghan, Mark, Carim, Sean, Deutschbauer, Adam M., & Arkin, Adam P. Dual-barcoded shotgun expression library sequencing for high-throughput characterization of functional traits in bacteria. United States. doi:10.1038/s41467-018-08177-8.
Mutalik, Vivek K., Novichkov, Pavel S., Price, Morgan N., Owens, Trenton K., Callaghan, Mark, Carim, Sean, Deutschbauer, Adam M., and Arkin, Adam P. Fri . "Dual-barcoded shotgun expression library sequencing for high-throughput characterization of functional traits in bacteria". United States. doi:10.1038/s41467-018-08177-8. https://www.osti.gov/servlets/purl/1508060.
@article{osti_1508060,
title = {Dual-barcoded shotgun expression library sequencing for high-throughput characterization of functional traits in bacteria},
author = {Mutalik, Vivek K. and Novichkov, Pavel S. and Price, Morgan N. and Owens, Trenton K. and Callaghan, Mark and Carim, Sean and Deutschbauer, Adam M. and Arkin, Adam P.},
abstractNote = {A major challenge in genomics is the knowledge gap between sequence and its encoded function. Gain-of-function methods based on gene overexpression are attractive avenues for phenotype-based functional screens, but are not easily applied in high-throughput across many experimental conditions. Here, we present Dual Barcoded Shotgun Expression Library Sequencing (Dub-seq), a method that uses random DNA barcodes to greatly increase experimental throughput. As a demonstration of this approach, we construct a Dub-seq library with Escherichia coli genomic DNA, performed 155 genome-wide fitness assays in 52 experimental conditions, and identified overexpression phenotypes for 813 genes. We show that Dub-seq data is reproducible, accurately recapitulates known biology, and identifies hundreds of novel gain-of-function phenotypes for E. coli genes, a subset of which we verified with assays of individual strains. Dub-seq provides complementary information to loss-of-function approaches and will facilitate rapid and systematic functional characterization of microbial genomes.},
doi = {10.1038/s41467-018-08177-8},
journal = {Nature Communications},
number = 1,
volume = 10,
place = {United States},
year = {2019},
month = {1}
}

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