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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 greatly increases the throughput of genome-wide overexpression assays. In Dub-seq, a shotgun expression library is cloned between dual random DNA barcodes and the precise breakpoints of DNA fragments are associated to the barcode sequences prior to performing assays. To assess the fitness of individual strains carrying these plasmids, we use DNA barcode sequencing (BarSeq), which is amenable to large-scale sample multiplexing. As a demonstration of this approach, we constructed a Dub-seq library with total Escherichia coli genomic DNA, performed 155 genome-wide fitness assays in 52 experimental conditions, and identified 813 genes with high-confidence overexpression phenotypes across 4,151 genes assayed. 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 such as transposon site sequencing or CRISPRi and willmore » facilitate rapid and systematic functional characterization of microbial genomes.ImportanceMeasuring the phenotypic consequences of overexpressing genes is a classic genetic approach for understanding protein function; for identifying drug targets, antibiotic and metal resistance mechanisms; and for optimizing strains for metabolic engineering. In microorganisms, these gain-of-function assays are typically done using laborious protocols with individually archived strains or in low-throughput following qualitative selection for a phenotype of interest, such as antibiotic resistance. However, many microbial genes are poorly characterized and the importance of a given gene may only be apparent under certain conditions. Therefore, more scalable approaches for gain-of-function assays are needed. Here, we present Dual Barcoded Shotgun Expression Library Sequencing (Dub-seq), a strategy that couples systematic gene overexpression with DNA barcode sequencing for large-scale interrogation of gene fitness under many experimental conditions at low cost. Dub-seq can be applied to many microorganisms and is a valuable new tool for large-scale gene function characterization.« less

Authors:
ORCiD logo [1];  [1];  [1];  [1];  [1]; ORCiD logo [2];  [3]; ORCiD logo [3]
+ Show Author Affiliations
  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 Laboratory (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
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.
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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. https://doi.org/10.1038/s41467-018-08177-8
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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. https://doi.org/10.1038/s41467-018-08177-8. https://www.osti.gov/servlets/purl/1508060.
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@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 greatly increases the throughput of genome-wide overexpression assays. In Dub-seq, a shotgun expression library is cloned between dual random DNA barcodes and the precise breakpoints of DNA fragments are associated to the barcode sequences prior to performing assays. To assess the fitness of individual strains carrying these plasmids, we use DNA barcode sequencing (BarSeq), which is amenable to large-scale sample multiplexing. As a demonstration of this approach, we constructed a Dub-seq library with total Escherichia coli genomic DNA, performed 155 genome-wide fitness assays in 52 experimental conditions, and identified 813 genes with high-confidence overexpression phenotypes across 4,151 genes assayed. 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 such as transposon site sequencing or CRISPRi and will facilitate rapid and systematic functional characterization of microbial genomes.ImportanceMeasuring the phenotypic consequences of overexpressing genes is a classic genetic approach for understanding protein function; for identifying drug targets, antibiotic and metal resistance mechanisms; and for optimizing strains for metabolic engineering. In microorganisms, these gain-of-function assays are typically done using laborious protocols with individually archived strains or in low-throughput following qualitative selection for a phenotype of interest, such as antibiotic resistance. However, many microbial genes are poorly characterized and the importance of a given gene may only be apparent under certain conditions. Therefore, more scalable approaches for gain-of-function assays are needed. Here, we present Dual Barcoded Shotgun Expression Library Sequencing (Dub-seq), a strategy that couples systematic gene overexpression with DNA barcode sequencing for large-scale interrogation of gene fitness under many experimental conditions at low cost. Dub-seq can be applied to many microorganisms and is a valuable new tool for large-scale gene function characterization.},
doi = {10.1038/s41467-018-08177-8},
journal = {Nature Communications},
number = 1,
volume = 10,
place = {United States},
year = {Fri Jan 18 00:00:00 EST 2019},
month = {Fri Jan 18 00:00:00 EST 2019}
}
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https://doi.org/10.1038/s41467-018-08177-8
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Expression of heterologous sigma factors enables functional screening of metagenomic and heterologous genomic libraries
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Artificial Gene Amplification in Escherichia coli Reveals Numerous Determinants for Resistance to Metal Toxicity
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Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms
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Functional metagenomics for enzyme discovery: challenges to efficient screening
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Developmental dynamics of the preterm infant gut microbiota and antibiotic resistome
journal, March 2016

TnAraOut, A transposon-based approach to identify and characterize essential bacterial genes
journal, July 2000

  • Judson, Nicholas; Mekalanos, John J.
  • Nature Biotechnology, Vol. 18, Issue 7
  • DOI: 10.1038/77305

Annotation Error in Public Databases: Misannotation of Molecular Function in Enzyme Superfamilies
journal, December 2009

The Complete Genome Sequence of Escherichia coli DH10B: Insights into the Biology of a Laboratory Workhorse
journal, February 2008

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  • Journal of Bacteriology, Vol. 190, Issue 7
  • DOI: 10.1128/jb.01695-07

Directional Cloning into Plasmid Vectors
journal, June 2006

  • Sambrook, Joseph; Russell, David W.
  • Cold Spring Harbor Protocols, Vol. 2006, Issue 1
  • DOI: 10.1101/pdb.prot3919
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    A Novel pH-Regulated, Unusual 603 bp Overlapping Protein Coding Gene pop Is Encoded Antisense to ompA in Escherichia coli O157:H7 (EHEC)
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    • Zehentner, Barbara; Ardern, Zachary; Kreitmeier, Michaela
    • Frontiers in Microbiology, Vol. 11
    • DOI: 10.3389/fmicb.2020.00377

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    • Fernández‐Cabezón, Lorena; Cros, Antonin; Nikel, Pablo I.
    • Biotechnology Journal, Vol. 14, Issue 9
    • DOI: 10.1002/biot.201800439
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