Artifact-Free Quantification and Sequencing of Rare Recombinant Viruses by Using Drop-Based Microfluidics

Ye Tao; Assaf Rotem; Huidan Zhang; Shelley K. Cockrell; Stephan A. Koehler; Connie B. Chang; Lloyd W. Ung; Paul G. Cantalupo; Yukun Ren; Jeffrey S. Lin; Andrew B. Feldman; Christiane E. Wobus; James M. Pipas; David A. Weitz

doi:10.1002/cbic.201500384

Artifact-Free Quantification and Sequencing of Rare Recombinant Viruses by Using Drop-Based Microfluidics

Ye Tao
, Assaf Rotem
, Huidan Zhang
, Shelley K. Cockrell
, Stephan A. Koehler
, Connie B. Chang
, Lloyd W. Ung
, Paul G. Cantalupo
, Yukun Ren
, Jeffrey S. Lin
, Andrew B. Feldman
, Christiane E. Wobus
, James M. Pipas
, David A. Weitz^*

^*Corresponding author for this work

Harvard University
School of Mechatronics Engineering, Harbin Institute of Technology
China Medical University
University of Pittsburgh
Montana State University
Johns Hopkins University Applied Physics Laboratory
Johns Hopkins University
University of Michigan, Ann Arbor

Research output: Contribution to journal › Article › peer-review

25 Scopus citations

Abstract

Recombination is an important driver in the evolution of viruses and thus is key to understanding viral epidemics and improving strategies to prevent future outbreaks. Characterization of rare recombinant subpopulations remains technically challenging because of artifacts such as artificial recombinants, known as chimeras, and amplification bias. To overcome this, we have developed a high-throughput microfluidic technique with a second verification step in order to amplify and sequence single recombinant viruses with high fidelity in picoliter drops. We obtained the first artifact-free estimate of in vitro recombination rate between murine norovirus strains MNV-1 and WU20 co-infecting a cell (P_rec=3.3×10^-4±2×10^-5) for a 1205nt region. Our approach represents a time- and cost-effective improvement over current methods, and can be adapted for genomic studies requiring artifact- and bias-free selective amplification, such as microbial pathogens, or rare cancer cells.

Original language	English
Pages (from-to)	2167-2171
Number of pages	5
Journal	ChemBioChem
Volume	16
Issue number	15
DOIs	https://doi.org/10.1002/cbic.201500384
State	Published - 1 Oct 2015
Externally published	Yes

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

SDG 3 Good Health and Well-being

Keywords

RT-PCR
drop-based microfluidics
error-free genomic amplification
sequence determination
viruses

Access to Document

10.1002/cbic.201500384

Cite this

Tao, Y., Rotem, A., Zhang, H., Cockrell, S. K., Koehler, S. A., Chang, C. B., Ung, L. W., Cantalupo, P. G., Ren, Y., Lin, J. S., Feldman, A. B., Wobus, C. E., Pipas, J. M., & Weitz, D. A. (2015). Artifact-Free Quantification and Sequencing of Rare Recombinant Viruses by Using Drop-Based Microfluidics. ChemBioChem, 16(15), 2167-2171. https://doi.org/10.1002/cbic.201500384

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title = "Artifact-Free Quantification and Sequencing of Rare Recombinant Viruses by Using Drop-Based Microfluidics",

abstract = "Recombination is an important driver in the evolution of viruses and thus is key to understanding viral epidemics and improving strategies to prevent future outbreaks. Characterization of rare recombinant subpopulations remains technically challenging because of artifacts such as artificial recombinants, known as chimeras, and amplification bias. To overcome this, we have developed a high-throughput microfluidic technique with a second verification step in order to amplify and sequence single recombinant viruses with high fidelity in picoliter drops. We obtained the first artifact-free estimate of in vitro recombination rate between murine norovirus strains MNV-1 and WU20 co-infecting a cell (Prec=3.3×10-4±2×10-5) for a 1205nt region. Our approach represents a time- and cost-effective improvement over current methods, and can be adapted for genomic studies requiring artifact- and bias-free selective amplification, such as microbial pathogens, or rare cancer cells.",

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author = "Ye Tao and Assaf Rotem and Huidan Zhang and Cockrell, \{Shelley K.\} and Koehler, \{Stephan A.\} and Chang, \{Connie B.\} and Ung, \{Lloyd W.\} and Cantalupo, \{Paul G.\} and Yukun Ren and Lin, \{Jeffrey S.\} and Feldman, \{Andrew B.\} and Wobus, \{Christiane E.\} and Pipas, \{James M.\} and Weitz, \{David A.\}",

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doi = "10.1002/cbic.201500384",

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AU - Koehler, Stephan A.

AU - Chang, Connie B.

AU - Ung, Lloyd W.

AU - Cantalupo, Paul G.

AU - Ren, Yukun

AU - Lin, Jeffrey S.

AU - Feldman, Andrew B.

AU - Wobus, Christiane E.

AU - Pipas, James M.

AU - Weitz, David A.

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AB - Recombination is an important driver in the evolution of viruses and thus is key to understanding viral epidemics and improving strategies to prevent future outbreaks. Characterization of rare recombinant subpopulations remains technically challenging because of artifacts such as artificial recombinants, known as chimeras, and amplification bias. To overcome this, we have developed a high-throughput microfluidic technique with a second verification step in order to amplify and sequence single recombinant viruses with high fidelity in picoliter drops. We obtained the first artifact-free estimate of in vitro recombination rate between murine norovirus strains MNV-1 and WU20 co-infecting a cell (Prec=3.3×10-4±2×10-5) for a 1205nt region. Our approach represents a time- and cost-effective improvement over current methods, and can be adapted for genomic studies requiring artifact- and bias-free selective amplification, such as microbial pathogens, or rare cancer cells.

KW - RT-PCR

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KW - error-free genomic amplification

KW - sequence determination

KW - viruses

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SN - 1439-4227

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JO - ChemBioChem

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Artifact-Free Quantification and Sequencing of Rare Recombinant Viruses by Using Drop-Based Microfluidics

Abstract

UN SDGs

Keywords

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