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 (P(rec) = 3.3 × 10(-4) ± 2 × 10(-5) ) for a 1205 nt 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.
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, 29 Oxford Street, Pierce 231, Cambridge, MA, 02138, USA.|School of Mechatronics Engineering, Harbin Institute of Technology, 92 West Dazhi Street, Nan Gang District, Harbin, 150001, China.
authors.forename: Ye
authors.initials: Y
authors.lastname: Tao
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, 29 Oxford Street, Pierce 231, Cambridge, MA, 02138, USA.
authors.forename: Assaf
authors.initials: A
authors.lastname: Rotem
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, 29 Oxford Street, Pierce 231, Cambridge, MA, 02138, USA.|Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, 92 Beier Road, Heping District, Shenyang, 110001, China.
authors.forename: Huidan
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, 4249 Fifth Avenue, Pittsburgh, PA, 15260, USA.
authors.forename: Shelley K
authors.initials: SK
authors.lastname: Cockrell
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, 29 Oxford Street, Pierce 231, Cambridge, MA, 02138, USA.
authors.forename: Stephan A
authors.initials: SA
authors.lastname: Koehler
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, 29 Oxford Street, Pierce 231, Cambridge, MA, 02138, USA.|Chemical and Biological Engineering Department, Montana State University, Bozeman, MT, 59717, USA.
authors.forename: Connie B
authors.initials: CB
authors.lastname: Chang
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, 29 Oxford Street, Pierce 231, Cambridge, MA, 02138, USA.
authors.forename: Lloyd W
authors.initials: LW
authors.lastname: Ung
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, 4249 Fifth Avenue, Pittsburgh, PA, 15260, USA.
authors.forename: Paul G
authors.initials: PG
authors.lastname: Cantalupo
authors.affiliation: School of Mechatronics Engineering, Harbin Institute of Technology, 92 West Dazhi Street, Nan Gang District, Harbin, 150001, China.
authors.forename: Yukun
authors.initials: Y
authors.lastname: Ren
authors.affiliation: Applied Physics Laboratory, Johns Hopkins University, 11100 Johns Hopkins Road, Laurel, MD, 20723, USA.
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Lin
authors.affiliation: Applied Physics Laboratory, Johns Hopkins University, 11100 Johns Hopkins Road, Laurel, MD, 20723, USA.|Department of Emergency Medicine, Johns Hopkins Medicine, 5801 Smith Avenue, Suite 3220, Baltimore, MD, 21209, USA.
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Feldman
authors.affiliation: Department of Microbiology and Immunology, University of Michigan, 1150 West Medical Center Drive, 5622 Medical Science II, Ann Arbor, MI, 48109, USA.
authors.forename: Christiane E
authors.initials: CE
authors.lastname: Wobus
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, 4249 Fifth Avenue, Pittsburgh, PA, 15260, USA.
authors.forename: James M
authors.initials: JM
authors.lastname: Pipas
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, 29 Oxford Street, Pierce 231, Cambridge, MA, 02138, USA. weitz@seas.harvard.edu.|Department of Physics, Harvard University, 29 Oxford Street, Pierce 231, Cambridge, MA, 02138, USA. weitz@seas.harvard.edu.
authors.forename: David A
authors.initials: DA
authors.lastname: Weitz
mesh_terms: D000818:Animals; D016477:Artifacts; D002478:Cells, Cultured; D005456:Fluorescent Dyes; D057166:High-Throughput Screening Assays; D051379:Mice; D044085:Microfluidics / Q000379:methods*; D010316:Particle Size; D011995:Recombination, Genetic / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017421:Sequence Analysis / Q000379:methods*; D014779:Virus Replication / Q000235:genetics; D014780:Viruses / Q000235:genetics*
chemical_list: D005456:Fluorescent Dyes
keywords: RT-PCR; drop-based microfluidics; error-free genomic amplification; sequence determination; viruses