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

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

doi:10.1002/cbic.201500384

Cited by 31 publications

(31 citation statements)

References 43 publications

(13 reference statements)

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“…120 In parallel to studying cellular mRNA molecules in droplets, the Weitz lab also applied droplet microfluidics to analyze single viral RNA templates and characterized mutations/ recombinations associated with it. 121 The mutations including recombinations that happen in viral genomes are an important aspect of host-pathogen interaction, since such rare recombinant viruses often cause epidemics and pandemics. Tao et al developed a droplet based microfluidic platform for specific identification and sequencing of rare recombinant Noroviruses emerging from co-infection of 2 strains of Norovirus (MNV-1 and WU20) in macrophage cells.…”

Section: Single-cell Dna and Genomic Studiesmentioning

confidence: 99%

Droplet-based microfluidics in drug discovery, transcriptomics and high-throughput molecular genetics

et al. 2016

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Droplet-based microfluidics enables assays to be carried out at very high throughput (up to thousands of samples per second) and enables researchers to work with very limited material, such as primary cells, patient's biopsies or expensive reagents. An additional strength of the technology is the possibility to perform large-scale genotypic or phenotypic screens at the single-cell level. Here we critically review the latest developments in antibody screening, drug discovery and highly multiplexed genomic applications such as targeted genetic workflows, single-cell RNAseq and single-cell ChIPseq. Starting with a comprehensive introduction for non-experts, we pinpoint current limitations, analyze how they might be overcome and give an outlook on exciting future applications.

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Section: Single-cell Dna and Genomic Studiesmentioning

confidence: 99%

Droplet-based microfluidics in drug discovery, transcriptomics and high-throughput molecular genetics

et al. 2016

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“…We have applied SMDB to the barcoding of single DNA molecules from virus and microbial genomes, but the principle of encapsulating and barcoding nucleic acids in microfluidic droplets is broadly applicable. For example, droplet microfluidics has been used to encapsulate, lyse, and amplify single viruses and cells 46 47 . The SMDB workflow we describe here could be combined with these methods to barcode the genomes of these organisms, to perform whole-genome single virus or cell sequencing.…”

Section: Discussionmentioning

confidence: 99%

Droplet barcoding for massively parallel single-molecule deep sequencing

et al. 2016

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The ability to accurately sequence long DNA molecules is important across biology, but existing sequencers are limited in read length and accuracy. Here, we demonstrate a method to leverage short-read sequencing to obtain long and accurate reads. Using droplet microfluidics, we isolate, amplify, fragment and barcode single DNA molecules in aqueous picolitre droplets, allowing the full-length molecules to be sequenced with multi-fold coverage using short-read sequencing. We show that this approach can provide accurate sequences of up to 10 kb, allowing us to identify rare mutations below the detection limit of conventional sequencing and directly link them into haplotypes. This barcoding methodology can be a powerful tool in sequencing heterogeneous populations such as viruses.

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“…The actual flow rates inside the microfluidic channels may be slightly different but have not been measured. Similar flow rate settings for drop sorting have been used in previous publications 19 31 32 .…”

Section: Methodsmentioning

confidence: 99%

A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders

Cui

Zhang

Schneider

et al. 2016

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Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (103–106). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution.

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

Cited by 31 publications

References 43 publications

Droplet-based microfluidics in drug discovery, transcriptomics and high-throughput molecular genetics

Droplet-based microfluidics in drug discovery, transcriptomics and high-throughput molecular genetics

Droplet barcoding for massively parallel single-molecule deep sequencing

A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders

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