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Microdroplet-based PCR enrichment for large-scale targeted sequencing
Open accessAbstract
Targeted enrichment of specific loci of the human genome is a promising approach to enable sequencing-based studies of genetic variation in large populations. Here we describe an enrichment approach based on microdroplet PCR, which enables 1.5 million amplifications in parallel. We sequenced six samples enriched by microdroplet or traditional singleplex PCR using primers targeting 435 exons of 47 genes. Both methods generated similarly high-quality data: 84% of the uniquely mapping reads fell within the targeted sequences; coverage was uniform across approximately 90% of targeted bases; sequence variants were called with >99% accuracy; and reproducibility between samples was high (r(2) = 0.9). We scaled the microdroplet PCR to 3,976 amplicons totaling 1.49 Mb of sequence, sequenced the resulting sample with both Illumina GAII and Roche 454, and obtained data with equally high specificity and sensitivity. Our results demonstrate that microdroplet technology is well suited for processing DNA for massively parallel enrichment of specific subsets of the human genome for targeted sequencing.
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“In genome research, an average depth of sequence coverage of 30-fold has been accepted as sufficient for single-nucleotide polymorphism (SNP) and nucleotide insertion or deletion (indel) detection (15–22).”
From Carrier testing for severe childhood recessive diseases by next-generation sequencing (2011) · cites this paper as [16]
Experimental — accuracy not yet measuredThe source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
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“Target capture and next-generation sequencing (NGS) have shown efficacy and, recently, scalability for resequencing human genomes and exomes, providing an alternative potential paradigm for comprehensive carrier testing (15–22).”
From Carrier testing for severe childhood recessive diseases by next-generation sequencing (2011) · cites this paper as [16]
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“Alternatively, microdroplet PCR should theoretically achieve a sensitivity of ~99%, albeit at higher cost (16, 47).”
From Carrier testing for severe childhood recessive diseases by next-generation sequencing (2011) · cites this paper as [16]
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“Preliminary experiments suggested that existing protocols for Agilent SureSelect hybrid capture (15) and RainDance microdroplet polymerase chain reaction (PCR) (16) but not Febit HybSelect microarray-based biochip capture (30) or Olink padlock probe ligation and PCR (31) yielded consistent target enrichment.”
From Carrier testing for severe childhood recessive diseases by next-generation sequencing (2011) · cites this paper as [16]
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“Recently, RainDance Technologies developed an alternative and fully integrated enrichment system using microdroplet PCR that can also be coupled to next-generation sequencing platforms.(15) The encapsulation of distinct PCR reactions in microdroplets enables the sensitive, specific and simultaneous amplification of up…”
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“Droplet microfluidics has shown promise in many different biological and chemical applications, including cell biology (1–3), drug screening (4–6), and nucleic acid analysis (7, 8) applications.”
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“The RainDance DeepSeq system is an example of an amplicon-based platform that is differentiated by its use of microdropletPCR, which allows for the simultaneous amplification of a large number of targeted regions without disruptions due to primer pair interactions.(8) In this study, we sought to leverage the advantage…”
MethodOil-Isolated Hydrogel Microstructures for Sensitive Bioassays On-Chip
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“Microdroplet PCR technology utilizes picoliter-sized droplets as individual reaction vessels to perform over one million unique PCR reactions per sample in less than one day [Tewhey et al., 2009].”
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Multiplex target capture with double-stranded DNA probes
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“The most widely used methods utilize multiplex PCR amplification [6-8], hybrid-capture [9-12], selective target circularization [13-21], and oligonucleotide-selective sequencing [22].”
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Molecular tools for companion diagnostics
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“Raindance emulsion microdroplet PCR technology was selected for sequence enrichment [19] and a list of 45 muscle disease causing genes submitted for primer design.”
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“These features have led to a growing number of applications of droplet microfluidics technology for DNA amplification and detection [5–7], library preparation [8,9], in vivo directed evolution [10–12], single-cell assays [13–15] and others [16–18].”
BackgroundTargeted sequencing with microfluidics
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“Keywords: AT-rich DNA, clinical samples, E.coli, enrichment, host DNA contamination, in-solution hybridisation, next generation sequencing, P.falciparum.”
Droplet microfluidics for kinetic studies of viral fusion
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Multiple pathogen biomarker detection using an encoded bead array in droplet PCR
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“Classically, PCR is used to analyse a small number of genes in combination with the Sanger method, or more recently, with second-generation high-throughput sequencing (Tewhey et al., 2009; reviewed in Metzker, 2010).”
MethodFemtosecond laser-patterned slippery surfaces on PET for liquid patterning and blood resistance
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InfluentialSelected technologies for measuring acquired genetic damage in humans
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“…pyrosequencing methods [Margulies et al., 2005; Russom et al., 2005], digital PCR enrichment using a droplet primer library for large-scale target sequencing [Tewhey et al., 2009], and microscale PCR that is promising for single molecule or single-cell sequencing [Blazej et al., 2006].”
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Microdroplets-on-chip: A review
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Detection and Surveillance of Harmful Algal Bloom Species and Toxins
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“Technologies such as chromosome separation or sorting with a microfluidic device, or droplet PCR to amplify genome fragments in large scale, are being developed [122–128].”
Genotyping by Sequencing Advancements in Barley
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Next Generation Sequencing—General Information about the Technology, Possibilities, and Limitations
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“More recent high-throughput PCR strategies, such as micro-droplet PCR by Raindance [6], nanoliter SmartChip reactions by WaferGen Biosystems and Access arrays by Fluidigm, could bring PCR enrichment into the * Correspondence: Steve.Lefever@UGent.be 1Center for Medical Genetics, Ghent University, De Pintelaan 185, 9000…”
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Sequencing the Cancer Methylome
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Sensitive and predictable separation of microfluidic droplets by size using in-line passive filter
Ding, R., Ung, W. L., Heyman, J. A., & Weitz, D. A. (2017) · Biomicrofluidics · doi:10.1063/1.4976723 · Cited by 17
Lv, J., Jiao, W., Guo, H., Liu, P., Wang, R., Zhang, L., Zeng, Q., Hu, X., Bao, Z., & Wang, S. (2018) · Genome Research · doi:10.1101/gr.235820.118 · Cited by 17
“Highly multiplexed PCR-based approaches, for example, microdroplet PCR (Tewhey et al. 2009) and AmpliSeq (Damiati et al. 2016), and sequence capture approaches like hybrid capture (Hodges et al. 2007; Gnirke et al. 2009) and molecular inversion 4These authors contributed equally to this work.”
Zeng, W., Li, S., & Fu, H. (2018) · Sensors and Actuators A Physical · doi:10.1016/j.sna.2018.01.013 · Cited by 16
Dealing with Pseudogenes in Molecular Diagnostics in the Next Generation Sequencing Era
Claes, K., Rosseel, T., & Leeneer, K. D. (2021) · Methods in molecular biology · doi:10.1007/978-1-0716-1503-4_22 · Cited by 18
Bagheri, M., Siavashi, M., & Mousavi, S. (2023) · Computers & Fluids · doi:10.1016/j.compfluid.2023.105969 · Cited by 16
Pinch-off droplet generator using microscale gigahertz acoustics
Zhou, Y., He, M., Zhang, H., Liu, B., Sun, C., Han, Z., & Duan, X. (2023) · Lab on a Chip · doi:10.1039/d3lc00515a · Cited by 16
Pel, J., Leung, A., Choi, W. W. Y., Despotovic, M., Ung, W. L., Shibahara, G., Gelinas, L., & Marziali, A. (2018) · PLoS ONE · doi:10.1371/journal.pone.0208283 · Cited by 16
“Techniques aimed at mitigating 60 multiplexing challenges include using droplets to reduce primer dimer formation (3), 61 integrating special primer adapters to enable tiling without sample splitting (4), or linking 62”
BackgroundCao, Z., Ye, Y., Li, G., Zhang, R., Dong, S., & Liu, Y. (2022) · Biosensors and Bioelectronics · doi:10.1016/j.bios.2022.114499 · Cited by 16
Ultra-small droplet generation via volatile component evaporation
Zhang, Q., Liu, X., Liu, D., & Gai, H. (2014) · Lab on a Chip · doi:10.1039/c3lc51183a · Cited by 16
A microfluidic device for on-chip agarose microbead generation with ultralow reagent consumption
Simon-Desbois, L., Padirac, A., Kaneda, S., Genot, A. J., Rondelez, Y., Hober, D., Collard, D., & Fujii, T. (2012) · Biomicrofluidics · doi:10.1063/1.4758460 · Cited by 16
Mixing inside droplet co-flowing with Newtonian and shear-thinning fluids in microchannel
Roshchin, D. E., & Патлажан, С. А. (2022) · International Journal of Multiphase Flow · doi:10.1016/j.ijmultiphaseflow.2022.104288 · Cited by 15
Evaluation of ultra-deep targeted sequencing for personalized breast cancer care
Harismendy, O., Schwab, R. B., Alakus, H., Yost, S. E., Matsui, H., Hasteh, F., Wallace, A. M., Park, H. L., Madlensky, L., Parker, B. A., et al. (2013) · Breast Cancer Research · doi:10.1186/bcr3584 · Cited by 15
“Tewhey R, Warner JB, Nakano M, Libby B, Medkova M, David PH, Kotsopoulos SK, Samuels ML, Hutchison JB, Larson JW, Topol EJ, Weiner MP, Harismendy O, Olson J, Link DR, Frazer KA: Microdroplet-based PCR enrichment for large-scale targeted sequencing.”
InfluentialSelf-citationmicroDuMIP: target-enrichment technique for microarray-based duplex molecular inversion probes
Yoon, J.-K., Ahn, J., Kim, H. S., Han, S. M., Jang, H. S., Lee, M. G., Lee, J. H., & Bang, D. (2014) · Nucleic Acids Research · doi:10.1093/nar/gku1188 · Cited by 15
“To capture genomic regions of interest for NGS, a variety of target enrichment strategies have been used such as microdroplet polymerase chain reaction (PCR) (2), solidphase capture (3) and solution-phase capture (4,5) methods.”
Identification and Analysis of Genes Associated with Inherited Retinal Diseases
Khan, M., Fadaie, Z., Cornelis, S. S., Cremers, F. P. M., & Roosing, S. (2018) · Methods in molecular biology · doi:10.1007/978-1-4939-8669-9_1 · Cited by 14
Li, Y., Shi, X., Zuo, Y., Tian, L., Liu, L., Shen, Z., Shen, J., Zhang, R., & Wang, S. (2022) · Microbiology Spectrum · doi:10.1128/spectrum.02297-22 · Cited by 17
Personalized medicine in hematology — A landmark from bench to bed
Badalian‐Very, G. (2014) · Computational and Structural Biotechnology Journal · doi:10.1016/j.csbj.2014.08.002 · Cited by 15
“on exons or targeted genomic sequences for drugable targets and predictive biomarkers for drug response, several questions could be addressed for a fraction of the cost of WGS (Table 2) [68–71, 132].”
BackgroundSteering particles by breaking symmetries
Bet, B., Samin, S., Georgiev, R., Eral, H. B., & van Roij, R. (2018) · Journal of Physics Condensed Matter · doi:10.1088/1361-648x/aabea9 · Cited by 14
Nedelkova, M., Maresca, M., Fu, J., Rostovskaya, M., Chenna, R., Thiede, C., Anastassiadis, K., Sarov, M., & Stewart, A. F. (2011) · Nucleic Acids Research · doi:10.1093/nar/gkr668 · Cited by 14
“target-enrichment strategies, based on polymerase chain reaction (PCR) (21), hybridization or molecular inversion probes (15) merely detect variations, without isolation of the intact allele as a clone that can be further analyzed to link polymorphisms over large regions or to be genetically manipulated for downstream…”
BackgroundExperiences with array-based sequence capture; toward clinical applications
Almomani, R., van der Heijden, J., Ariyürek, Y., Lai, Y., Bakker, E., van Galen, M., Breuning, M. H., & den Dunnen, J. T. (2010) · European Journal of Human Genetics · doi:10.1038/ejhg.2010.145 · Cited by 14
High-Throughput Resequencing of Maize Landraces at Genomic Regions Associated with Flowering Time
Jamann, T., Sood, S., Wisser, R. J., & Holland, J. B. (2017) · PLoS ONE · doi:10.1371/journal.pone.0168910 · Cited by 14
Adaptive sequencing using nanopores and deep learning of mitochondrial DNA
Danilevsky, A., Polsky, A. L., & Shomron, N. (2022) · Briefings in Bioinformatics · doi:10.1093/bib/bbac251 · Cited by 19
Assessing Limit of Detection in Clinical Sequencing
Starks, E. R., Swanson, L., Docking, R., Bosdet, I., Munro, S., Moore, R. A., & Karsan, A. (2021) · Journal of Molecular Diagnostics · doi:10.1016/j.jmoldx.2020.12.010 · Cited by 13
Thinking Outside the Box: Fostering Innovation and Non–Hypothesis-Driven Research at NIH
Aragon, R. (2011) · Science Translational Medicine · doi:10.1126/scitranslmed.3001742 · Cited by 13
Lan, H., & Khismatullin, D. B. (2014) · Physical Review E · doi:10.1103/physreve.90.012705 · Cited by 13
Recent development of microfluidics-based platforms for respiratory virus detection
Shi, J., Zhang, Y., & Yang, M. (2023) · Biomicrofluidics · doi:10.1063/5.0135778 · Cited by 15
Yu, H., van Karnebeek, C., Sinclair, G. B., Hill, A., Cui, H., Zhang, V. W., & Wong, L. C. (2013) · Molecular Genetics and Metabolism · doi:10.1016/j.ymgme.2013.09.018 · Cited by 13
Edge effects in calling variants from targeted amplicon sequencing
Satya, R. V., & DiCarlo, J. (2014) · BMC Genomics · doi:10.1186/1471-2164-15-1073 · Cited by 12
“multiplex PCR reactions (AmpliSeqTM from Life Technologies, GeneRead from QIAGEN), or using thousands of single-plex PCR reactions accomplished using microfluidics [3].”
MethodJiang, Y., Du, L. X., Li, Y., Mu, Q., Cui, Z.-X., Zhou, J. H., & Wu, W. (2018) · The Analyst · doi:10.1039/c8an00035b · Cited by 12
Simultaneous Droplet Generation with In-Series Droplet T-Junctions Induced by Gravity-Induced Flow
Bajgiran, K. R., Cordova, A. S., Elkhanoufi, R., Dorman, J. A., & Melvin, A. T. (2021) · Micromachines · doi:10.3390/mi12101211 · Cited by 11
Sipos, B., Massingham, T., Stütz, A. M., & Goldman, N. (2012) · PLoS ONE · doi:10.1371/journal.pone.0043359 · Cited by 12
“Nevertheless, this procedure is more scalable than molecular cloning due to the recent developments in microfluidics technologies which enable the manipulation of reactants in very small volumes and might offer a practical solution for performing single molecule PCR in a safe and high-throughput way [27].”
BackgroundDynamic self-assembly of charged colloidal strings and walls in simple fluid flows
Abe, Y., Zhang, B., Gordillo, L., Karim, A. M., Francis, L. F., & Cheng, X. (2017) · Soft Matter · doi:10.1039/c6sm02524b · Cited by 11
Fixation Effects on Variant Calling in a Clinical Resequencing Panel
Parker, J., Yap, S. Q., Starks, E. R., Slind, J., Swanson, L., Docking, R., Fuller, M., Zhou, C., WALKER, B. A. M., Filipenko, D., et al. (2019) · Journal of Molecular Diagnostics · doi:10.1016/j.jmoldx.2019.03.005 · Cited by 11
Metadata
- Authors
- Tewhey, R., Warner, J. B., Nakano, M., Libby, B., Medkova, M., David, P. H., Kotsopoulos, S. K., Samuels, M. L., Hutchison, J. B., Larson, J. W., Topol, E. J., Weiner, M. P., Harismendy, O., Olson, J., Link, D. R., & Frazer, K. A.
- Affiliations
- Scripps Research Institute (US)
- Massachusetts Institute of Technology (US)
- University of California San Diego (US)
- Corresponding author
- Link, Darren R
- Frazer, Kelly A
- ORCID
- Venue
- Nature Biotechnology
- Published
- 2009-11
- Volume
- 27
- Issue
- 11
- Pages
- 1025-1031
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC2779736
- OpenAlex
- W2164112721
- Cited by
- 454
- References
- 27
- Citations by year
- 2026: 3 · 2025: 9 · 2024: 14 · 2023: 18 · 2022: 13 · 2021: 13 · 2020: 11 · 2019: 13 · 2018: 20 · 2017: 34 · 2016: 25 · 2015: 31 · 2014: 42 · 2013: 49 · 2012: 58
- FWCI
- 31.84
- Type
- journal article
- Language
- en
- Update notices
- erratum (2010-02-01)
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- eScholarship (California Digital Library) (submittedVersion)
- Funders
- National Institutes of Health (1r21ca125693-01)
- National Heart, Lung, and Blood Institute (P50HL077107)
- National Cancer Institute (1R21CA125693-01, R21 CA125693)
- National Center for Research Resources (1U54RR025204-01, UL1RR025774)
- Topics
- Physical Sciences › Chemical Engineering › Fluid Flow and Transfer Processes › Innovative Microfluidic and Catalytic Techniques Innovation
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genomics and Rare Diseases
- Keywords
- target enrichment, microfluidics, targeted sequencing, exome sequencing, variant calling, human genome
- MeSH terms
- Base Sequence, Humans, Mutation, Reproducibility of Results, Polymerase Chain Reaction, Sequence Analysis, DNA, Microfluidics
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