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 sequence coverage depth of 30-fold has been accepted as sufficient for detecting single-nucleotide polymorphisms and nucleotide insertions or deletions.
“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]
The Results and Discussion section on coverage uniformity reports that 25-fold average coverage gave at least five reads for about 90% of targeted bases, and the paper evaluates SNP calls. It does not state that 30-fold coverage is an accepted sufficiency threshold or establish that depth for indel detection. (LLM, conf=0.83)
Experimental — accuracy not yet measuredTarget capture and next-generation sequencing have shown efficacy and, recently, scalability for resequencing human genomes and exomes.
“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]
The Abstract and scale-up Results report effective next-generation sequencing after targeted enrichment of human genomic regions, scaled to 1.49 Mb. The Discussion describes larger-scale exon capture reported by other studies. This paper does not demonstrate scalable capture and resequencing of whole human genomes or exomes; its own experiments cover subsets of them. (LLM, conf=0.87)
Experimental — accuracy not yet measuredMicrodroplet PCR should theoretically achieve a sensitivity of about 99%, albeit at higher cost.
“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]
The Discussion reports that more than 99.6% of amplicons were successfully amplified, which supports a roughly 99% figure for amplification success, not necessarily sensitivity. It separately reports a 94.5% variant-detection rate for microdroplet PCR. Our copy does not establish the claimed higher cost relative to an alternative. (LLM, conf=0.86)
Experimental — accuracy not yet measuredPreliminary experiments suggested that Agilent SureSelect hybrid capture and RainDance microdroplet polymerase chain reaction, but not Febit HybSelect microarray-based capture or Olink padlock probe ligation and PCR, yielded consistent target enrichment.
“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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Vyawahare, S., Griffiths, A. D., & Merten, C. A. (2010) · Chemistry & Biology · doi:10.1016/j.chembiol.2010.09.007 · Cited by 169 · see citing works
A glimpse into past, present, and future DNA sequencing
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“After thermal cycling, the emulsion is broken down and the products are subjected to library construction [61].”
BackgroundDiagnostics based on nucleic acid sequence variant profiling: PCR, hybridization, and NGS approaches
Khodakov, D. A., Wang, C., & Zhang, D. Y. (2016) · Advanced Drug Delivery Reviews · doi:10.1016/j.addr.2016.04.005 · Cited by 155 · see citing works
The next-generation sequencing technology and application
Zhou, X., Ren, L., Meng, Q., Li, Y., Yu, Y., & Yu, J. M. (2010) · Protein & Cell · doi:10.1007/s13238-010-0065-3 · Cited by 151 · see citing works
“Two methods of target-region resequencing are widely used: PCR-based candidate gene (Dracatos et al., 2009; Goossens et al., 2009; Harismendy and Frazer, 2009; Tewhey et al., 2009) and whole exome approaches (Hodges et al.”
MethodUtilization of next-generation sequencing platforms in plant genomics and genetic variant discovery
Deschamps, S., & Campbell, M. A. (2009) · Molecular Breeding · doi:10.1007/s11032-009-9357-9 · Cited by 149 · see citing works
Self-priming compartmentalization digital LAMP for point-of-care
Zhu, Q., Gao, Y., Yu, B., Ren, H., Qiu, L., Han, S., Jin, W., Jin, Q., & Mu, Y. (2012) · Lab on a Chip · doi:10.1039/c2lc40774d · Cited by 147 · see citing works
Ballew, B. J., Vijai, J., De, S., Sarek, G., Vannier, J., Stracker, T. H., Schrader, K. A., Small, T. N., O'Reilly, R., Manschreck, C., et al. (2013) · PLoS Genetics · doi:10.1371/journal.pgen.1003695 · Cited by 145 · see citing works
Digital PCR on an integrated self-priming compartmentalization chip
Zhu, Q., Qiu, L., Yu, B., Xu, Y., Gao, Y., Pan, T., Tian, Q., Song, Q., Jin, W., Jin, Q., et al. (2014) · Lab on a Chip · doi:10.1039/c3lc51327k · Cited by 144 · see citing works
Ocwieja, K., Sherrill-Mix, S., Mukherjee, R., Custers-Allen, R., David, P. H., Brown, M., Wang, S., Link, D., Olson, J., Travers, K., et al. (2012) · Nucleic Acids Research · doi:10.1093/nar/gks753 · Cited by 138 · see citing works
“We also used end point PCR and analysis on ethidium-bromide-stained gels to confirm that the relative ratios of transcripts containing junctions to A3, A4a, A4b and A4c were different in HOS and T cells (Figure 4B).”
InfluentialSelf-citationExisting and Emerging Technologies for Tumor Genomic Profiling
MacConaill, L. E. (2013) · Journal of Clinical Oncology · doi:10.1200/jco.2012.46.5948 · Cited by 138 · see citing works
Multiphase flow in microfluidics: From droplets and bubbles to the encapsulated structures
Sattari, A., Hanafizadeh, P., & Hoorfar, M. (2020) · Advances in Colloid and Interface Science · doi:10.1016/j.cis.2020.102208 · Cited by 141 · see citing works
Miniaturization of biological assays — Overview on microwell devices for single-cell analyses
Lindström, S., & Svahn, H. A. (2010) · Biochimica et Biophysica Acta (BBA) - General Subjects · doi:10.1016/j.bbagen.2010.04.009 · Cited by 135 · see citing works
The PhyloPythiaS Web Server for Taxonomic Assignment of Metagenome Sequences
Patil, K. R., Roune, L., & McHardy, A. C. (2012) · PLoS ONE · doi:10.1371/journal.pone.0038581 · Cited by 133 · see citing works
“In the current study, we analyzed two datasets, one sequenced with Sanger and another with Illumina sequencing technology [21].”
UroMark—a urinary biomarker assay for the detection of bladder cancer
Feber, A., Dhami, P., Dong, L., de Winter, P., Tan, W. S., Martínez‐Fernández, M., Paul, D. S., Hynes-Allen, A., Rezaee, S., Gurung, P. M. S., et al. (2017) · Clinical Epigenetics · doi:10.1186/s13148-016-0303-5 · Cited by 131 · see citing works
“For example, the microdroplet-based PCR amplification of bisulfite-converted DNA followed by NGS of the amplified target loci (termed RainDrop BS-Seq) enables the sensitive, specific and simultaneous amplification of up to 4000 bisulfite-converted target loci [26].”
Voelkerding, K. V., Dames, S. A., & Durtschi, J. (2010) · Journal of Molecular Diagnostics · doi:10.2353/jmoldx.2010.100043 · Cited by 130 · see citing works
A ferrobotic system for automated microfluidic logistics
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Sequence-based marker development in wheat: Advances and applications to breeding
Paux, E., Sourdille, P., Mackay, I., & Feuillet, C. (2011) · Biotechnology Advances · doi:10.1016/j.biotechadv.2011.09.015 · Cited by 122 · see citing works
Liao, G. J. W., Lun, F. M. F., Zheng, Y. W. L., Chan, K. C. A., Leung, T. Y., Lau, T. K., Chiu, R. W. K., & Lo, Y. D. (2010) · Clinical Chemistry · doi:10.1373/clinchem.2010.154336 · Cited by 120 · see citing works
Nanoliter Multiplex PCR Arrays on a SlipChip
Shen, F., Du, W., Davydova, E. K., Karymov, M. A., Pandey, J., & Ismagilov, R. F. (2010) · Analytical Chemistry · doi:10.1021/ac1007249 · Cited by 115 · see citing works
The physics of 2D microfluidic droplet ensembles
Beatus, T., Bar‐Ziv, R., & Tlusty, T. (2012) · Physics Reports · doi:10.1016/j.physrep.2012.02.003 · Cited by 111 · see citing works
“…embryos (Funfak et al., 2007), PCR reactions of a single template molecule (Beer et al., 2007, 2008; Kiss et al., 2008; Schaerli et al., 2008; Tewhey et al., 2009), scanning of protein crystallization conditions (Lau et al., 2007; Leng and Salmon, 2009; Shim et al., 2007; Zheng et al., 2005,…”
BackgroundDetecting biomarkers with microdroplet technology
Taly, V., Pekin, D., Abed, A. I. E., & Laurent‐Puig, P. (2012) · Trends in Molecular Medicine · doi:10.1016/j.molmed.2012.05.001 · Cited by 110 · see citing works
Gasc, C., Peyretaillade, É., & Peyret, P. (2016) · Nucleic Acids Research · doi:10.1093/nar/gkw309 · Cited by 114 · see citing works
Moon, B., Jones, S. G., Hwang, D. K., & Tsai, S. S. H. (2015) · Lab on a Chip · doi:10.1039/c5lc00217f · Cited by 113 · see citing works
Perkins, G., Lu, H., Garlan, F., & Taly, V. (2016) · Advances in clinical chemistry · doi:10.1016/bs.acc.2016.10.001 · Cited by 113 · see citing works
Microfluidic platform for combinatorial synthesis in picolitre droplets
Theberge, A. B., Mayot, E., Harrak, A. E., Kleinschmidt, F., Huck, W. T. S., & Griffiths, A. D. (2012) · Lab on a Chip · doi:10.1039/c2lc21019c · Cited by 111 · see citing works
Lin, X., Tang, W., Ahmad, S., Lü, J., Colby, C. C., Zhu, J., & Yu, Q. (2012) · Hearing Research · doi:10.1016/j.heares.2012.01.004 · Cited by 108 · see citing works
Clinical application of amplicon-based next-generation sequencing in cancer
Chang, F., & Li, M. M. (2013) · Cancer Genetics · doi:10.1016/j.cancergen.2013.10.003 · Cited by 107 · see citing works
“The droplet PCR reactions are then destabilized and the PCR products are released and purified for sequencing (21).”
BackgroundOverview of Target Enrichment Strategies
Kozarewa, I., Armisen, J., Gardner, A. F., Slatko, B. E., & Hendrickson, C. L. (2015) · Current Protocols in Molecular Biology · doi:10.1002/0471142727.mb0721s112 · Cited by 102 · see citing works
Medlin, L. K., & Orozco, J. (2017) · Sensors · doi:10.3390/s17051184 · Cited by 104 · see citing works
“Field-induced coalescence of these droplet pairs results in the two droplets merging into a single PCR droplet, which is collected and processed as an emulsion PCR reaction [99].”
MethodAdvances in microfluidics in combating infectious diseases
Tay, A. K. P., Pavesi, A., Yazdi, S. R., Lim, C. T., & Warkiani, M. E. (2016) · Biotechnology Advances · doi:10.1016/j.biotechadv.2016.02.002 · Cited by 101 · see citing works
“Currently, droplet microfluidic PCR is also being tested (Tewhey et al., 2009).”
Droplet-Based Microfluidics: Enabling Impact on Drug Discovery
Dressler, O. J., Maceiczyk, R. M., Chang, S., & deMello, A. J. (2013) · SLAS DISCOVERY · doi:10.1177/1087057113510401 · Cited by 98 · see citing works
Detection of low prevalence somatic mutations in solid tumors with ultra-deep targeted sequencing
Harismendy, O., Schwab, R. B., Bao, L., Olson, J., Rozenzhak, S., Kotsopoulos, S. K., Pond, S. J. K., Crain, B., Chee, M. S., Messer, K. S., et al. (2011) · Genome biology · doi:10.1186/gb-2011-12-12-r124 · Cited by 98 · see citing works
“The RDT1000 generates each PCR droplet by merging a single DNA template droplet with a single primer droplet [13].”
MethodResultInfluentialSelf-citationA novel three-round multiplex PCR for SNP genotyping with next generation sequencing
Chen, K., Zhou, Y., Li, K., Qi, L.-X., Zhang, Q., Wang, M., & Xiao, J. (2016) · Analytical and Bioanalytical Chemistry · doi:10.1007/s00216-016-9536-6 · Cited by 97 · see citing works
Under-three minute PCR: Probing the limits of fast amplification
Wheeler, E. K., Hara, C. A., Frank, J., DeOtte, J. R., Hall, S., Benett, W. J., Spadaccini, C. M., & Beer, N. R. (2011) · The Analyst · doi:10.1039/c1an15365j · Cited by 97 · see citing works
Target Enrichment Approaches for Next-Generation Sequencing Applications in Oncology
Singh, R. R. (2022) · Diagnostics · doi:10.3390/diagnostics12071539 · Cited by 100 · see citing works
“They involve varied approaches, such as droplet microfluidics, where the PCR amplification, barcoding, and library preparation occur in microscopic droplets, which act as microreactors [18,43], and multilayered soft lithographics microfluidics systems, which use microfluidics in a cartridge to miniaturize and conduct…”
Zargar, S. M., Raatz, B., Sonah, H., MuslimaNazir, Bhat, J. A., Dar, Z. A., Agrawal, G. K., & Rakwal, R. (2015) · Journal of Crop Science and Biotechnology · doi:10.1007/s12892-015-0037-5 · Cited by 95 · see citing works
“Tewhey and co-workers in 2009 reported sequencing PCR amplicons from primers targeting 435 exons of 47 genes in six samples simultaneously using a microdroplet PCR platform from RainDance Technologies (Lexington, USA) (Tewhey et al. 2009).”
MethodComparison of Three Targeted Enrichment Strategies on the SOLiD Sequencing Platform
Hedges, D. J., Guettouche, T., Yang, S., Bademci, G., Diaz, A., Andersen, A., Hulme, W. F., Linker, S. B., Mehta, A., Edwards, Y. J. K., et al. (2011) · PLoS ONE · doi:10.1371/journal.pone.0018595 · Cited by 94 · see citing works
“Genomic enrichment via massively parallel PCR was conducted at Raindance Technologies, as previously described [6].”
MethodReview of massively parallel DNA sequencing technologies
Moorthie, S. A., Mattocks, C., & Wright, C. F. (2011) · The HUGO Journal · doi:10.1007/s11568-011-9156-3 · Cited by 93 · see citing works
“…from RainDance Technologies which uses an emPCR approach to simultaneously amplify up to 4,000 short DNA sequences in separate microdroplets (Tewhey et al. 2009), and the Access Array from Fluidigm which uses proprietary microfluidics to setup an array of 2,304 PCRs (48 samples 9 48 assays).”
Droplet-based microfluidics at the femtolitre scale
Leman, M., Abouakil, F., Griffiths, A. D., & Tabeling, P. (2014) · Lab on a Chip · doi:10.1039/c4lc01122h · Cited by 92 · see citing works
Trans genomic capture and sequencing of primate exomes reveals new targets of positive selectionPreprint
George, R. D., McVicker, G., Diederich, R., Ng, S. B. H., MacKenzie, A. P., Swanson, W. J., Shendure, J., & Thomas, J. H. (2011) · Genome Research · doi:10.1101/gr.121327.111 · Cited by 89 · see citing works
“New methods for targeted enrichment of the human exome allow high-coverage sequence to be generated for the coding fraction of the genome (Albert et al. 2007; Gnirke et al. 2009; Tewhey et al. 2009; Turner et al. 2009).”
Jones, M. A., Bhide, S., Chin, E., Ng, B. G., Rhodenizer, D., Zhang, V. W., Sun, J., Tanner, A. K., Freeze, H. H., & Hegde, M. (2011) · Genetics in Medicine · doi:10.1097/gim.0b013e318226fbf2 · Cited by 91 · see citing works
Cryptic species in plant-parasitic nematodes
Palomares‐Rius, J. E., Cantalapiedra‐Navarrete, C., & Castillo, P. (2014) · Nematology · doi:10.1163/15685411-00002831 · Cited by 90 · see citing works
Analyzing the cancer methylome through targeted bisulfite sequencing
Lee, E.-J., Luo, J., Wilson, J. M., & Shi, H. (2012) · Cancer Letters · doi:10.1016/j.canlet.2012.10.040 · Cited by 89 · see citing works
“PCR amplification bias is significantly decreased due to the nature of microdroplet emulsion PCR reactions [59].”
Shen, X., Liang, D., Feng, Y., Chen, M. Y., & Zhang, P. (2013) · Molecular Biology and Evolution · doi:10.1093/molbev/mst122 · Cited by 88 · see citing works
Höijer, I., Tsai, Y., Clark, T. A., Kotturi, P., Dahl, N., Stattin, E., Bondeson, M.-L., Feuk, L., Gyllensten, U. B., & Ameur, A. (2018) · Human Mutation · doi:10.1002/humu.23580 · Cited by 88 · see citing works
Novel Insight into Mutational Landscape of Head and Neck Squamous Cell Carcinoma
Gaykalova, D. A., Mambo, E., Choudhary, A. K., Houghton, J., Buddavarapu, K., Sanford, T., Darden, W., Adai, A. T., Hadd, A. G., Latham, G. J., et al. (2014) · PLoS ONE · doi:10.1371/journal.pone.0093102 · Cited by 87 · see citing works
“Somatic mutation calling The sequence read data generated from the GAIIx were demultiplexed, adapter and primer sequences were removed and trimmed to retain high quality data (q20 or higher) [37,39,40].”
MethodRapid and continuous magnetic separation in droplet microfluidic devices
Brouzes, E. R., Kruse, T., Kimmerling, R. J., & Strey, H. H. (2014) · Lab on a Chip · doi:10.1039/c4lc01327a · Cited by 87 · see citing works
From tubes to drops: droplet-based microfluidics for ultrahigh-throughput biology
Tran, T. M., Lan, F., Thompson, C. S., & Abate, A. R. (2013) · Journal of Physics D Applied Physics · doi:10.1088/0022-3727/46/11/114004 · Cited by 86 · see citing works
Advances in clinical next-generation sequencing: target enrichment and sequencing technologies
Ballester, L. Y., Luthra, R., Kanagal‐Shamanna, R., & Singh, R. R. (2015) · Expert Review of Molecular Diagnostics · doi:10.1586/14737159.2016.1133298 · Cited by 86 · see citing works
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“Therefore, Sanger Sequencing (Genewiz), RainDance analysis (28) and RNAseq (29) were performed to determine whether any of these cell lines had NOTCH3 mutations.”
Hopp, K., Heyer, C. M., Sieben, C. J., Henke, S. A., Sundsbak, J. L., Patel, S., Patel, P., Consugar, M. B., Czarnecki, P. G., Gliem, T. J., et al. (2011) · Human Molecular Genetics · doi:10.1093/hmg/ddr151 · Cited by 84 · see citing works
Zellmer, A. J., Hanes, M. M., Hird, S. M., & Carstens, B. C. (2012) · Systematic Biology · doi:10.1093/sysbio/sys048 · Cited by 79 · see citing works
“…for systems where high levels of paralogy are expected, other methods of library construction may be preferable, such as PCR amplicon sequencing (e.g., Binladen et al. 2007; Meyer et al. 2008; Tewhey et al. 2009) or target enrichment (e.g., Albert et al. 2007; Okou et al. 2007; Gnirke et al. 2009).”
Smith, C. A., Li, X., Mize, T. H., Sharpe, T., Graziani, E. I., Abell, C., & Huck, W. T. S. (2013) · Analytical Chemistry · doi:10.1021/ac400453t · Cited by 79 · see citing works
Fluorogenic DNA sequencing in PDMS microreactors
Sims, P. A., Greenleaf, W. J., Duan, H., & Xie, X. S. (2011) · Nature Methods · doi:10.1038/nmeth.1629 · Cited by 79 · see citing works
Targeted resequencing of candidate genes using selector probes
Johansson, H., Isaksson, M., Falk-Sörqvist, E., Roos, F., Stenberg, J., Sjöblom, T., Botling, J., Micke, P., Edlund, K., Fredriksson, S., et al. (2010) · Nucleic Acids Research · doi:10.1093/nar/gkq1005 · Cited by 76 · see citing works
A scalable self-priming fractal branching microchannel net chip for digital PCR
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Scheible, M. K. R., Loreille, O. M., Just, R. S., & Irwin, J. A. (2014) · Forensic Science International Genetics · doi:10.1016/j.fsigen.2014.04.010 · Cited by 75 · see citing works
- 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 › Engineering › Biomedical Engineering › 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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