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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]
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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“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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1 of the 27 references couldn’t be matched to a known paper.
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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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