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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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“For sequencing of PTPN11, all coding regions of PTPN11 were amplified using RainDance microdroplet digital PCR enrichment (RainDance, Billerica, MA) as previously described (38, 39) followed by followed by Illumina HiSeq massively parallel sequencing.”
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High-quality DNA sequence capture of 524 disease candidate genes
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“Target-enrichment sequencing strategies, based on polymerase chain reaction (PCR) (Tewhey et al. 2009), hybridization or molecular inversion probes (Mamanova et al.”
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“It has been extensively used for genomic analysis, including sequencing [5] and digital PCR [6].”
Chemical Transfection of Cells in Picoliter Aqueous Droplets in Fluorocarbon Oil
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“Many of the active droplet actuation methods have been utilized towards the demonstration of chemical and biological assays, including polymerase chain reaction (PCR) [18–22] and immunoassays [23–26].”
MethodMicrofluidic approaches for systems and synthetic biology
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“The enriched DNA samples generated sequencing data of quality comparable to what is obtained with traditional approaches while drastically increasing the experimental throughput [4 ].”
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“The merged RainDance library/PCRmix is deposited into a PCR tube and amplified.(24) After amplification, the PCR fragments are concatenated and sonicated to obtain the desired length inserts for NGS libraries.”
MethodDing, Y., Choo, J., & deMello, A. J. (2017) · Microfluidics and Nanofluidics · doi:10.1007/s10404-017-1889-4 · Cited by 52
“A key feature in this respect is the ability to perform rapid DNA amplification (via the polymerase chain reaction or PCR) within millions of individual droplets in a parallel fashion (Tewhey et al. 2009; Markey et al. 2010; Hindson et al. 2011).”
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“In this setup, each reaction droplet is a reaction well in isolation with a lipid capsule and contains a small amount of template DNA along with one primer pair and the PCR reagents (polymerase, dNTPs, and buffers) [87].”
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“Amplicons corresponding to functionally relevant regions of these genes were sequenced on an Illumina/Solexa Genome Analyzer II platform.”
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Extended Blood Group Molecular Typing and Next-Generation Sequencing
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“For example, microdroplet-based PCRs are performed in a large number of microdroplets, each of which encloses all reagents required for an independent PCR reaction for a specific target [53].”
Kanigowska, P., Shen, Y., Zheng, Y., Rosser, S. J., & Cai, Y. (2015) · SLAS TECHNOLOGY · doi:10.1177/2211068215593754 · Cited by 49
Enrichment of genomic DNA for polymorphism detection in a non‐model highly polyploid crop plant
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“Recently, miniaturization of PCR has shown potential to overcome this limitation, resulting in the development of novel LOC microfluidicbased and droplet-based amplification technologies (Khandurina et al. 2000; Tewhey et al. 2009).”
BackgroundEfficient and Cost Effective Population Resequencing by Pooling and In-Solution Hybridization
Bansal, V., Tewhey, R., LeProust, E. M., & Schork, N. J. (2011) · PLoS ONE · doi:10.1371/journal.pone.0018353 · Cited by 52
“The availability of highthroughput target capture methods combined with the massive throughput of next-generation sequencing platforms has made it possible to interrogate thousands of genomic loci in a cost effective manner[5,6].”
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MethodMultiplex Target Enrichment Using DNA Indexing for Ultra-High Throughput SNP Detection
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“This approach can be used to enrich targets within the human genome [18].”
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On-Demand Droplet Capture and Release Using Microwell-Assisted Surface Acoustic Waves
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“loaded on microfluidic chip to compartmentalize the PCR reactions by single primer pairs[87].”
MethodRecent advances in droplet‐based microfluidics in liquid biopsy for cancer diagnosis
Shi, J., Zhang, Y., Fan, Y., Liu, Y., & Yang, M. (2024) · Droplet · doi:10.1002/dro2.92 · Cited by 44
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“Although microfluidic droplet-based PCR reactions have been used to amplify target molecules for sequencing (15), the MESA approach is unique in that the droplet PCR reactions are not used to directly amplify the material to be sequenced.”
Scalable and deep profiling of mRNA targets for individual microRNAs with chimeric eCLIP
Manakov, S. A., Shishkin, A. A., Yee, B. A., Shen, K. A., Cox, D. C., Park, S. S., Foster, H. M., Chapman, K. B., Yeo, G. W., & Van Nostrand, E. L. (2022) · doi:10.1101/2022.02.13.480296 · Cited by 43
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“dPCR has been successfully performed in a variety of formats, such as multiwell plates [1, 3] , an emulsion PCR [25] [26] [27] , microdroplets [19, [28] [29] [30] [31] , microfluidic chambers [32] , a spinning disk platform [33] , and a SlipChip [34, 35] , and these can be summarized as chip-based or droplet-based dPC…”
MethodApplication of microdroplet PCR for large-scale targeted bisulfite sequencingPreprint
Komori, H. K., LaMere, S. A., Torkamani, A., Hart, T., Kotsopoulos, S. K., Warner, J. B., Samuels, M. L., Olson, J., Head, S. R., Ordoukhanian, P. T., et al. (2011) · Genome Research · doi:10.1101/gr.116863.110 · Cited by 43
“The authors from RainDance Technologies declare competing financial interests; RainDance Technologies is a for-profit company offering a sequence enrichment product for bisulfite-converted DNA.”
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“We used RainDance technology for sequence enrichment (35) and TruSeq DNA Sample preparation Kit V2 (Illumina, catalog number FC-121–2001) for library preparation.”
MethodMicrofluidics for genome-wide studies involving next generation sequencing
Ma, S., Murphy, T. W., & Lu, C. (2017) · Biomicrofluidics · doi:10.1063/1.4978426 · Cited by 42
Clever PCR: more genotyping, smaller volumes
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Droplet-based microfluidic washing module for magnetic particle-based assays
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Particles and microfluidics merged: perspectives of highly sensitive diagnostic detection
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A multi-volume microfluidic device with no reagent loss for low-cost digital PCR application
Si, H., Xu, G., Jing, F., Sun, P., Zhao, D., & Wu, D. (2020) · Sensors and Actuators B Chemical · doi:10.1016/j.snb.2020.128197 · Cited by 41
Targeted Screening and Validation of Copy Number Variations
Ceulemans, S., van der Ven, K., & Del‐Favero, J. (2011) · Methods in molecular biology · doi:10.1007/978-1-61779-507-7_15 · Cited by 42
Chen, H., & Zhou, Q. (2023) · Oncology Reports · doi:10.3892/or.2023.8543 · Cited by 44
“…multiple separate reaction pools running simultaneously to cover the large genomic space of designed gene panels, considering the limited multiplex‑ability of PCR in a single reaction pool up to 2,000 bp of genomic breadth (57,58), which may be challenging when the total cfDNA quantity is limited.”
Prakash, R., Pabbaraju, K., Wong, S., Wong, A. A., Tellier, R., & Kaler, K. (2014) · Micromachines · doi:10.3390/mi6010063 · Cited by 40
“PCR micro-devices [6–9], handling the PCR sample volume in form of rapidly dispensed, discrete microliter or smaller droplets has become a preferred method of choice for PCR micro-devices [10,11].”
MethodMikrotröpfchen in Mikrofluidiksystemen: eine Technik für Entdeckungen in der Chemie und Biologie
Theberge, A. B., Courtois, F., Schaerli, Y., Fischlechner, M., Abell, C., Hollfelder, F., & Huck, W. T. S. (2010) · Angewandte Chemie · doi:10.1002/ange.200906653 · Cited by 40
Modeling of Newtonian droplet formation in power-law non-Newtonian fluids in a flow-focusing device
Chen, Q., Li, J., Song, Y., Christopher, D. M., & Li, X. (2020) · Heat and Mass Transfer · doi:10.1007/s00231-020-02899-6 · Cited by 40
Dames, S. A., Durtschi, J., Geiersbach, K. B., Stephens, J., & Voelkerding, K. V. (2010) · PubMed Central · Cited by 39
Massive parallel sequencing applied to the molecular autopsy in sudden cardiac death in the young
Brión, M., Sobrino, B., Martínez, M. A. L., Blanco‐Verea, A., & Carracedo, Á. (2015) · Forensic Science International Genetics · doi:10.1016/j.fsigen.2015.07.010 · Cited by 39
Next-generation sequencing applied to molecular diagnostics
Natrajan, R., & Reis-Filho, J. S. (2011) · Expert Review of Molecular Diagnostics · doi:10.1586/erm.11.18 · Cited by 41
Sivakumaran, T. A., Husami, A., Kissell, D., Zhang, W., Keddache, M. A., Black, A. P., Tinkle, B. T., Greinwald, J. H., & Zhang, K. (2013) · Otolaryngology · doi:10.1177/0194599813482294 · Cited by 39
“We used RainDance’s microdroplet PCR-based method to enrich the target regions.(10) Using the standard enrichment protocol, we found that more than 50% of the quality filtered reads were not mapped to our target genes and that these reads were resulting from nontarget genomic DNA fragments.”
BackgroundMethodOptical separation of droplets on a microfluidic platform
Jung, J. H., Lee, K. H., Lee, K. S., Ha, B. H., Oh, Y. S., & Sung, H. J. (2013) · Microfluidics and Nanofluidics · doi:10.1007/s10404-013-1263-0 · Cited by 39
Janiesch, J., Weiss, M., Kannenberg, G., Hannabuss, J., Surrey, T., Platzman, I., & Spatz, J. P. (2015) · Analytical Chemistry · doi:10.1021/ac504736e · Cited by 39
Reduced Representation Methods for Subgenomic Enrichment and Next-Generation Sequencing
Good, J. M. (2011) · Methods in molecular biology · doi:10.1007/978-1-61779-228-1_5 · Cited by 39
CRISPR-Cap: multiplexed double-stranded DNA enrichment based on the CRISPR system
Lee, J., Lim, H., Jang, H. S., Hwang, B., Lee, J. H., Cho, J., Lee, J. H., & Bang, D. (2018) · Nucleic Acids Research · doi:10.1093/nar/gky820 · Cited by 39
“Based on the sequencing and qPCR results, we found that the average sequencing depth of the bla gene relative to the average sequencing depth of the other target genes increased as the bla copy number increased.”
BackgroundMethodInfluentialUsing high-throughput barcode sequencing to efficiently map connectomes
Peikon, I. D., Kebschull, J. M., Vagin, V. V., Ravens, D. I., Sun, Y.-C., Brouzes, E. R., Junior, I. R. C., Bressan, D., & Zador, A. M. (2017) · Nucleic Acids Research · doi:10.1093/nar/gkx292 · Cited by 39
“Stability through thermocycling was further increased by adding Tetronic 1307 (BASF) at 1% weight to the RT-PCR mixture (28).”
Thermal microdevices for biological and biomedical applications
Jain, A., & Goodson, K. E. (2011) · Journal of Thermal Biology · doi:10.1016/j.jtherbio.2011.02.006 · Cited by 40
“Application of droplet microfluidics based PCR for large-scale targeted sequencing has been reported (Tewhey et al., 2009).”
Matvienko, M., Kozik, A., Froenicke, L., Lavelle, D. O., Martineau, B., Perroud, B., & Michelmore, R. W. (2013) · PLoS ONE · doi:10.1371/journal.pone.0055913 · Cited by 37
“Therefore, it may be possible to make qualitative inferences about differences in gene expression that could be validated by gene-specific approaches such as qPCR.”
BackgroundInfluentialRecent advances in microfluidic technologies for biochemistry and molecular biology
Cho, S., Kang, D.-K., Choo, J. B., deMello, A. J., & Chang, S. (2011) · BMB Reports · doi:10.5483/bmbrep.2011.44.11.705 · Cited by 37
“5 million amplifications in parallel (19).”
Li, X., Zhang, D., Zhang, H., Guan, Z., Song, Y., Liu, R., Zhu, Z., & Yang, C. J. (2018) · Analytical Chemistry · doi:10.1021/acs.analchem.7b04040 · Cited by 36
Programmable Functionalization of Surfactant‐Stabilized Microfluidic Droplets via DNA‐Tags
Jahnke, K., Weiss, M., Frey, C., Antona, S., Janiesch, J., Platzman, I., Göpfrich, K., & Spatz, J. P. (2019) · Advanced Functional Materials · doi:10.1002/adfm.201808647 · Cited by 37
Temperature-Tolerant COLD-PCR Reduces Temperature Stringency and Enables Robust Mutation Enrichment
Castellanos-Rizaldos, E., Liu, P., Milbury, C. A., Guha, M., Brisci, A., Cremonesi, L., Ferrari, M., Mamon, H. J., & Makrigiorgos, G. M. (2012) · Clinical Chemistry · doi:10.1373/clinchem.2012.183095 · Cited by 37
Genetics of hearing loss in africans: use of next generation sequencing is the best way forward
Lebeko, K., Bosch, J., Nziale, J. J. N., Dandara, C., & Wonkam, A. (2015) · Pan African Medical Journal · doi:10.11604/pamj.2015.20.383.5230 · Cited by 36
“Eleven of these mutations were novel MYO7A mutations [66].”
BackgroundHigh-Resolution Phenotypic Landscape of the RNA Polymerase II Trigger Loop
Qiu, C., Erinne, O. C., Dave, J. M., Cui, P., Jin, H., Muthukrishnan, N., Tang, L. K., Babu, S. G., Lam, K. C., Vandeventer, P. J., et al. (2016) · PLoS Genetics · doi:10.1371/journal.pgen.1006321 · Cited by 36
Wooderchak‐Donahue, W. L., O’Fallon, B. D., Furtado, L. V., Durtschi, J., Plant, P., Ridge, P. G., Rope, A. F., Yetman, A. T., & Bayrak‐Toydemir, P. (2012) · BMC Medical Genomics · doi:10.1186/1755-8794-5-50 · Cited by 35
“In this work we compare two competing enrichment protocols, an emulsion-PCR based technique from RainDance technologies [18] and a method utilizing solution-based RNA-bait hybrid capture marketed as the SureSelect Target Enrichment Platform [19].”
MethodNext-generation sequencing entering the clinical arena
Haas, J., Katus, H. A., & Meder, B. (2011) · Molecular and Cellular Probes · doi:10.1016/j.mcp.2011.08.005 · Cited by 34
How do novel molecular genetic markers influence treatment decisions in acute myeloid leukemia?
Patel, J. P., & Levine, R. L. (2012) · Hematology · doi:10.1182/asheducation.v2012.1.28.3797923 · Cited by 37
Optical manipulation and control of real-time PCR in cell encapsulating microdroplets by IR laser
Hettiarachchi, K., Kim, H., & Faris, G. W. (2012) · Microfluidics and Nanofluidics · doi:10.1007/s10404-012-1016-5 · Cited by 35
“…for hundreds or thousands of emulsion PCR assays to be run concurrently (Nakano et al. 2003; Dorfman et al. 2005; Beer et al. 2007; Ohashi et al. 2007; Kumaresan et al. 2008; Mazutis et al. 2009; Schaerli et al. 2009; Tewhey et al. 2009; Wang and Burns 2009; Zhang et al. 2009; Hatch et al. 2011).”
BackgroundDealing with Pseudogenes in Molecular Diagnostics in the Next-Generation Sequencing Era
Claes, K., & Leeneer, K. D. (2014) · Methods in molecular biology · doi:10.1007/978-1-4939-0835-6_21 · Cited by 35
Comparison of the jet breakup and droplet formation between non-Newtonian and Newtonian fluids
Mousavi, S., Siavashi, M., & Bagheri, M. (2023) · Journal of Non-Newtonian Fluid Mechanics · doi:10.1016/j.jnnfm.2023.105093 · Cited by 34
A three-dimensional electrode for highly efficient electrocoalescence-based droplet merging
Guzman, A. R., Kim, H. S., de Figueiredo, P., & Han, A. (2015) · Biomedical Microdevices · doi:10.1007/s10544-014-9921-x · Cited by 34
Liquid-capped encoded microcapsules for multiplex assays
Song, Y., Jeong, Y., Kwon, T., Lee, D., Oh, D. Y., Park, T.-J., Kim, J., Kim, J., & Kwon, S. (2016) · Lab on a Chip · doi:10.1039/c6lc01268j · Cited by 34
Emerman, A. B., Bowman, S., Barry, A. J., Henig, N., Patel, K. M., Gardner, A. F., & Hendrickson, C. L. (2017) · Current Protocols in Molecular Biology · doi:10.1002/cpmb.39 · Cited by 34
“Partitioning individual amplification reactions into emulsion droplets can alleviate some of these constraints and improve target uniformity (Tewhey et al., 2009), but this approach requires investment in a digital droplet PCR machine.”
MethodSummerer, D., Schracke, N., Wu, H., Cheng, Y., Bau, S., Stähler, C., Stähler, P. F., & Beier, M. (2010) · Genomics · doi:10.1016/j.ygeno.2010.01.006 · Cited by 34
Novel droplet platforms for the detection of disease biomarkers
Zec, H. C., Shin, D. J., & Wang, T. (2014) · Expert Review of Molecular Diagnostics · doi:10.1586/14737159.2014.945437 · Cited by 33
“5 million amplifications in parallel, was described [63].”
BackgroundMethodOn-chip background noise reduction for cell-based assays in droplets
Mary, P., Chen, A., Chen, I., Abate, A. R., & Weitz, D. A. (2011) · Lab on a Chip · doi:10.1039/c1lc20159j · Cited by 33
Leveraging the power of long reads for targeted sequencingPreprint
Iyer, S. V., Goodwin, S., & McCombie, W. R. (2024) · Genome Research · doi:10.1101/gr.279168.124 · Cited by 36
Detection and Diagnostic Utilization of Cellular and Cell-Free Tumor DNA
Dudley, J. C., & Diehn, M. (2020) · Annual Review of Pathology Mechanisms of Disease · doi:10.1146/annurev-pathmechdis-012419-032604 · Cited by 31
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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