Digital Droplet Multiple Displacement Amplification (ddMDA) for Whole Genome Sequencing of Limited DNA Samples
| Autor: | Anup K. Singh, Robert J. Meagher, Yooli Kim Light, Minsoung Rhee |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
0301 basic medicine
Microfluidics Sequence assembly lcsh:Medicine 02 engineering and technology Biochemistry Sequencing by hybridization Genomic library Genome Sequencing DNA sequencing DNA libraries lcsh:Science Multidisciplinary Bacterial Genomics MALBAC Microbial Genetics Genomics 021001 nanoscience & nanotechnology 3. Good health Nucleic acids Engineering and Technology Fluidics 0210 nano-technology Nucleic Acid Amplification Techniques Research Article DNA Bacterial DNA nanoball sequencing Microbial Genomics Mycology Biology Research and Analysis Methods Microbiology 03 medical and health sciences Escherichia coli Genetics Bacterial Genetics Fungal Genetics Molecular Biology Techniques Sequencing Techniques Molecular Biology Fungal Genomics lcsh:R Multiple displacement amplification Biology and Life Sciences Computational Biology Bacteriology Nucleic acid amplification technique Sequence Analysis DNA DNA Comparative Genomics Genome Analysis Genomic Libraries Molecular biology DNA sequencer 030104 developmental biology lcsh:Q Genome Bacterial |
| Zdroj: | PLoS ONE PLoS ONE, Vol 11, Iss 5, p e0153699 (2016) |
| ISSN: | 1932-6203 |
| Popis: | Multiple displacement amplification (MDA) is a widely used technique for amplification of DNA from samples containing limited amounts of DNA (e.g., uncultivable microbes or clinical samples) before whole genome sequencing. Despite its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. Here, we present a microfluidic digital droplet MDA (ddMDA) technique where partitioning of the template DNA into thousands of sub-nanoliter droplets, each containing a small number of DNA fragments, greatly reduces the competition among DNA fragments for primers and polymerase thereby greatly reducing amplification bias. Consequently, the ddMDA approach enabled a more uniform coverage of amplification over the entire length of the genome, with significantly lower bias and non-specific amplification than conventional MDA. For a sample containing 0.1 pg/μL of E. coli DNA (equivalent of ~3/1000 of an E. coli genome per droplet), ddMDA achieves a 65-fold increase in coverage in de novo assembly, and more than 20-fold increase in specificity (percentage of reads mapping to E. coli) compared to the conventional tube MDA. ddMDA offers a powerful method useful for many applications including medical diagnostics, forensics, and environmental microbiology. |
| Databáze: | OpenAIRE |
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