Digital Droplet Multiple Displacement Amplification (ddMDA) for Whole Genome Sequencing of Limited DNA Samples
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{"title"=>"Digital Droplet Multiple Displacement Amplification (ddMDA) for Whole Genome Sequencing of Limited DNA Samples", "type"=>"journal", "authors"=>[{"first_name"=>"Minsoung", "last_name"=>"Rhee", "scopus_author_id"=>"15063577500"}, {"first_name"=>"Yooli K.", "last_name"=>"Light", "scopus_author_id"=>"10044759400"}, {"first_name"=>"Robert J.", "last_name"=>"Meagher", "scopus_author_id"=>"7101710710"}, {"first_name"=>"Anup K.", "last_name"=>"Singh", "scopus_author_id"=>"14023532400"}], "year"=>2016, "source"=>"PloS one", "identifiers"=>{"issn"=>"19326203", "scopus"=>"2-s2.0-85016146681", "pui"=>"617358526", "doi"=>"10.1371/journal.pone.0153699", "isbn"=>"10.1371/journal.pone.0153699", "sgr"=>"85016146681", "pmid"=>"27144304"}, "id"=>"71742031-9c31-3d15-af8d-a6d0114e1475", "abstract"=>"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.", "link"=>"http://www.mendeley.com/research/digital-droplet-multiple-displacement-amplification-ddmda-whole-genome-sequencing-limited-dna-sample", "reader_count"=>42, "reader_count_by_academic_status"=>{"Unspecified"=>2, "Professor > Associate Professor"=>4, "Researcher"=>10, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>16, "Student > Postgraduate"=>1, "Student > Master"=>4, "Other"=>1, "Student > Bachelor"=>2, "Professor"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>2, "Professor > Associate Professor"=>4, "Researcher"=>10, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>16, "Student > Postgraduate"=>1, "Student > Master"=>4, "Other"=>1, "Student > Bachelor"=>2, "Professor"=>1}, "reader_count_by_subject_area"=>{"Engineering"=>3, "Unspecified"=>2, "Environmental Science"=>1, "Biochemistry, Genetics and Molecular Biology"=>7, "Agricultural and Biological Sciences"=>20, "Medicine and Dentistry"=>1, "Chemistry"=>4, "Immunology and Microbiology"=>1, "Earth and Planetary Sciences"=>1, "Computer Science"=>2}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>3}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Chemistry"=>{"Chemistry"=>4}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>1}, "Earth and Planetary Sciences"=>{"Earth and Planetary Sciences"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>20}, "Computer Science"=>{"Computer Science"=>2}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>7}, "Unspecified"=>{"Unspecified"=>2}, "Environmental Science"=>{"Environmental Science"=>1}}, "reader_count_by_country"=>{"Norway"=>1, "Switzerland"=>1}, "group_count"=>4}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/5083201"], "description"=>"<p>(A) The ddMDA procedures as a high quality alternative to the conventional tube MDA. The MDA ready <i>E</i>. <i>coli</i> samples were partitioned into millions of picoliter droplets using a microfluidic droplet generator. Upon collection, droplets were tightly sealed for isothermal incubation at 30°C for 18 hours. DNA amplicons were then purified, cleaned, and prepared for the following sequencing. (B) Denatured and fragmented whole genomes consist of highly amplifiable (yellow) and weakly amplifiable (red) sequences. During tube MDA, yellow fragments are preferred and repeatedly amplified with a high gain until it reaches a concentration plateau, whereas red fragments are less preferred and barely amplify. For ddMDA, DNA fragments are randomly partitioned into picoliter droplets, resulting in different subsets of the template DNA. When a droplet contains yellow fragments, the amplification kinetics favor the yellow fragments, ending up with significant biases on amplification. The enzyme will amplify red fragments at a slower rate only in the absence of yellow fragments. The overall gain of ddMDA is always lower than tube MDA because of the volume constraint. Every droplet is uniquely composed of fragments and ends up with a different amplification gain after MDA. (C) A fluorescence micrograph showing ddMDA endpoint with the initial template DNA concentration of 100 pg/μL. Having started with different parts of the <i>E</i>. <i>coli</i> genome, individual droplets expressed discrete levels of amplification by showing different sizes of DNA amplicon aggregates and different fluorescent signals. The scale bar shows 100μm.</p>", "links"=>[], "tags"=>["coli", "sample", "Whole Genome Sequencing", "MDA", "DNA fragments", "ddMDA", "Digital Droplet Multiple Displacement Amplification", "template DNA", "genome", "amplification bias", "Limited DNA Samples Multiple displacement amplification"], "article_id"=>3248608, "categories"=>["Biophysics", "Biochemistry", "Medicine", "Microbiology", "Cell Biology", "Genetics", "Molecular Biology", "Biological Sciences not elsewhere classified", "Mathematical Sciences not elsewhere classified", "Cancer", "Infectious Diseases"], "users"=>["Minsoung Rhee", "Yooli K. Light", "Robert J. Meagher", "Anup K. Singh"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0153699.g001", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/The_working_principles_of_ddMDA_/3248608", "title"=>"The working principles of ddMDA.", "pos_in_sequence"=>2, "defined_type"=>1, "published_date"=>"2016-05-04 09:37:03"}
  • {"files"=>["https://ndownloader.figshare.com/files/5083189"], "description"=>"<div><p>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 <i>E</i>. <i>coli</i> 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.</p></div>", "links"=>[], "tags"=>["coli", "sample", "Whole Genome Sequencing", "MDA", "DNA fragments", "ddMDA", "Digital Droplet Multiple Displacement Amplification", "template DNA", "genome", "amplification bias", "Limited DNA Samples Multiple displacement amplification"], "article_id"=>3248593, "categories"=>["Biophysics", "Biochemistry", "Medicine", "Microbiology", "Cell Biology", "Genetics", "Molecular Biology", "Biological Sciences not elsewhere classified", "Mathematical Sciences not elsewhere classified", "Cancer", "Infectious Diseases"], "users"=>["Minsoung Rhee", "Yooli K. Light", "Robert J. Meagher", "Anup K. Singh"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0153699", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Digital_Droplet_Multiple_Displacement_Amplification_ddMDA_for_Whole_Genome_Sequencing_of_Limited_DNA_Samples/3248593", "title"=>"Digital Droplet Multiple Displacement Amplification (ddMDA) for Whole Genome Sequencing of Limited DNA Samples", "pos_in_sequence"=>1, "defined_type"=>6, "published_date"=>"2016-05-04 09:37:03"}
  • {"files"=>["https://ndownloader.figshare.com/files/5083216"], "description"=>"<p>(A) From the outermost circle, ddMDA for 100 pg/μL (dark purple), tube MDA for 100 pg/μL (bright purple), 10 pg/μL (dark brown), and 10 pg/μL (bright brown), respectively. GC contents across the genome were depicted in black in the innermost circle and reads from genomic DNA were illustrated in green as a reference. (B) From the outermost circle, ddMDA for 1 pg/μL (dark red), tube MDA for 1 pg/μL (bright red), 0.1 pg/μL (dark blue), and 0.1 pg/μL (bright blue), respectively. (C) GC contents (top) and amplification read depths over the entire E. coli genome for ddMDA (middle) and tube MDA (bottom) at the DNA concentration of 10 pg/μL. The right panels show zoomed-in plots of the dotted-line box region of the genome for close-up visualization.</p>", "links"=>[], "tags"=>["coli", "sample", "Whole Genome Sequencing", "MDA", "DNA fragments", "ddMDA", "Digital Droplet Multiple Displacement Amplification", "template DNA", "genome", "amplification bias", "Limited DNA Samples Multiple displacement amplification"], "article_id"=>3248620, "categories"=>["Biophysics", "Biochemistry", "Medicine", "Microbiology", "Cell Biology", "Genetics", "Molecular Biology", "Biological Sciences not elsewhere classified", "Mathematical Sciences not elsewhere classified", "Cancer", "Infectious Diseases"], "users"=>["Minsoung Rhee", "Yooli K. Light", "Robert J. Meagher", "Anup K. Singh"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0153699.g002", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Comparison_of_whole_genome_coverage_of_assembled_contigs_mapped_onto_i_E_i_i_coli_i_K12_genome_sequences_for_ddMDA_and_tube_MDA_/3248620", "title"=>"Comparison of whole genome coverage of assembled contigs mapped onto <i>E</i>. <i>coli</i> K12 genome sequences for ddMDA and tube MDA.", "pos_in_sequence"=>3, "defined_type"=>1, "published_date"=>"2016-05-04 09:37:03"}
  • {"files"=>["https://ndownloader.figshare.com/files/5083234"], "description"=>"<p>(A) The fraction of reads correctly mapped to <i>E</i>. <i>coli</i> genome depending on the DNA concentration and the MDA reaction volume. Blue and red columns show ddMDA and tube MDA results, respectively. The green line shows the fold change of the % mapping from tube MDA to ddMDA (= ddMDA/tubeMDA). It stayed around 1 for high concentrations (10–100 pg/μL) while it considerably increased for low concentrations (0.1–1 pg/μL). (B) The fraction of an <i>E</i>. <i>coli</i> genome covered by one or more sequencing reads depending on the DNA concentration and the MDA reaction volume. Tube MDA and ddMDA showed little difference at high concentrations but the fold change significantly increased at low concentrations. (C) The fraction of an <i>E</i>. <i>coli</i> genome covered by contigs during de novo assembly. While the advantage of ddMDA over tube MDA was still limited at high concentrations but the fold change significantly increased at low concentrations. (D) Lorenz curves depict the amplification bias in read coverage across the <i>E</i>. <i>coli</i> genome. Each curve was calculated by evaluating the read depth for each base and using the resultant cumulative distribution function for read depth to determine the cumulative proportion of total genome coverage (y-axis) accounted for by the cumulative proportion of bases (x-axis). The ideal Lorentz curve (black dotted line) for a distribution in which all of the bases have the same coverage and a Lorenz curve for gDNA were plotted for comparison. Other solid curves show ddMDA curves while dotted curves indicate tube MDA. (Upper Left) For 100 pg/μL, ddMDA in dark purple and tube MDA in bright purple. (Bottom Left) For 10 pg/μL, ddMDA in dark brown and tube MDA in bright brown. (Upper Right) For 1 pg/μL, ddMDA in dark red and tube MDA in bright red. (Bottom Right) For 0.1 pg/μL, ddMDA in dark blue and tube MDA in bright blue.</p>", "links"=>[], "tags"=>["coli", "sample", "Whole Genome Sequencing", "MDA", "DNA fragments", "ddMDA", "Digital Droplet Multiple Displacement Amplification", "template DNA", "genome", "amplification bias", "Limited DNA Samples Multiple displacement amplification"], "article_id"=>3248638, "categories"=>["Biophysics", "Biochemistry", "Medicine", "Microbiology", "Cell Biology", "Genetics", "Molecular Biology", "Biological Sciences not elsewhere classified", "Mathematical Sciences not elsewhere classified", "Cancer", "Infectious Diseases"], "users"=>["Minsoung Rhee", "Yooli K. Light", "Robert J. Meagher", "Anup K. Singh"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0153699.g003", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Improvement_of_the_overall_quality_of_amplification_by_ddMDA_/3248638", "title"=>"Improvement of the overall quality of amplification by ddMDA.", "pos_in_sequence"=>4, "defined_type"=>1, "published_date"=>"2016-05-04 09:37:03"}
  • {"files"=>["https://ndownloader.figshare.com/files/5083240"], "description"=>"<p>Total number of reads included all sequencing reads mapped and unmapped to <i>E</i>. <i>coli</i> genome. % Reads mapped to genome corresponds to the percentage of sequencing reads that are specifically aligned to <i>E</i>. <i>coli</i> genome. % Genome covered by reads refers to the percentage of <i>E</i>. <i>coli</i> genome covered by one or more sequencing reads. % Genome covered by assembly indicated the percentage of <i>E</i>. <i>coli</i> genome covered by contigs. Gini indices were calculated based on the cumulative distribution of sequencing reads.</p>", "links"=>[], "tags"=>["coli", "sample", "Whole Genome Sequencing", "MDA", "DNA fragments", "ddMDA", "Digital Droplet Multiple Displacement Amplification", "template DNA", "genome", "amplification bias", "Limited DNA Samples Multiple displacement amplification"], "article_id"=>3248647, "categories"=>["Biophysics", "Biochemistry", "Medicine", "Microbiology", "Cell Biology", "Genetics", "Molecular Biology", "Biological Sciences not elsewhere classified", "Mathematical Sciences not elsewhere classified", "Cancer", "Infectious Diseases"], "users"=>["Minsoung Rhee", "Yooli K. Light", "Robert J. Meagher", "Anup K. Singh"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0153699.t001", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Statistics_of_sequence_mapping_and_assembly_of_i_E_i_i_coli_i_K12_MG1655_samples_prepared_with_ddMDA_and_conventional_tube_MDA_at_different_initial_copy_numbers_/3248647", "title"=>"Statistics of sequence mapping and assembly of <i>E</i>. <i>coli</i> K12 MG1655 samples prepared with ddMDA and conventional tube MDA at different initial copy numbers.", "pos_in_sequence"=>5, "defined_type"=>3, "published_date"=>"2016-05-04 09:37:03"}

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Relative Metric

{"start_date"=>"2016-01-01T00:00:00Z", "end_date"=>"2016-12-31T00:00:00Z", "subject_areas"=>[]}
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