Massively Parallel Haplotyping on Microscopic Beads for the High-Throughput Phase Analysis of Single Molecules
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{"title"=>"Massively parallel haplotyping on microscopic beads for the high-throughput phase analysis of single molecules", "type"=>"journal", "authors"=>[{"first_name"=>"Jérôme", "last_name"=>"Boulanger", "scopus_author_id"=>"13907902100"}, {"first_name"=>"Leila", "last_name"=>"Muresan", "scopus_author_id"=>"12804453100"}, {"first_name"=>"Irene", "last_name"=>"Tiemann-Boege", "scopus_author_id"=>"6506615248"}], "year"=>2012, "source"=>"PLoS ONE", "identifiers"=>{"scopus"=>"2-s2.0-84860459640", "doi"=>"10.1371/journal.pone.0036064", "pui"=>"364721010", "issn"=>"19326203", "pmid"=>"22558329", "isbn"=>"1932-6203 (Electronic) 1932-6203 (Linking)", "sgr"=>"84860459640"}, "id"=>"c1b89309-638b-34e6-978a-ca644f6cf1a1", "abstract"=>"In spite of the many advances in haplotyping methods, it is still very difficult to characterize rare haplotypes in tissues and different environmental samples or to accurately assess the haplotype diversity in large mixtures. This would require a haplotyping method capable of analyzing the phase of single molecules with an unprecedented throughput. Here we describe such a haplotyping method capable of analyzing in parallel hundreds of thousands single molecules in one experiment. In this method, multiple PCR reactions amplify different polymorphic regions of a single DNA molecule on a magnetic bead compartmentalized in an emulsion drop. The allelic states of the amplified polymorphisms are identified with fluorescently labeled probes that are then decoded from images taken of the arrayed beads by a microscope. This method can evaluate the phase of up to 3 polymorphisms separated by up to 5 kilobases in hundreds of thousands single molecules. We tested the sensitivity of the method by measuring the number of mutant haplotypes synthesized by four different commercially available enzymes: Phusion, Platinum Taq, Titanium Taq, and Phire. The digital nature of the method makes it highly sensitive to detecting haplotype ratios of less than 1:10,000. We also accurately quantified chimera formation during the exponential phase of PCR by different DNA polymerases.", "link"=>"http://www.mendeley.com/research/massively-parallel-haplotyping-microscopic-beads-highthroughput-phase-analysis-single-molecules", "reader_count"=>18, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>2, "Researcher"=>4, "Student > Ph. D. Student"=>5, "Student > Master"=>5, "Student > Bachelor"=>1, "Professor"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>2, "Researcher"=>4, "Student > Ph. D. Student"=>5, "Student > Master"=>5, "Student > Bachelor"=>1, "Professor"=>1}, "reader_count_by_subject_area"=>{"Engineering"=>2, "Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>1, "Agricultural and Biological Sciences"=>13, "Immunology and Microbiology"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>2}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>13}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>1}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"Austria"=>1, "Belgium"=>1}, "group_count"=>1}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/644623"], "description"=>"<p>The intensity was normalized for the two fluorphores used per polymorphism. The high peak on the left represents empty beads without an amplification product.</p>", "links"=>[], "tags"=>["fluorescence", "experiments", "amplified"], "article_id"=>315117, "categories"=>["Biological Sciences", "Biochemistry", "Genetics", "Evolutionary Biology"], "users"=>["Jérôme Boulanger", "Leila Muresan", "Irene Tiemann-Boege"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036064.g002", "stats"=>{"downloads"=>1, "page_views"=>14, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Distribution_of_the_fluorescence_intensity_of_the_same_target_SNP_1_common_in_all_three_experiments_amplified_with_one_two_or_three_targets_/315117", "title"=>"Distribution of the fluorescence intensity of the same target (SNP 1) common in all three experiments amplified with one, two or three targets.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-04-30 01:25:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/644905"], "description"=>"1<p>Number of beads counted for a specific sequence string in a total of 500,000 beads. The “0” in the sequence string represents an empty position for which only background florescence was recorded. Beads positives for more than one allele (two alleles per SNP) derived from multi-template reactions were removed from the data.</p>2<p>Ratio of alleles obtained for heterozygous DNA.</p>3<p>Sum of beads informative for the queried SNPs relative to the total number of beads with a product. The sum of different types of drop-outs (“0” in sequence string) are shown for the multiplex reactions.</p>", "links"=>[], "tags"=>["haplotyping", "targeting", "snp"], "article_id"=>315400, "categories"=>["Biological Sciences", "Biochemistry", "Genetics", "Evolutionary Biology"], "users"=>["Jérôme Boulanger", "Leila Muresan", "Irene Tiemann-Boege"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036064.t001", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Bead_emulsion_haplotyping_BEH_targeting_1_2_or_3_SNP_regions_/315400", "title"=>"Bead-emulsion haplotyping (BEH) targeting 1, 2, or 3 SNP regions.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-04-30 01:30:00"}
  • {"files"=>["https://ndownloader.figshare.com/files/332358", "https://ndownloader.figshare.com/files/332514", "https://ndownloader.figshare.com/files/332546", "https://ndownloader.figshare.com/files/332591", "https://ndownloader.figshare.com/files/332617", "https://ndownloader.figshare.com/files/332655", "https://ndownloader.figshare.com/files/332690", "https://ndownloader.figshare.com/files/332745", "https://ndownloader.figshare.com/files/332807"], "description"=>"<div><p>In spite of the many advances in haplotyping methods, it is still very difficult to characterize rare haplotypes in tissues and different environmental samples or to accurately assess the haplotype diversity in large mixtures. This would require a haplotyping method capable of analyzing the phase of single molecules with an unprecedented throughput. Here we describe such a haplotyping method capable of analyzing in parallel hundreds of thousands single molecules in one experiment. In this method, multiple PCR reactions amplify different polymorphic regions of a single DNA molecule on a magnetic bead compartmentalized in an emulsion drop. The allelic states of the amplified polymorphisms are identified with fluorescently labeled probes that are then decoded from images taken of the arrayed beads by a microscope. This method can evaluate the phase of up to 3 polymorphisms separated by up to 5 kilobases in hundreds of thousands single molecules. We tested the sensitivity of the method by measuring the number of mutant haplotypes synthesized by four different commercially available enzymes: Phusion, Platinum Taq, Titanium Taq, and Phire. The digital nature of the method makes it highly sensitive to detecting haplotype ratios of less than 1∶10,000. We also accurately quantified chimera formation during the exponential phase of PCR by different DNA polymerases.</p> </div>", "links"=>[], "tags"=>["massively", "parallel", "haplotyping", "microscopic", "beads", "high-throughput", "molecules"], "article_id"=>125635, "categories"=>["Biological Sciences", "Biochemistry", "Genetics", "Evolutionary Biology"], "users"=>["Jérôme Boulanger", "Leila Muresan", "Irene Tiemann-Boege"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0036064.s001", "https://dx.doi.org/10.1371/journal.pone.0036064.s002", "https://dx.doi.org/10.1371/journal.pone.0036064.s003", "https://dx.doi.org/10.1371/journal.pone.0036064.s004", "https://dx.doi.org/10.1371/journal.pone.0036064.s005", "https://dx.doi.org/10.1371/journal.pone.0036064.s006", "https://dx.doi.org/10.1371/journal.pone.0036064.s007", "https://dx.doi.org/10.1371/journal.pone.0036064.s008", "https://dx.doi.org/10.1371/journal.pone.0036064.s009"], "stats"=>{"downloads"=>7, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Massively_Parallel_Haplotyping_on_Microscopic_Beads_for_the_High_Throughput_Phase_Analysis_of_Single_Molecules/125635", "title"=>"Massively Parallel Haplotyping on Microscopic Beads for the High-Throughput Phase Analysis of Single Molecules", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2012-04-30 01:33:55"}
  • {"files"=>["https://ndownloader.figshare.com/files/644818"], "description"=>"<p>A. Comparison of the fraction of false recombinants measured in 400–2200 bp templates amplified with Phusion polymerase using either 35 or 15 PCR cycles. The fractions are averages estimated from at least 5 independent experiments using different template lengths (error bars are the estimated standard deviation). B. Fraction of false recombinants measured in 422 base pair PCR products amplified for 15 cycles by different polymerases. The fractions are averages estimated from three independent experiments and error bars are the estimated standard deviations.</p>", "links"=>[], "tags"=>["chimeras", "formed"], "article_id"=>315311, "categories"=>["Biological Sciences", "Biochemistry", "Genetics", "Evolutionary Biology"], "users"=>["Jérôme Boulanger", "Leila Muresan", "Irene Tiemann-Boege"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036064.g004", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Assessment_of_chimeras_formed_during_PCR_/315311", "title"=>"Assessment of chimeras formed during PCR.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-04-30 01:28:31"}
  • {"files"=>["https://ndownloader.figshare.com/files/644702"], "description"=>"<p>The left panel is a schematic of the location of the amplified SNP region relative to the template. The right panel represents the sum of the fluorescence intensity obtained for both alleles for the possible haplotypes. The error bars are the standard deviation of the fluorescence intensity. Shown above each pair of haplotypes is the observed allelic ratio of the captured haplotypes. A. Assay of the same two SNPs (422 bp apart) on templates of different lengths. B. Assay of different SNPs pairs 400, 1200, and 2200 base pairs apart on the same 2733 bp template.</p>", "links"=>[], "tags"=>["template", "lengths", "snp"], "article_id"=>315197, "categories"=>["Biological Sciences", "Biochemistry", "Genetics", "Evolutionary Biology"], "users"=>["Jérôme Boulanger", "Leila Muresan", "Irene Tiemann-Boege"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036064.g003", "stats"=>{"downloads"=>0, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_BEH_on_different_template_lengths_and_SNP_positions_/315197", "title"=>"BEH on different template lengths and SNP positions.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-04-30 01:26:37"}
  • {"files"=>["https://ndownloader.figshare.com/files/644540"], "description"=>"<p>In step 1, a region of several kilobases containing several polymorphic sites is amplified from genomic DNA heterozygous for the tested SNPs. In step 2, single amplicons are hybridized to microscopic beads covered with a primer that has a universal tail common to the 5′ end of the multiplexing primers (shown in red). PCR products from 3 small regions containing the polymorphisms are produced within the aqueous compartment of an emulsion droplet and bound to the bead. In step 3, the beads are washed and labeled by allele-specific extensions of fluorescent probes specific for one of the polymorphic sites. In step 4, unextended probes are washed off and the fluorescent beads are arrayed on a slide. In step 5, the array is scanned with a microscope followed by subsequent washing, probing and imaging cycles to screen additional polymorphisms (up to 4 different alleles; 2SNPs can be analyzed simultaneously). In step 6, a series of imaging and data analysis steps are performed to assess the initial haplotype of ∼10<sup>5</sup> molecules.</p>", "links"=>[], "tags"=>["bead-emulsion", "haplotyping"], "article_id"=>315031, "categories"=>["Biological Sciences", "Biochemistry", "Genetics", "Evolutionary Biology"], "users"=>["Jérôme Boulanger", "Leila Muresan", "Irene Tiemann-Boege"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036064.g001", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Schematics_of_bead_emulsion_haplotyping_BEH_/315031", "title"=>"Schematics of bead-emulsion haplotyping (BEH).", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-04-30 01:23:51"}

PMC Usage Stats | Further Information

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Relative Metric

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