Targeted RNA-Sequencing with Competitive Multiplex-PCR Amplicon Libraries
Publication Date
November 13, 2013
Journal
PLOS ONE
Authors
Thomas M. Blomquist, Erin L. Crawford, Jennie L. Lovett, Jiyoun Yeo, et al
Volume
8
Issue
11
Pages
e79120
DOI
https://dx.plos.org/10.1371/journal.pone.0079120
Publisher URL
http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0079120
PubMed
http://www.ncbi.nlm.nih.gov/pubmed/24236095
PubMed Central
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3827295
Europe PMC
http://europepmc.org/abstract/MED/24236095
Web of Science
000327254700093
Scopus
84893156322
Mendeley
http://www.mendeley.com/research/targeted-rnasequencing-competitive-multiplex-pcr-amplicon-libraries
Events
Loading … Spinner

Mendeley | Further Information

{"title"=>"Targeted RNA-Sequencing with Competitive Multiplex- PCR Amplicon Libraries", "type"=>"journal", "authors"=>[{"first_name"=>"Thomas M.", "last_name"=>"Blomquist", "scopus_author_id"=>"57201070399"}, {"first_name"=>"Erin L.", "last_name"=>"Crawford", "scopus_author_id"=>"7201680003"}, {"first_name"=>"Jennie L.", "last_name"=>"Lovett", "scopus_author_id"=>"55891283400"}, {"first_name"=>"Jiyoun", "last_name"=>"Yeo", "scopus_author_id"=>"55830121900"}, {"first_name"=>"Lauren M.", "last_name"=>"Stanoszek", "scopus_author_id"=>"55628291000"}, {"first_name"=>"Albert", "last_name"=>"Levin", "scopus_author_id"=>"7403194355"}, {"first_name"=>"Jia", "last_name"=>"Li", "scopus_author_id"=>"55782286900"}, {"first_name"=>"Mei", "last_name"=>"Lu", "scopus_author_id"=>"26642898800"}, {"first_name"=>"Leming", "last_name"=>"Shi", "scopus_author_id"=>"7401640714"}, {"first_name"=>"Kenneth", "last_name"=>"Muldrew", "scopus_author_id"=>"9238461300"}, {"first_name"=>"James C.", "last_name"=>"Willey", "scopus_author_id"=>"7006815899"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"sgr"=>"84893156322", "doi"=>"10.1371/journal.pone.0079120", "pui"=>"372232447", "pmid"=>"24236095", "scopus"=>"2-s2.0-84893156322", "issn"=>"19326203", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)"}, "id"=>"bd37e44b-eecb-38fd-9d4e-3eba1ce7ea96", "abstract"=>"Whole transcriptome RNA-sequencing is a powerful tool, but is costly and yields complex data sets that limit its utility in molecular diagnostic testing. A targeted quantitative RNA-sequencing method that is reproducible and reduces the number of sequencing reads required to measure transcripts over the full range of expression would be better suited to diagnostic testing. Toward this goal, we developed a competitive multiplex PCR-based amplicon sequencing library preparation method that a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. To determine the utility of this method, we intentionally selected PCR conditions that would cause transcript amplification products (amplicons) to converge toward equimolar concentrations (normalization) during library preparation. We then tested whether this approach would enable accurate and reproducible quantification of each transcript across multiple library preparations, and at the same time reduce (through normalization) total sequencing reads required for quantification of transcript targets across a large range of expression. We demonstrate excellent reproducibility (R² = 0.997) with 97% accuracy to detect 2-fold change using External RNA Controls Consortium (ERCC) reference materials; high inter-day, inter-site and inter-library concordance (R² = 0.97-0.99) using FDA Sequencing Quality Control (SEQC) reference materials; and cross-platform concordance with both TaqMan qPCR (R²  = 0.96) and whole transcriptome RNA-sequencing following \"traditional\" library preparation using Illumina NGS kits (R² = 0.94). Using this method, sequencing reads required to accurately quantify more than 100 targeted transcripts expressed over a 10⁷-fold range was reduced more than 10,000-fold, from 2.3×10⁹ to 1.4×10⁵ sequencing reads. These studies demonstrate that the competitive multiplex-PCR amplicon library preparation method presented here provides the quality control, reproducibility, and reduced sequencing reads necessary for development and implementation of targeted quantitative RNA-sequencing biomarkers in molecular diagnostic testing.", "link"=>"http://www.mendeley.com/research/targeted-rnasequencing-competitive-multiplex-pcr-amplicon-libraries", "reader_count"=>69, "reader_count_by_academic_status"=>{"Unspecified"=>2, "Professor > Associate Professor"=>3, "Researcher"=>27, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>17, "Student > Postgraduate"=>1, "Other"=>7, "Student > Master"=>5, "Lecturer"=>1, "Professor"=>4}, "reader_count_by_user_role"=>{"Unspecified"=>2, "Professor > Associate Professor"=>3, "Researcher"=>27, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>17, "Student > Postgraduate"=>1, "Other"=>7, "Student > Master"=>5, "Lecturer"=>1, "Professor"=>4}, "reader_count_by_subject_area"=>{"Unspecified"=>3, "Environmental Science"=>1, "Biochemistry, Genetics and Molecular Biology"=>14, "Mathematics"=>1, "Agricultural and Biological Sciences"=>43, "Medicine and Dentistry"=>4, "Arts and Humanities"=>1, "Chemistry"=>1, "Immunology and Microbiology"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>4}, "Chemistry"=>{"Chemistry"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>43}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>14}, "Mathematics"=>{"Mathematics"=>1}, "Unspecified"=>{"Unspecified"=>3}, "Environmental Science"=>{"Environmental Science"=>1}, "Arts and Humanities"=>{"Arts and Humanities"=>1}}, "reader_count_by_country"=>{"Sweden"=>1, "Hong Kong"=>1, "United States"=>3, "Japan"=>1, "Denmark"=>1, "South Africa"=>2, "United Kingdom"=>1, "Germany"=>1}, "group_count"=>4}

Scopus | Further Information

{"@_fa"=>"true", "link"=>[{"@_fa"=>"true", "@ref"=>"self", "@href"=>"https://api.elsevier.com/content/abstract/scopus_id/84893156322"}, {"@_fa"=>"true", "@ref"=>"author-affiliation", "@href"=>"https://api.elsevier.com/content/abstract/scopus_id/84893156322?field=author,affiliation"}, {"@_fa"=>"true", "@ref"=>"scopus", "@href"=>"https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84893156322&origin=inward"}, {"@_fa"=>"true", "@ref"=>"scopus-citedby", "@href"=>"https://www.scopus.com/inward/citedby.uri?partnerID=HzOxMe3b&scp=84893156322&origin=inward"}], "prism:url"=>"https://api.elsevier.com/content/abstract/scopus_id/84893156322", "dc:identifier"=>"SCOPUS_ID:84893156322", "eid"=>"2-s2.0-84893156322", "dc:title"=>"Targeted RNA-Sequencing with Competitive Multiplex- PCR Amplicon Libraries", "dc:creator"=>"Blomquist T.M.", "prism:publicationName"=>"PLoS ONE", "prism:eIssn"=>"19326203", "prism:volume"=>"8", "prism:issueIdentifier"=>"11", "prism:pageRange"=>nil, "prism:coverDate"=>"2013-11-13", "prism:coverDisplayDate"=>"13 November 2013", "prism:doi"=>"10.1371/journal.pone.0079120", "citedby-count"=>"21", "affiliation"=>[{"@_fa"=>"true", "affilname"=>"The University of Toledo", "affiliation-city"=>"Toledo", "affiliation-country"=>"United States"}], "pubmed-id"=>"24236095", "prism:aggregationType"=>"Journal", "subtype"=>"ar", "subtypeDescription"=>"Article", "article-number"=>"e79120", "source-id"=>"10600153309", "openaccess"=>"1", "openaccessFlag"=>true}

Facebook

  • {"url"=>"http%3A%2F%2Fjournals.plos.org%2Fplosone%2Farticle%3Fid%3D10.1371%252Fjournal.pone.0079120", "share_count"=>0, "like_count"=>0, "comment_count"=>0, "click_count"=>0, "total_count"=>0}

Counter

  • {"month"=>"11", "year"=>"2013", "pdf_views"=>"142", "xml_views"=>"8", "html_views"=>"595"}
  • {"month"=>"12", "year"=>"2013", "pdf_views"=>"45", "xml_views"=>"1", "html_views"=>"246"}
  • {"month"=>"1", "year"=>"2014", "pdf_views"=>"52", "xml_views"=>"0", "html_views"=>"363"}
  • {"month"=>"2", "year"=>"2014", "pdf_views"=>"42", "xml_views"=>"1", "html_views"=>"252"}
  • {"month"=>"3", "year"=>"2014", "pdf_views"=>"37", "xml_views"=>"2", "html_views"=>"318"}
  • {"month"=>"4", "year"=>"2014", "pdf_views"=>"69", "xml_views"=>"1", "html_views"=>"348"}
  • {"month"=>"5", "year"=>"2014", "pdf_views"=>"36", "xml_views"=>"0", "html_views"=>"296"}
  • {"month"=>"6", "year"=>"2014", "pdf_views"=>"28", "xml_views"=>"0", "html_views"=>"253"}
  • {"month"=>"7", "year"=>"2014", "pdf_views"=>"33", "xml_views"=>"0", "html_views"=>"250"}
  • {"month"=>"8", "year"=>"2014", "pdf_views"=>"25", "xml_views"=>"2", "html_views"=>"228"}
  • {"month"=>"9", "year"=>"2014", "pdf_views"=>"25", "xml_views"=>"2", "html_views"=>"226"}
  • {"month"=>"10", "year"=>"2014", "pdf_views"=>"26", "xml_views"=>"1", "html_views"=>"256"}
  • {"month"=>"11", "year"=>"2014", "pdf_views"=>"26", "xml_views"=>"1", "html_views"=>"329"}
  • {"month"=>"12", "year"=>"2014", "pdf_views"=>"14", "xml_views"=>"1", "html_views"=>"223"}
  • {"month"=>"1", "year"=>"2015", "pdf_views"=>"18", "xml_views"=>"0", "html_views"=>"164"}
  • {"month"=>"2", "year"=>"2015", "pdf_views"=>"19", "xml_views"=>"0", "html_views"=>"163"}
  • {"month"=>"3", "year"=>"2015", "pdf_views"=>"22", "xml_views"=>"0", "html_views"=>"232"}
  • {"month"=>"4", "year"=>"2015", "pdf_views"=>"35", "xml_views"=>"1", "html_views"=>"177"}
  • {"month"=>"5", "year"=>"2015", "pdf_views"=>"20", "xml_views"=>"0", "html_views"=>"180"}
  • {"month"=>"6", "year"=>"2015", "pdf_views"=>"17", "xml_views"=>"0", "html_views"=>"153"}
  • {"month"=>"7", "year"=>"2015", "pdf_views"=>"10", "xml_views"=>"0", "html_views"=>"120"}
  • {"month"=>"8", "year"=>"2015", "pdf_views"=>"22", "xml_views"=>"0", "html_views"=>"138"}
  • {"month"=>"9", "year"=>"2015", "pdf_views"=>"13", "xml_views"=>"1", "html_views"=>"144"}
  • {"month"=>"10", "year"=>"2015", "pdf_views"=>"15", "xml_views"=>"1", "html_views"=>"169"}
  • {"month"=>"11", "year"=>"2015", "pdf_views"=>"10", "xml_views"=>"0", "html_views"=>"136"}
  • {"month"=>"12", "year"=>"2015", "pdf_views"=>"8", "xml_views"=>"0", "html_views"=>"100"}
  • {"month"=>"1", "year"=>"2016", "pdf_views"=>"26", "xml_views"=>"0", "html_views"=>"100"}
  • {"month"=>"2", "year"=>"2016", "pdf_views"=>"6", "xml_views"=>"0", "html_views"=>"102"}
  • {"month"=>"3", "year"=>"2016", "pdf_views"=>"24", "xml_views"=>"0", "html_views"=>"105"}
  • {"month"=>"4", "year"=>"2016", "pdf_views"=>"18", "xml_views"=>"0", "html_views"=>"114"}
  • {"month"=>"5", "year"=>"2016", "pdf_views"=>"10", "xml_views"=>"0", "html_views"=>"67"}
  • {"month"=>"6", "year"=>"2016", "pdf_views"=>"23", "xml_views"=>"0", "html_views"=>"100"}
  • {"month"=>"7", "year"=>"2016", "pdf_views"=>"18", "xml_views"=>"0", "html_views"=>"82"}
  • {"month"=>"8", "year"=>"2016", "pdf_views"=>"14", "xml_views"=>"0", "html_views"=>"76"}
  • {"month"=>"9", "year"=>"2016", "pdf_views"=>"16", "xml_views"=>"0", "html_views"=>"60"}
  • {"month"=>"10", "year"=>"2016", "pdf_views"=>"29", "xml_views"=>"0", "html_views"=>"104"}
  • {"month"=>"11", "year"=>"2016", "pdf_views"=>"8", "xml_views"=>"0", "html_views"=>"134"}
  • {"month"=>"12", "year"=>"2016", "pdf_views"=>"16", "xml_views"=>"0", "html_views"=>"118"}
  • {"month"=>"1", "year"=>"2017", "pdf_views"=>"14", "xml_views"=>"0", "html_views"=>"78"}
  • {"month"=>"2", "year"=>"2017", "pdf_views"=>"21", "xml_views"=>"0", "html_views"=>"82"}
  • {"month"=>"3", "year"=>"2017", "pdf_views"=>"16", "xml_views"=>"0", "html_views"=>"112"}
  • {"month"=>"4", "year"=>"2017", "pdf_views"=>"17", "xml_views"=>"0", "html_views"=>"108"}
  • {"month"=>"5", "year"=>"2017", "pdf_views"=>"16", "xml_views"=>"1", "html_views"=>"78"}
  • {"month"=>"6", "year"=>"2017", "pdf_views"=>"8", "xml_views"=>"0", "html_views"=>"86"}
  • {"month"=>"7", "year"=>"2017", "pdf_views"=>"12", "xml_views"=>"3", "html_views"=>"110"}
  • {"month"=>"8", "year"=>"2017", "pdf_views"=>"15", "xml_views"=>"2", "html_views"=>"98"}
  • {"month"=>"9", "year"=>"2017", "pdf_views"=>"19", "xml_views"=>"1", "html_views"=>"67"}
  • {"month"=>"10", "year"=>"2017", "pdf_views"=>"27", "xml_views"=>"1", "html_views"=>"84"}
  • {"month"=>"11", "year"=>"2017", "pdf_views"=>"24", "xml_views"=>"2", "html_views"=>"125"}
  • {"month"=>"12", "year"=>"2017", "pdf_views"=>"9", "xml_views"=>"3", "html_views"=>"39"}
  • {"month"=>"1", "year"=>"2018", "pdf_views"=>"20", "xml_views"=>"3", "html_views"=>"55"}
  • {"month"=>"2", "year"=>"2018", "pdf_views"=>"27", "xml_views"=>"1", "html_views"=>"50"}
  • {"month"=>"3", "year"=>"2018", "pdf_views"=>"15", "xml_views"=>"3", "html_views"=>"71"}
  • {"month"=>"4", "year"=>"2018", "pdf_views"=>"19", "xml_views"=>"0", "html_views"=>"56"}
  • {"month"=>"5", "year"=>"2018", "pdf_views"=>"21", "xml_views"=>"2", "html_views"=>"54"}
  • {"month"=>"6", "year"=>"2018", "pdf_views"=>"13", "xml_views"=>"1", "html_views"=>"31"}
  • {"month"=>"7", "year"=>"2018", "pdf_views"=>"10", "xml_views"=>"3", "html_views"=>"36"}
  • {"month"=>"8", "year"=>"2018", "pdf_views"=>"20", "xml_views"=>"1", "html_views"=>"46"}
  • {"month"=>"9", "year"=>"2018", "pdf_views"=>"15", "xml_views"=>"0", "html_views"=>"43"}
  • {"month"=>"10", "year"=>"2018", "pdf_views"=>"13", "xml_views"=>"2", "html_views"=>"45"}
  • {"month"=>"11", "year"=>"2018", "pdf_views"=>"7", "xml_views"=>"3", "html_views"=>"35"}
  • {"month"=>"12", "year"=>"2018", "pdf_views"=>"12", "xml_views"=>"0", "html_views"=>"14"}
  • {"month"=>"1", "year"=>"2019", "pdf_views"=>"14", "xml_views"=>"0", "html_views"=>"31"}
  • {"month"=>"2", "year"=>"2019", "pdf_views"=>"17", "xml_views"=>"0", "html_views"=>"35"}
  • {"month"=>"3", "year"=>"2019", "pdf_views"=>"20", "xml_views"=>"0", "html_views"=>"34"}
  • {"month"=>"4", "year"=>"2019", "pdf_views"=>"10", "xml_views"=>"0", "html_views"=>"31"}
  • {"month"=>"5", "year"=>"2019", "pdf_views"=>"27", "xml_views"=>"0", "html_views"=>"45"}
  • {"month"=>"6", "year"=>"2019", "pdf_views"=>"8", "xml_views"=>"0", "html_views"=>"13"}
  • {"month"=>"7", "year"=>"2019", "pdf_views"=>"6", "xml_views"=>"0", "html_views"=>"26"}
  • {"month"=>"8", "year"=>"2019", "pdf_views"=>"16", "xml_views"=>"0", "html_views"=>"19"}
  • {"month"=>"9", "year"=>"2019", "pdf_views"=>"8", "xml_views"=>"0", "html_views"=>"27"}
  • {"month"=>"10", "year"=>"2019", "pdf_views"=>"17", "xml_views"=>"0", "html_views"=>"23"}
  • {"month"=>"11", "year"=>"2019", "pdf_views"=>"4", "xml_views"=>"1", "html_views"=>"15"}
  • {"month"=>"12", "year"=>"2019", "pdf_views"=>"24", "xml_views"=>"0", "html_views"=>"32"}
  • {"month"=>"1", "year"=>"2020", "pdf_views"=>"12", "xml_views"=>"1", "html_views"=>"28"}
  • {"month"=>"2", "year"=>"2020", "pdf_views"=>"10", "xml_views"=>"2", "html_views"=>"40"}
  • {"month"=>"3", "year"=>"2020", "pdf_views"=>"13", "xml_views"=>"1", "html_views"=>"50"}
  • {"month"=>"4", "year"=>"2020", "pdf_views"=>"48", "xml_views"=>"0", "html_views"=>"47"}
  • {"month"=>"5", "year"=>"2020", "pdf_views"=>"36", "xml_views"=>"2", "html_views"=>"55"}
  • {"month"=>"6", "year"=>"2020", "pdf_views"=>"14", "xml_views"=>"1", "html_views"=>"50"}
  • {"month"=>"7", "year"=>"2020", "pdf_views"=>"10", "xml_views"=>"0", "html_views"=>"29"}
  • {"month"=>"8", "year"=>"2020", "pdf_views"=>"12", "xml_views"=>"0", "html_views"=>"32"}
  • {"month"=>"9", "year"=>"2020", "pdf_views"=>"13", "xml_views"=>"0", "html_views"=>"25"}
  • {"month"=>"10", "year"=>"2020", "pdf_views"=>"17", "xml_views"=>"0", "html_views"=>"23"}
  • {"month"=>"11", "year"=>"2020", "pdf_views"=>"15", "xml_views"=>"1", "html_views"=>"30"}
  • {"month"=>"12", "year"=>"2020", "pdf_views"=>"2", "xml_views"=>"1", "html_views"=>"0"}

Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1278557"], "description"=>"<p>Each sample comprising multiple native targets was mixed in multiple ratios with a competitive internal standard (IS) mixture (depicted in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079120#pone-0079120-g001\" target=\"_blank\"><b>Figure 1</b></a>). The goal is for each combined ratio of native material and competitive IS mixture (<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079120#pone-0079120-g001\" target=\"_blank\"><b>Figure 1A</b></a>) to have a subset of targets in “balance” or equivalence (i.e. as close to a 1∶1 relationship of NT:IS as possible). The closer to equivalence, the fewer sequencing reads required across all library preparations to meet criteria outlined in Statistical Methods section for valid measurement of a given target. Preparation of multiple sequencing libraries with competitive IS mixture spiked-in over the range of expression will result in a greater decrease in required sequencing reads than what is obtainable with only one spike-in ratio (as depicted in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079120#pone-0079120-g002\" target=\"_blank\"><b>Figure 2</b></a> and <b><a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079120#pone.0079120.s012\" target=\"_blank\">Animation S1</a></b>). <b>a)</b> Actual sequencing reads data for 26 ERCC (n = 104) and 100 endogenous (n = 400) cDNA targets across samples A-D. X-axis represents the abundance of each target in a library preparation normalized to the lowest abundance target (set to 10°). Y-axis is in units of normalized sequencing reads (coverage) required to sequence the lowest abundance target at least once. <b>b)</b> Tabular summary of panel A where the number of sequencing reads represents the sum of all sequencing reads to observe all targets at least fifteen times. As discussed in the results section, fifteen sequencing reads is sufficient to achieve a type 1 error rate less than 0.05, and a type 2 error rate of less than 0.20. The required number of traditional RNA-sequencing reads is calculated based on an assumed relationship between target copies present prior to library preparation. Fold-reduction in required sequencing reads by competitive amplicon library preparation is the quotient of calculated traditional RNA-sequencing and measured competitive amplicon library preparation sequencing reads.</p>", "links"=>[], "tags"=>["amplicon", "reduced", "targeted", "rna-sequencing", "reads"], "article_id"=>848896, "categories"=>["Biological Sciences"], "users"=>["Thomas M. Blomquist", "Erin L. Crawford", "Jennie L. Lovett", "Jiyoun Yeo", "Lauren M. Stanoszek", "Albert Levin", "Jia Li", "Mei Lu", "Leming Shi", "Kenneth Muldrew", "James C. Willey"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0079120.g003", "stats"=>{"downloads"=>1, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Competitive_amplicon_library_preparation_reduced_required_targeted_RNA_sequencing_reads_up_to_10_000_fold_/848896", "title"=>"Competitive amplicon library preparation reduced required targeted RNA-sequencing reads up to 10,000-fold.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-11-13 02:58:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/1278551"], "description"=>"<p><b>a)</b> Competitive amplicon library preparation workflow. An internal standard (IS) is a single-stranded or double-stranded DNA template that a) is homologous to a specific native target (NT) at the primer sequences (green shaded regions) and therefore competes for amplification with the NT but b) contains one or more base substitutions (depicted by asterisks) internal to the primer sites and therefore is distinguishable from the NT during post-sequencing data analysis. The corresponding IS template for each native target is in a fixed relationship relative to the IS for the other genes in an internal standard mixture, and the concentration of each IS in the mixture is known. The native sample and competitive internal standard mixture are combined in varying ratios and processed according to the flow diagram (details available in Methods section). <b>b)</b> Linearity of titration between competitive IS relative to native targets in samples A-D (samples used are described in detail in Methods section). With competitive methodologies, it is important to demonstrate that as the abundance of competitive internal standard mixture changes relative to native material (x-axis), the ratio of measured native material to competitive internal standards changes in direct proportion (y-axis), and that the slope is near to 1.00. The proportional relationship among native targets in the original sample is preserved during amplification and sequencing because i) the competition between each NT and its respective IS enables calculation of the original concentration for each NT, and ii) the IS are in a fixed relationship relative to each other (<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079120#pone-0079120-g002\" target=\"_blank\"><b>Figure 2</b></a> and <b><a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079120#pone.0079120.s012\" target=\"_blank\">Animation S1</a></b>). Native targets for which values could not be measured across at least three dilution points are not shown. Upper panel: Linearity of cross titrating competitive Internal Standard Mixture with constant amount of genomic DNA (gDNA) for 123 targets (10 titration points). Dotted lines represent 95% prediction interval for NT:IS ratio values. Middle panel: Linearity of cross titrating competitive Internal Standard Mixture with constant amount of endogenous cDNA native targets from samples A–D (same targets as assessed in gDNA; upper panel) (12 titration points). Lower panel: Linearity of cross-titrating competitive Internal Standard Mixture with constant amount of 26 ERCC native targets from samples A–D (5 titration points). Each titration and assay measurement represents a single technical replicate.</p>", "links"=>[], "tags"=>["amplicon", "workflow"], "article_id"=>848890, "categories"=>["Biological Sciences"], "users"=>["Thomas M. Blomquist", "Erin L. Crawford", "Jennie L. Lovett", "Jiyoun Yeo", "Lauren M. Stanoszek", "Albert Levin", "Jia Li", "Mei Lu", "Leming Shi", "Kenneth Muldrew", "James C. Willey"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0079120.g001", "stats"=>{"downloads"=>0, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Competitive_amplicon_library_preparation_workflow_and_data_analysis_overview_/848890", "title"=>"Competitive amplicon library preparation workflow and data analysis overview.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-11-13 02:58:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/1278565"], "description"=>"<p><b>a</b>) Comparison of TaqMan qPCR with competitive amplicon library preparation (n = 146) for samples A and B without correction for systematic biases. Data is normalized to a median relative abundance. <b>b</b>) Comparison of Illumina RNA-Sequencing with competitive amplicon library preparation (n = 170) for samples A and B without correction for systematic biases. Data is normalized to a median relative abundance. For a) and b), Spearman’s rank correlation coefficient is noted (r<sub>s</sub>). The average of differences for measurements of samples A and B between competitive amplicon library preparation and TaqMan qPCR (<b><a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079120#pone.0079120.s001\" target=\"_blank\">Figure S1</a></b>) or Illumina RNA-sequencing (<b><a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079120#pone.0079120.s002\" target=\"_blank\">Figure S2</a></b>) was determined for each endogenous target; and to illustrate the systematic bias away from the regression line, data points for MMP2 have been highlighted in orange. This difference was subtracted from TaqMan qPCR or Illumina RNA-sequencing measurements for samples C and D and plotted (X-axis). Competitive amplicon library preparation measurements of C and D are plotted on the Y-axis. <b>c)</b> Comparison of TaqMan qPCR with competitive amplicon library preparation (n = 146) for samples C and D with correction for platform and assay specific bias. <b>d)</b> Comparison of Illumina RNA-Sequencing with competitive amplicon library preparation (n = 170) for samples C and D with correction for platform and assay specific bias.</p>", "links"=>[], "tags"=>["cross-platform", "amplicon", "taqman", "qpcr", "illumina"], "article_id"=>848904, "categories"=>["Biological Sciences"], "users"=>["Thomas M. Blomquist", "Erin L. Crawford", "Jennie L. Lovett", "Jiyoun Yeo", "Lauren M. Stanoszek", "Albert Levin", "Jia Li", "Mei Lu", "Leming Shi", "Kenneth Muldrew", "James C. Willey"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0079120.g006", "stats"=>{"downloads"=>0, "page_views"=>50, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Cross_platform_comparison_of_competitive_amplicon_library_preparation_with_TaqMan_qPCR_and_Illumina_RNA_Sequencing_/848904", "title"=>"Cross-platform comparison of competitive amplicon library preparation with TaqMan qPCR and Illumina RNA-Sequencing.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-11-13 02:58:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/1278559"], "description"=>"<p><b>a)</b> Measured signal abundance of ERCC targets in samples A, B, C and D. X-axis units are derived from Ambion product literature for the known concentration of ERCC spike-in controls (n = 104). <b>b)</b> Difference plots of data in panel A ordered numerically by ERCC ID. Each ERCC target depicted was measured at least once in all four samples A–D. For purposes of clarity, ERCC-170 is highlighted orange in panels A and B (n = 104). <b>c)</b> Samples C and D represent a 3∶1 and 1∶3 mixture, respectively, of samples A and B. These ratios were used to calculate expected measurements for samples C and D (X-axis). Actual measurements of samples C and D are plotted on the Y-axis (n = 52). <b>d)</b> Coefficient of variation (CV) in measurements of ERCC targets in samples A-D, for those assays with at least two IS dilution points. Red line depicts expected CV based on a Poisson sampling (n = 95). <b>e)</b> ROC curves to detect fold change with corresponding area under the curve (AUC) with 95% confidence intervals. ROC curves are derived from the comparison of differential ratio subpools of ERCC targets in samples: A vs. B, A vs. C, A vs. D, B vs. C, B vs. D and C vs. D. Results for 1.1-fold change represent a range of differential ratio subpools [1.05–1.174] (controls n = 100, tests n = 96); 1.25-fold change [1.175–1.374] (controls n = 163, tests n = 163); 1.5-fold change [1.375–1.74] (controls n = 229, tests n = 227); 2.0-fold change [1.75–2.49] (controls n = 229, tests n = 223); ≥4.0-fold change [2.5–10.0] (controls n = 286, tests n = 290).</p>", "links"=>[], "tags"=>["amplicon", "ercc"], "article_id"=>848898, "categories"=>["Biological Sciences"], "users"=>["Thomas M. Blomquist", "Erin L. Crawford", "Jennie L. Lovett", "Jiyoun Yeo", "Lauren M. Stanoszek", "Albert Levin", "Jia Li", "Mei Lu", "Leming Shi", "Kenneth Muldrew", "James C. Willey"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0079120.g004", "stats"=>{"downloads"=>0, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Performance_of_competitive_amplicon_library_preparation_with_ERCC_Reference_Materials_/848898", "title"=>"Performance of competitive amplicon library preparation with ERCC Reference Materials.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-11-13 02:58:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/1278554"], "description"=>"<p><b>a)</b> Depicted are two native targets (NT) within a hypothetical cDNA sample. One NT is in high abundance, 10<sup>8</sup> copies (“Abundant” NT), while another is in low abundance, 10<sup>2</sup> copies (“Rare” NT), representing a one million-fold difference in abundance between targets. This hypothetical cDNA sample is combined with a mixture of internal standards (IS) with a fixed relationship of concentrations at 10<sup>5</sup> copies. <b>b)</b> Depicted is the competitive multiplex-PCR library preparation for panel A. The PCR amplification plots for both the “Abundant” and “Rare” NT are separated for purposes of clarity, but occur in the same reaction. During competitive multiplex-PCR, each NT competes equally with its respective competitive IS for dNTPs, polymerase and a limiting concentration of primers. Because the starting concentration of each target’s primer-pair is the same, each competitive reaction will plateau around the same end-point concentration (∼10<sup>9</sup> copies). <b>c)</b> The equal competition between each NT and respective IS preserves the proportional relationship between NT in the original sample, allowing for measurement of native target abundance without signal compression (also see <b><a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079120#pone.0079120.s012\" target=\"_blank\">Animation S1</a></b>). Yet, a 10<sup>6</sup> fold range of templates is reduced to 10<sup>3</sup> after competitive multiplex-PCR library preparation resulting in a 1,000-fold reduction in oversampling/sequencing of the high abundance target.</p>", "links"=>[], "tags"=>["depiction", "amplicon"], "article_id"=>848893, "categories"=>["Biological Sciences"], "users"=>["Thomas M. Blomquist", "Erin L. Crawford", "Jennie L. Lovett", "Jiyoun Yeo", "Lauren M. Stanoszek", "Albert Levin", "Jia Li", "Mei Lu", "Leming Shi", "Kenneth Muldrew", "James C. Willey"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0079120.g002", "stats"=>{"downloads"=>2, "page_views"=>24, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Schematic_depiction_of_how_competitive_amplicon_library_preparation_reduces_oversampling_/848893", "title"=>"Schematic depiction of how competitive amplicon library preparation reduces oversampling.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-11-13 02:58:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/1278567", "https://ndownloader.figshare.com/files/1278568", "https://ndownloader.figshare.com/files/1278569", "https://ndownloader.figshare.com/files/1278570", "https://ndownloader.figshare.com/files/1278571", "https://ndownloader.figshare.com/files/1278572", "https://ndownloader.figshare.com/files/1278573", "https://ndownloader.figshare.com/files/1278574", "https://ndownloader.figshare.com/files/1278575", "https://ndownloader.figshare.com/files/1278576", "https://ndownloader.figshare.com/files/1278577", "https://ndownloader.figshare.com/files/1278578"], "description"=>"<div><p>Whole transcriptome RNA-sequencing is a powerful tool, but is costly and yields complex data sets that limit its utility in molecular diagnostic testing. A targeted quantitative RNA-sequencing method that is reproducible and reduces the number of sequencing reads required to measure transcripts over the full range of expression would be better suited to diagnostic testing. Toward this goal, we developed a competitive multiplex PCR-based amplicon sequencing library preparation method that a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. To determine the utility of this method, we intentionally selected PCR conditions that would cause transcript amplification products (amplicons) to converge toward equimolar concentrations (normalization) during library preparation. We then tested whether this approach would enable accurate and reproducible quantification of each transcript across multiple library preparations, and at the same time reduce (through normalization) total sequencing reads required for quantification of transcript targets across a large range of expression. We demonstrate excellent reproducibility (R<sup>2</sup> = 0.997) with 97% accuracy to detect 2-fold change using External RNA Controls Consortium (ERCC) reference materials; high inter-day, inter-site and inter-library concordance (R<sup>2</sup> = 0.97–0.99) using FDA Sequencing Quality Control (SEQC) reference materials; and cross-platform concordance with both TaqMan qPCR (R<sup>2</sup> = 0.96) and whole transcriptome RNA-sequencing following “traditional” library preparation using Illumina NGS kits (R<sup>2</sup> = 0.94). Using this method, sequencing reads required to accurately quantify more than 100 targeted transcripts expressed over a 10<sup>7</sup>-fold range was reduced more than 10,000-fold, from 2.3×10<sup>9</sup> to 1.4×10<sup>5</sup> sequencing reads. These studies demonstrate that the competitive multiplex-PCR amplicon library preparation method presented here provides the quality control, reproducibility, and reduced sequencing reads necessary for development and implementation of targeted quantitative RNA-sequencing biomarkers in molecular diagnostic testing.</p></div>", "links"=>[], "tags"=>["rna-sequencing", "multiplex-pcr", "amplicon"], "article_id"=>848906, "categories"=>["Biological Sciences"], "users"=>["Thomas M. Blomquist", "Erin L. Crawford", "Jennie L. Lovett", "Jiyoun Yeo", "Lauren M. Stanoszek", "Albert Levin", "Jia Li", "Mei Lu", "Leming Shi", "Kenneth Muldrew", "James C. Willey"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0079120.s001", "https://dx.doi.org/10.1371/journal.pone.0079120.s002", "https://dx.doi.org/10.1371/journal.pone.0079120.s003", "https://dx.doi.org/10.1371/journal.pone.0079120.s004", "https://dx.doi.org/10.1371/journal.pone.0079120.s005", "https://dx.doi.org/10.1371/journal.pone.0079120.s006", "https://dx.doi.org/10.1371/journal.pone.0079120.s007", "https://dx.doi.org/10.1371/journal.pone.0079120.s008", "https://dx.doi.org/10.1371/journal.pone.0079120.s009", "https://dx.doi.org/10.1371/journal.pone.0079120.s010", "https://dx.doi.org/10.1371/journal.pone.0079120.s011", "https://dx.doi.org/10.1371/journal.pone.0079120.s012"], "stats"=>{"downloads"=>7, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Targeted_RNA_Sequencing_with_Competitive_Multiplex_PCR_Amplicon_Libraries_/848906", "title"=>"Targeted RNA-Sequencing with Competitive Multiplex-PCR Amplicon Libraries", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-11-13 02:58:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/1278561"], "description"=>"<p><b>a–d)</b> Absolute signal abundance of cDNA targets in sample A in units of copies per library preparation measured on separate days, at different sites (OU = Ohio University; UTMC = University of Toledo Medical Center), and between different reverse transcription preparations (RT1 and RT2). <b>a)</b> Inter-day effect (n = 88). <b>b</b>) Inter-day and Inter-site effect (n = 81). <b>c)</b> Inter-day and Inter-library effect (n = 92). <b>d)</b> Inter-day, Inter-site and Inter-library effect (n = 80). <b>e–f)</b> Samples C and D represent a 3∶1 and 1∶3 mixture, respectively, of total RNA from samples A and B. These ratios were used to calculate expected measurements for samples C and D (X-axis) from measurements of A and B. Plotted on the Y-axis are actual measurements of samples C (n = 86) and D (n = 90).</p>", "links"=>[], "tags"=>["amplicon", "endogenous", "cdna"], "article_id"=>848900, "categories"=>["Biological Sciences"], "users"=>["Thomas M. Blomquist", "Erin L. Crawford", "Jennie L. Lovett", "Jiyoun Yeo", "Lauren M. Stanoszek", "Albert Levin", "Jia Li", "Mei Lu", "Leming Shi", "Kenneth Muldrew", "James C. Willey"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0079120.g005", "stats"=>{"downloads"=>1, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Performance_of_competitive_amplicon_library_preparation_with_endogenous_cDNA_targets_/848900", "title"=>"Performance of competitive amplicon library preparation with endogenous cDNA targets.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-11-13 02:58:24"}

PMC Usage Stats | Further Information

  • {"unique-ip"=>"35", "full-text"=>"33", "pdf"=>"14", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"8", "supp-data"=>"28", "cited-by"=>"0", "year"=>"2013", "month"=>"11"}
  • {"unique-ip"=>"22", "full-text"=>"27", "pdf"=>"8", "abstract"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"13", "supp-data"=>"36", "cited-by"=>"0", "year"=>"2013", "month"=>"12"}
  • {"unique-ip"=>"30", "full-text"=>"33", "pdf"=>"13", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"4", "supp-data"=>"6", "cited-by"=>"0", "year"=>"2014", "month"=>"1"}
  • {"unique-ip"=>"24", "full-text"=>"28", "pdf"=>"8", "abstract"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"9", "supp-data"=>"2", "cited-by"=>"0", "year"=>"2014", "month"=>"2"}
  • {"unique-ip"=>"19", "full-text"=>"23", "pdf"=>"6", "abstract"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"6", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2014", "month"=>"3"}
  • {"unique-ip"=>"15", "full-text"=>"12", "pdf"=>"10", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"5", "supp-data"=>"2", "cited-by"=>"0", "year"=>"2014", "month"=>"5"}
  • {"unique-ip"=>"14", "full-text"=>"18", "pdf"=>"6", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"6", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2014", "month"=>"6"}
  • {"unique-ip"=>"23", "full-text"=>"21", "pdf"=>"12", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"10", "supp-data"=>"2", "cited-by"=>"0", "year"=>"2014", "month"=>"4"}
  • {"unique-ip"=>"14", "full-text"=>"13", "pdf"=>"4", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"5", "supp-data"=>"3", "cited-by"=>"0", "year"=>"2015", "month"=>"4"}
  • {"unique-ip"=>"28", "full-text"=>"27", "pdf"=>"16", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"20", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2015", "month"=>"5"}
  • {"unique-ip"=>"10", "full-text"=>"9", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"4", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2015", "month"=>"6"}
  • {"unique-ip"=>"30", "full-text"=>"27", "pdf"=>"13", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"22", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2015", "month"=>"7"}
  • {"unique-ip"=>"17", "full-text"=>"17", "pdf"=>"7", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"5", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2015", "month"=>"3"}
  • {"unique-ip"=>"19", "full-text"=>"20", "pdf"=>"6", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"4", "supp-data"=>"2", "cited-by"=>"0", "year"=>"2015", "month"=>"2"}
  • {"unique-ip"=>"17", "full-text"=>"18", "pdf"=>"7", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2015", "month"=>"8"}
  • {"unique-ip"=>"34", "full-text"=>"32", "pdf"=>"9", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2015", "month"=>"9"}
  • {"unique-ip"=>"32", "full-text"=>"31", "pdf"=>"8", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"21", "supp-data"=>"3", "cited-by"=>"0", "year"=>"2015", "month"=>"10"}
  • {"unique-ip"=>"28", "full-text"=>"25", "pdf"=>"10", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"6", "supp-data"=>"3", "cited-by"=>"1", "year"=>"2014", "month"=>"7"}
  • {"unique-ip"=>"21", "full-text"=>"16", "pdf"=>"7", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"4", "supp-data"=>"9", "cited-by"=>"0", "year"=>"2014", "month"=>"8"}
  • {"unique-ip"=>"51", "full-text"=>"34", "pdf"=>"14", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"9", "supp-data"=>"19", "cited-by"=>"0", "year"=>"2014", "month"=>"9"}
  • {"unique-ip"=>"35", "full-text"=>"34", "pdf"=>"13", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"4", "supp-data"=>"3", "cited-by"=>"0", "year"=>"2014", "month"=>"10"}
  • {"unique-ip"=>"22", "full-text"=>"28", "pdf"=>"7", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"4", "cited-by"=>"0", "year"=>"2016", "month"=>"2"}
  • {"unique-ip"=>"31", "full-text"=>"24", "pdf"=>"8", "abstract"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"7", "supp-data"=>"3", "cited-by"=>"1", "year"=>"2014", "month"=>"11"}
  • {"unique-ip"=>"21", "full-text"=>"16", "pdf"=>"9", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"2", "cited-by"=>"0", "year"=>"2014", "month"=>"12"}
  • {"unique-ip"=>"17", "full-text"=>"14", "pdf"=>"5", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"4", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2015", "month"=>"1"}
  • {"unique-ip"=>"29", "full-text"=>"23", "pdf"=>"12", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"6", "supp-data"=>"7", "cited-by"=>"2", "year"=>"2015", "month"=>"11"}
  • {"unique-ip"=>"33", "full-text"=>"22", "pdf"=>"20", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"2", "cited-by"=>"0", "year"=>"2015", "month"=>"12"}
  • {"unique-ip"=>"41", "full-text"=>"32", "pdf"=>"15", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"12", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2016", "month"=>"1"}
  • {"unique-ip"=>"36", "full-text"=>"39", "pdf"=>"13", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"9", "supp-data"=>"4", "cited-by"=>"0", "year"=>"2016", "month"=>"3"}
  • {"unique-ip"=>"21", "full-text"=>"17", "pdf"=>"6", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2016", "month"=>"4"}
  • {"unique-ip"=>"19", "full-text"=>"18", "pdf"=>"7", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"5"}
  • {"unique-ip"=>"23", "full-text"=>"24", "pdf"=>"7", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"1", "cited-by"=>"1", "year"=>"2016", "month"=>"6"}
  • {"unique-ip"=>"15", "full-text"=>"9", "pdf"=>"11", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"8", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"7"}
  • {"unique-ip"=>"27", "full-text"=>"28", "pdf"=>"7", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"4", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2016", "month"=>"8"}
  • {"unique-ip"=>"19", "full-text"=>"24", "pdf"=>"6", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"8", "supp-data"=>"14", "cited-by"=>"1", "year"=>"2016", "month"=>"9"}
  • {"unique-ip"=>"28", "full-text"=>"26", "pdf"=>"7", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"10", "supp-data"=>"18", "cited-by"=>"0", "year"=>"2016", "month"=>"10"}
  • {"unique-ip"=>"14", "full-text"=>"15", "pdf"=>"12", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2016", "month"=>"11"}
  • {"unique-ip"=>"11", "full-text"=>"11", "pdf"=>"8", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2016", "month"=>"12"}
  • {"unique-ip"=>"20", "full-text"=>"20", "pdf"=>"5", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"4", "supp-data"=>"0", "cited-by"=>"1", "year"=>"2017", "month"=>"1"}
  • {"unique-ip"=>"22", "full-text"=>"22", "pdf"=>"4", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"6", "supp-data"=>"2", "cited-by"=>"0", "year"=>"2017", "month"=>"2"}
  • {"unique-ip"=>"25", "full-text"=>"24", "pdf"=>"6", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"13", "supp-data"=>"2", "cited-by"=>"0", "year"=>"2017", "month"=>"3"}
  • {"unique-ip"=>"22", "full-text"=>"22", "pdf"=>"6", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"4", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2017", "month"=>"4"}
  • {"unique-ip"=>"26", "full-text"=>"29", "pdf"=>"8", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"7", "supp-data"=>"2", "cited-by"=>"0", "year"=>"2017", "month"=>"5"}
  • {"unique-ip"=>"19", "full-text"=>"22", "pdf"=>"5", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"6"}
  • {"unique-ip"=>"21", "full-text"=>"22", "pdf"=>"3", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"8", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"7"}
  • {"unique-ip"=>"24", "full-text"=>"29", "pdf"=>"8", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"3", "cited-by"=>"0", "year"=>"2017", "month"=>"8"}
  • {"unique-ip"=>"26", "full-text"=>"28", "pdf"=>"5", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"15", "supp-data"=>"4", "cited-by"=>"0", "year"=>"2017", "month"=>"9"}
  • {"unique-ip"=>"37", "full-text"=>"31", "pdf"=>"7", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"5", "supp-data"=>"5", "cited-by"=>"0", "year"=>"2017", "month"=>"10"}
  • {"unique-ip"=>"19", "full-text"=>"20", "pdf"=>"4", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"7", "supp-data"=>"3", "cited-by"=>"0", "year"=>"2017", "month"=>"11"}
  • {"unique-ip"=>"22", "full-text"=>"23", "pdf"=>"6", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"15", "supp-data"=>"3", "cited-by"=>"0", "year"=>"2017", "month"=>"12"}
  • {"unique-ip"=>"31", "full-text"=>"24", "pdf"=>"8", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"12", "cited-by"=>"0", "year"=>"2018", "month"=>"1"}
  • {"unique-ip"=>"1", "full-text"=>"1", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"2"}
  • {"unique-ip"=>"25", "full-text"=>"31", "pdf"=>"7", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"3"}
  • {"unique-ip"=>"27", "full-text"=>"24", "pdf"=>"4", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"6", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"1"}
  • {"unique-ip"=>"26", "full-text"=>"20", "pdf"=>"2", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"8", "supp-data"=>"6", "cited-by"=>"0", "year"=>"2018", "month"=>"11"}
  • {"unique-ip"=>"19", "full-text"=>"19", "pdf"=>"5", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"7", "supp-data"=>"3", "cited-by"=>"0", "year"=>"2018", "month"=>"5"}
  • {"unique-ip"=>"26", "full-text"=>"27", "pdf"=>"5", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"4", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"6"}
  • {"unique-ip"=>"13", "full-text"=>"16", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2018", "month"=>"4"}
  • {"unique-ip"=>"29", "full-text"=>"25", "pdf"=>"4", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"6", "cited-by"=>"0", "year"=>"2018", "month"=>"7"}
  • {"unique-ip"=>"19", "full-text"=>"19", "pdf"=>"5", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"24", "cited-by"=>"1", "year"=>"2018", "month"=>"8"}
  • {"unique-ip"=>"29", "full-text"=>"19", "pdf"=>"2", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"10", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2018", "month"=>"10"}
  • {"unique-ip"=>"20", "full-text"=>"17", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"4", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2018", "month"=>"12"}
  • {"unique-ip"=>"27", "full-text"=>"19", "pdf"=>"4", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"13", "supp-data"=>"2", "cited-by"=>"0", "year"=>"2018", "month"=>"9"}
  • {"unique-ip"=>"17", "full-text"=>"9", "pdf"=>"3", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"7", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"2"}
  • {"unique-ip"=>"24", "full-text"=>"23", "pdf"=>"3", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"4", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"3"}
  • {"unique-ip"=>"21", "full-text"=>"22", "pdf"=>"2", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"4"}
  • {"unique-ip"=>"23", "full-text"=>"29", "pdf"=>"3", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"5"}
  • {"unique-ip"=>"20", "full-text"=>"12", "pdf"=>"3", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"7", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"8"}
  • {"unique-ip"=>"23", "full-text"=>"20", "pdf"=>"7", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"1", "cited-by"=>"1", "year"=>"2019", "month"=>"9"}
  • {"unique-ip"=>"14", "full-text"=>"13", "pdf"=>"4", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"10"}
  • {"unique-ip"=>"20", "full-text"=>"27", "pdf"=>"12", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"2", "cited-by"=>"2", "year"=>"2019", "month"=>"12"}
  • {"unique-ip"=>"22", "full-text"=>"24", "pdf"=>"4", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2020", "month"=>"2"}
  • {"unique-ip"=>"22", "full-text"=>"25", "pdf"=>"8", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2020", "month"=>"3"}
  • {"unique-ip"=>"17", "full-text"=>"17", "pdf"=>"5", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"6", "cited-by"=>"0", "year"=>"2020", "month"=>"4"}
  • {"unique-ip"=>"20", "full-text"=>"22", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2020", "month"=>"5"}
  • {"unique-ip"=>"29", "full-text"=>"33", "pdf"=>"8", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"2", "cited-by"=>"0", "year"=>"2020", "month"=>"6"}
  • {"unique-ip"=>"8", "full-text"=>"9", "pdf"=>"3", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2020", "month"=>"7"}
  • {"unique-ip"=>"8", "full-text"=>"7", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"12", "cited-by"=>"0", "year"=>"2020", "month"=>"8"}
  • {"unique-ip"=>"3", "full-text"=>"3", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"8", "cited-by"=>"0", "year"=>"2020", "month"=>"9"}
  • {"unique-ip"=>"16", "full-text"=>"18", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2020", "month"=>"10"}

Relative Metric

{"start_date"=>"2013-01-01T00:00:00Z", "end_date"=>"2013-12-31T00:00:00Z", "subject_areas"=>[{"subject_area"=>"/Biology and life sciences", "average_usage"=>[269, 466, 588, 697, 800, 896, 988, 1076, 1165, 1254, 1340, 1417]}, {"subject_area"=>"/Biology and life sciences/Biochemistry", "average_usage"=>[266, 468, 593, 703, 804, 903, 993, 1084, 1171, 1256, 1339, 1422, 1492]}, {"subject_area"=>"/Biology and life sciences/Computational biology", "average_usage"=>[295, 511, 651, 775, 882, 992, 1100, 1201, 1304, 1400, 1486, 1570, 1650]}]}
Loading … Spinner
There are currently no alerts