Cost-Effective Sequencing of Full-Length cDNA Clones Powered by a De Novo-Reference Hybrid Assembly
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{"title"=>"Cost-effective sequencing of full-length cDNA clones powered by a de novo-reference hybrid assembly", "type"=>"journal", "authors"=>[{"first_name"=>"Reginaldo M.", "last_name"=>"Kuroshu", "scopus_author_id"=>"35337279700"}, {"first_name"=>"Junichi", "last_name"=>"Watanabe", "scopus_author_id"=>"57189705985"}, {"first_name"=>"Sumio", "last_name"=>"Sugano", "scopus_author_id"=>"35354458800"}, {"first_name"=>"Shinichi", "last_name"=>"Morishita", "scopus_author_id"=>"7102349168"}, {"first_name"=>"Yutaka", "last_name"=>"Suzuki", "scopus_author_id"=>"55626680600"}, {"first_name"=>"Masahiro", "last_name"=>"Kasahara", "scopus_author_id"=>"55806768200"}], "year"=>2010, "source"=>"PLoS ONE", "identifiers"=>{"pui"=>"359494554", "pmid"=>"20479877", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "issn"=>"19326203", "doi"=>"10.1371/journal.pone.0010517", "scopus"=>"2-s2.0-77956270455", "sgr"=>"77956270455"}, "id"=>"24838415-4a7f-37ca-b9bf-e464a740e938", "abstract"=>"BACKGROUND: Sequencing full-length cDNA clones is important to determine gene structures including alternative splice forms, and provides valuable resources for experimental analyses to reveal the biological functions of coded proteins. However, previous approaches for sequencing cDNA clones were expensive or time-consuming, and therefore, a fast and efficient sequencing approach was demanded.\\n\\nMETHODOLOGY: We developed a program, MuSICA 2, that assembles millions of short (36-nucleotide) reads collected from a single flow cell lane of Illumina Genome Analyzer to shotgun-sequence approximately 800 human full-length cDNA clones. MuSICA 2 performs a hybrid assembly in which an external de novo assembler is run first and the result is then improved by reference alignment of shotgun reads. We compared the MuSICA 2 assembly with 200 pooled full-length cDNA clones finished independently by the conventional primer-walking using Sanger sequencers. The exon-intron structure of the coding sequence was correct for more than 95% of the clones with coding sequence annotation when we excluded cDNA clones insufficiently represented in the shotgun library due to PCR failure (42 out of 200 clones excluded), and the nucleotide-level accuracy of coding sequences of those correct clones was over 99.99%. We also applied MuSICA 2 to full-length cDNA clones from Toxoplasma gondii, to confirm that its ability was competent even for non-human species.\\n\\nCONCLUSIONS: The entire sequencing and shotgun assembly takes less than 1 week and the consumables cost only approximately US$3 per clone, demonstrating a significant advantage over previous approaches.", "link"=>"http://www.mendeley.com/research/costeffective-sequencing-fulllength-cdna-clones-powered-novoreference-hybrid-assembly", "reader_count"=>51, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>4, "Researcher"=>16, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>10, "Student > Postgraduate"=>4, "Other"=>3, "Student > Master"=>6, "Student > Bachelor"=>2, "Lecturer"=>1, "Professor"=>2}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>4, "Researcher"=>16, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>10, "Student > Postgraduate"=>4, "Other"=>3, "Student > Master"=>6, "Student > Bachelor"=>2, "Lecturer"=>1, "Professor"=>2}, "reader_count_by_subject_area"=>{"Engineering"=>1, "Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>6, "Agricultural and Biological Sciences"=>37, "Medicine and Dentistry"=>1, "Computer Science"=>5}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>37}, "Computer Science"=>{"Computer Science"=>5}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>6}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"Canada"=>1, "United States"=>2, "Japan"=>1, "China"=>1, "Brazil"=>1, "Italy"=>1, "United Kingdom"=>2, "Germany"=>1}, "group_count"=>2}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/851187"], "description"=>"<p>Clones in the simulated datasets were binned according to their per-clone sequence coverage. The bins were of every 10-fold. Note that every full-length cDNA clone was counted 10 times as it appeared with 10 different sequence coverage. We calculated the percentage of clones (left Y-axis) classified as consistent (or inconsistent) in terms of CDS structure for each bin. The number of clones in each bin is also shown (right Y-axis). Per-clone sequence coverage of 30-fold was sufficient to produce a CDS-consistent assembly in 95% of the cases, showing that uniform coverage distribution is highly desirable to achieve better efficiency.</p>", "links"=>[], "tags"=>["clone", "reconstruction", "cds"], "article_id"=>521645, "categories"=>["Genetics", "Infectious Diseases"], "users"=>["Reginaldo M. Kuroshu", "Junichi Watanabe,", "Sumio Sugano", "Shinichi Morishita", "Yutaka Suzuki", "Masahiro Kasahara"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0010517.g005", "stats"=>{"downloads"=>3, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Relationship_between_the_sequence_coverage_for_each_clone_and_the_reconstruction_accuracy_in_terms_of_CDS_structure_consistency_/521645", "title"=>"Relationship between the sequence coverage for each clone and the reconstruction accuracy in terms of CDS structure consistency.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-05-07 00:27:25"}
  • {"files"=>["https://ndownloader.figshare.com/files/851277"], "description"=>"<p><b>A</b>: The total number of base pairs in the CDS of the reference full-length cDNA clones successfully reconstructed with regard to CDS structure. Note that library 1 and 1+2 have different sets of PCR-amplified reference full-length cDNA clones; <b>B:</b> the number of matched base pairs; <b>C:</b> the number of mismatched base pairs; <b>D:</b> the number of indels (insertions/deletions).</p>", "links"=>[], "tags"=>["coding"], "article_id"=>521737, "categories"=>["Genetics", "Infectious Diseases"], "users"=>["Reginaldo M. Kuroshu", "Junichi Watanabe,", "Sumio Sugano", "Shinichi Morishita", "Yutaka Suzuki", "Masahiro Kasahara"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0010517.t004", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Base_accuracy_of_consistent_coding_sequences_/521737", "title"=>"Base accuracy of consistent coding sequences.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2010-05-07 00:28:57"}
  • {"files"=>["https://ndownloader.figshare.com/files/851393"], "description"=>"<p>The number of contigs is presented for each step in the assembly process. Ideally, the figures converge to the number of PCR-amplified full-length cDNA clones in each library as it goes down the process. However, the actual number of PCR-amplified full-length cDNA clones is not known except for library 1 (158 clones). Less-amplified clones were often reconstructed well but their bands in a gel electrophoresis picture were too faint to identify, making it difficult to experimentally measure the number of PCR-amplified full-length cDNA clones without aligning the shotgun reads. In the last step, the N50 contig size for library 1 decreased because some contigs from the previous step were not associated with any Sanger read and were then discarded to avoid false positives. For library 3, the number of contigs increased in the last step because multiple Sanger reads were associated to the same contig, which was possibly an error that could be eliminated if the output was manually examined.</p>", "links"=>[], "tags"=>["computational biology/genomics", "genetics and genomics/bioinformatics", "genetics and genomics/gene discovery", "genetics and genomics/genomics"], "article_id"=>521850, "categories"=>["Genetics", "Infectious Diseases"], "users"=>["Reginaldo M. Kuroshu", "Junichi Watanabe,", "Sumio Sugano", "Shinichi Morishita", "Yutaka Suzuki", "Masahiro Kasahara"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0010517.t001", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Hybrid_assembly_statistics_for_individual_libraries_/521850", "title"=>"Hybrid assembly statistics for individual libraries.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2010-05-07 00:30:50"}
  • {"files"=>["https://ndownloader.figshare.com/files/851091"], "description"=>"<p>The histograms show the sequence coverage distribution of clones in Library 1 and 1+2. First, we aligned the shotgun reads against the finished reference clone sequences, allowing up to 3 mismatches. Next we calculated the sequence coverage (X-axis) for each clone as follows: (# of reads aligned with the clone)×(read length (36 bp))/(the length of the clone). Y-axis shows the number of clones. Every clone is colored according to its consistency of the CDS structure; a red bar shows the proportion of the inconsistent clones in that range. Clones that were not successfully amplified by PCR are not shown in the histogram, as they always had little sequence coverage by definition. Most of the clones classified as inconsistent had sequence coverage lower than 50×, suggesting that inconsistency might have arisen due to lower sequence coverage.</p>", "links"=>[], "tags"=>["histogram", "clone"], "article_id"=>521544, "categories"=>["Genetics", "Infectious Diseases"], "users"=>["Reginaldo M. Kuroshu", "Junichi Watanabe,", "Sumio Sugano", "Shinichi Morishita", "Yutaka Suzuki", "Masahiro Kasahara"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0010517.g004", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_A_histogram_showing_relationship_between_the_sequence_coverage_for_each_clone_and_their_assembly_result_classifications_/521544", "title"=>"A histogram showing relationship between the sequence coverage for each clone and their assembly result classifications.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-05-07 00:25:44"}
  • {"files"=>["https://ndownloader.figshare.com/files/423057", "https://ndownloader.figshare.com/files/423063", "https://ndownloader.figshare.com/files/423080", "https://ndownloader.figshare.com/files/423094", "https://ndownloader.figshare.com/files/423106", "https://ndownloader.figshare.com/files/423116", "https://ndownloader.figshare.com/files/423141", "https://ndownloader.figshare.com/files/423150", "https://ndownloader.figshare.com/files/423163", "https://ndownloader.figshare.com/files/423172", "https://ndownloader.figshare.com/files/423182", "https://ndownloader.figshare.com/files/423189", "https://ndownloader.figshare.com/files/423201"], "description"=>"<div><h3>Background</h3><p>Sequencing full-length cDNA clones is important to determine gene structures including alternative splice forms, and provides valuable resources for experimental analyses to reveal the biological functions of coded proteins. However, previous approaches for sequencing cDNA clones were expensive or time-consuming, and therefore, a fast and efficient sequencing approach was demanded.</p><h3>Methodology</h3><p>We developed a program, MuSICA 2, that assembles millions of short (36-nucleotide) reads collected from a single flow cell lane of Illumina Genome Analyzer to shotgun-sequence ∼800 human full-length cDNA clones. MuSICA 2 performs a hybrid assembly in which an external <em>de novo</em> assembler is run first and the result is then improved by reference alignment of shotgun reads. We compared the MuSICA 2 assembly with 200 pooled full-length cDNA clones finished independently by the conventional primer-walking using Sanger sequencers. The exon-intron structure of the coding sequence was correct for more than 95% of the clones with coding sequence annotation when we excluded cDNA clones insufficiently represented in the shotgun library due to PCR failure (42 out of 200 clones excluded), and the nucleotide-level accuracy of coding sequences of those correct clones was over 99.99%. We also applied MuSICA 2 to full-length cDNA clones from <em>Toxoplasma gondii</em>, to confirm that its ability was competent even for non-human species.</p><h3>Conclusions</h3><p>The entire sequencing and shotgun assembly takes less than 1 week and the consumables cost only ∼US$3 per clone, demonstrating a significant advantage over previous approaches.</p></div>", "links"=>[], "tags"=>["cost-effective", "sequencing", "full-length", "cdna", "clones", "powered", "hybrid"], "article_id"=>143540, "categories"=>["Genetics", "Cancer"], "users"=>["Reginaldo M. Kuroshu", "Junichi Watanabe,", "Sumio Sugano", "Shinichi Morishita", "Yutaka Suzuki", "Masahiro Kasahara"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0010517.s001", "https://dx.doi.org/10.1371/journal.pone.0010517.s002", "https://dx.doi.org/10.1371/journal.pone.0010517.s003", "https://dx.doi.org/10.1371/journal.pone.0010517.s004", "https://dx.doi.org/10.1371/journal.pone.0010517.s005", "https://dx.doi.org/10.1371/journal.pone.0010517.s006", "https://dx.doi.org/10.1371/journal.pone.0010517.s007", "https://dx.doi.org/10.1371/journal.pone.0010517.s008", "https://dx.doi.org/10.1371/journal.pone.0010517.s009", "https://dx.doi.org/10.1371/journal.pone.0010517.s010", "https://dx.doi.org/10.1371/journal.pone.0010517.s011", "https://dx.doi.org/10.1371/journal.pone.0010517.s012", "https://dx.doi.org/10.1371/journal.pone.0010517.s013"], "stats"=>{"downloads"=>30, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Cost_Effective_Sequencing_of_Full_Length_cDNA_Clones_Powered_by_a_De_Novo_Reference_Hybrid_Assembly/143540", "title"=>"Cost-Effective Sequencing of Full-Length cDNA Clones Powered by a <em>De Novo</em>-Reference Hybrid Assembly", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2010-05-07 00:59:00"}
  • {"files"=>["https://ndownloader.figshare.com/files/851435"], "description"=>"<p><b>A:</b> The number of the reference full-length cDNA clones used in the experiment. the original number was 200, but one of them was excluded due to chimerism; <b>B:</b> the number of reference full-length cDNA clones successfully amplified by PCR. Note that it does not include non-reference full-length cDNA clones; <b>C:</b> the number of successfully amplified reference full-length cDNA clones with CDS annotation. Non-reference and/or non-amplified full-length cDNA clones were excluded; <b>D:</b> the number of full-length cDNA clones that were reconstructed consistently in terms of CDS structure. See <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010517#pone-0010517-g002\" target=\"_blank\">Fig. 2</a> for CDS structure consistency; <b>E:</b> the number of full-length cDNA clones that were not reconstructed consistently with regard to CDS structure. This always equals to C–D.</p>", "links"=>[], "tags"=>["full-length", "cdna"], "article_id"=>521890, "categories"=>["Genetics", "Infectious Diseases"], "users"=>["Reginaldo M. Kuroshu", "Junichi Watanabe,", "Sumio Sugano", "Shinichi Morishita", "Yutaka Suzuki", "Masahiro Kasahara"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0010517.t002", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Assembly_statistics_and_accuracy_evaluation_for_human_full_length_cDNA_clones_/521890", "title"=>"Assembly statistics and accuracy evaluation for human full-length cDNA clones.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2010-05-07 00:31:30"}
  • {"files"=>["https://ndownloader.figshare.com/files/851357"], "description"=>"<p>The definitions of the columns are exactly the same as in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010517#pone-0010517-t002\" target=\"_blank\">Table 2</a>.</p>", "links"=>[], "tags"=>["full-length", "cdna"], "article_id"=>521807, "categories"=>["Genetics", "Infectious Diseases"], "users"=>["Reginaldo M. Kuroshu", "Junichi Watanabe,", "Sumio Sugano", "Shinichi Morishita", "Yutaka Suzuki", "Masahiro Kasahara"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0010517.t006", "stats"=>{"downloads"=>1, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Assembly_statistics_and_accuracy_evaluation_for_Toxoplasma_gondii_full_length_cDNA_clones_/521807", "title"=>"Assembly statistics and accuracy evaluation for <i>Toxoplasma gondii</i> full-length cDNA clones.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2010-05-07 00:30:07"}
  • {"files"=>["https://ndownloader.figshare.com/files/850885"], "description"=>"<p>Target cDNA clones are prepared (for example, 800 clones per flow cell). The clones are individually amplified by PCR. The amplicons are mixed in equal volumes, and then fragmented by nebulization. The fragments of appropriate size for sequencing are size-fractionated by gel electrophoresis, followed by sequence adaptor ligation. A number of 36-bp reads are collected using the Illumina GA. The obtained reads are assembled by an external <i>de novo</i> assembler such as Velvet. The assembled contigs are then aligned against the reference genome sequence to merge overlapping contigs. Missing exons are filled by aligning raw reads against small gaps between the merged contigs. Missing introns (links between exons) are recovered by spliced alignment of the raw reads. The final contigs are associated with individual full-length cDNA clones by Sanger reads from either/both ends of the clones.</p>", "links"=>[], "tags"=>["full-length", "cdna", "clone", "sequencing", "musica"], "article_id"=>521327, "categories"=>["Genetics", "Infectious Diseases"], "users"=>["Reginaldo M. Kuroshu", "Junichi Watanabe,", "Sumio Sugano", "Shinichi Morishita", "Yutaka Suzuki", "Masahiro Kasahara"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0010517.g001", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Whole_full_length_cDNA_clone_sequencing_process_using_MuSICA_2_/521327", "title"=>"Whole full-length cDNA clone sequencing process using MuSICA 2.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-05-07 00:22:07"}
  • {"files"=>["https://ndownloader.figshare.com/files/851468"], "description"=>"<p><b>A:</b> The total number of base pairs in the reference full-length cDNA clones. Note that non-reference clones are excluded; <b>B:</b> the total number of base pairs in the output contigs; <b>C:</b> the number of base pairs matched to the reference full-length cDNA clones in the alignments (B); <b>D:</b> the number of base pairs mismatched to the reference full-length cDNA clones in the alignments (B); <b>E:</b> the number of base pairs inserted (e.g., a reconstructed contig is longer than the reference clone) in the alignments (B); <b>F:</b> the number of base pairs deleted in the alignments (B).</p>", "links"=>[], "tags"=>["musica"], "article_id"=>521925, "categories"=>["Genetics", "Infectious Diseases"], "users"=>["Reginaldo M. Kuroshu", "Junichi Watanabe,", "Sumio Sugano", "Shinichi Morishita", "Yutaka Suzuki", "Masahiro Kasahara"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0010517.t003", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Base_accuracy_of_MuSICA_2_assembly_/521925", "title"=>"Base accuracy of MuSICA 2 assembly.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2010-05-07 00:32:05"}
  • {"files"=>["https://ndownloader.figshare.com/files/851018"], "description"=>"<p>The diagram shows the accuracy evaluation process for the 200 human full-length cDNA clones in library 1 as well as the 800 human full-length cDNA clones in library 1+2. For library 1+2, the contigs associated to the clones with unknown sequences had an average length of 1,716 bp.</p>", "links"=>[], "tags"=>["clone"], "article_id"=>521477, "categories"=>["Genetics", "Infectious Diseases"], "users"=>["Reginaldo M. Kuroshu", "Junichi Watanabe,", "Sumio Sugano", "Shinichi Morishita", "Yutaka Suzuki", "Masahiro Kasahara"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0010517.g003", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_clone_fate_for_library_1_and_1_2_/521477", "title"=>"The clone fate for library 1 and 1+2.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-05-07 00:24:37"}
  • {"files"=>["https://ndownloader.figshare.com/files/851320"], "description"=>"<p>Shotgun reads in library 1 and 1+2 were randomly sampled to simulate datasets of different sequence coverage. Each simulated dataset was then assembled using MuSICA 2, and the CDS structure consistency for the 199 reference full-length cDNA clones was evaluated. Full data corresponds to 100% sequence coverage. The definition of CDS structure consistency is exactly same as in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010517#pone-0010517-t002\" target=\"_blank\">Table 2</a>. Note that the purity filtered reads, not the raw reads, were used for input to MuSICA 2, which accounts for a slight difference between <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010517#pone-0010517-t002\" target=\"_blank\">Table 2</a> and the number of CDS-consistent/inconsistent clones at 100% sequence coverage.</p>", "links"=>[], "tags"=>["cds", "consistency"], "article_id"=>521779, "categories"=>["Genetics", "Infectious Diseases"], "users"=>["Reginaldo M. Kuroshu", "Junichi Watanabe,", "Sumio Sugano", "Shinichi Morishita", "Yutaka Suzuki", "Masahiro Kasahara"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0010517.t005", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Relationship_between_CDS_consistency_and_the_sequence_coverage_/521779", "title"=>"Relationship between CDS consistency and the sequence coverage.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2010-05-07 00:29:39"}
  • {"files"=>["https://ndownloader.figshare.com/files/850952"], "description"=>"<p>The reconstructed full-length cDNA is <i>consistent</i> with the reference gene if the exon-intron structure of the true CDS was completely contained in the exon-intron structure of the reference gene. <i>Inconsistency</i> was caused by different exon boundaries, sequence gaps or missing links between exons (i.e., introns). Sequence gaps were categorized into three types: (1) a gap in the middle of exons, (2) the 5′-end (or 3′-end) of the cDNA clone was missing, or (3) a link between two adjacent exons was missing. Note that in the case (3) we cannot exclude a possibility that some exon(s) between them is missing; therefore, the output transcript needs manual finishing in such a case.</p>", "links"=>[], "tags"=>["schematic", "cds"], "article_id"=>521409, "categories"=>["Genetics", "Infectious Diseases"], "users"=>["Reginaldo M. Kuroshu", "Junichi Watanabe,", "Sumio Sugano", "Shinichi Morishita", "Yutaka Suzuki", "Masahiro Kasahara"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0010517.g002", "stats"=>{"downloads"=>1, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_schematic_picture_of_CDS_consistency_/521409", "title"=>"The schematic picture of CDS consistency.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-05-07 00:23:29"}

PMC Usage Stats | Further Information

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