Consistent Errors in First Strand cDNA Due to Random Hexamer Mispriming
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{"title"=>"Consistent errors in first strand cDNA due to random hexamer mispriming", "type"=>"journal", "authors"=>[{"first_name"=>"Thomas P.", "last_name"=>"Van Gurp", "scopus_author_id"=>"55085013800"}, {"first_name"=>"Lauren M.", "last_name"=>"McIntyre", "scopus_author_id"=>"7101992431"}, {"first_name"=>"Koen J F", "last_name"=>"Verhoeven", "scopus_author_id"=>"6603749681"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"sgr"=>"84893581526", "doi"=>"10.1371/journal.pone.0085583", "issn"=>"19326203", "pui"=>"372325117", "isbn"=>"1932-6203", "pmid"=>"24386481", "scopus"=>"2-s2.0-84893581526"}, "id"=>"94bbd1b1-7034-399e-b47e-d691218174af", "abstract"=>"Priming of random hexamers in cDNA synthesis is known to show sequence bias, but in addition it has been suggested recently that mismatches in random hexamer priming could be a cause of mismatches between the original RNA fragment and observed sequence reads. To explore random hexamer mispriming as a potential source of these errors, we analyzed two independently generated RNA-seq datasets of synthetic ERCC spikes for which the reference is known. First strand cDNA synthesized by random hexamer priming on RNA showed consistent position and nucleotide-specific mismatch errors in the first seven nucleotides. The mismatch errors found in both datasets are consistent in distribution and thermodynamically stable mismatches are more common. This strongly indicates that RNA-DNA mispriming of specific random hexamers causes these errors. Due to their consistency and specificity, mispriming errors can have profound implications for downstream applications if not dealt with properly.", "link"=>"http://www.mendeley.com/research/consistent-errors-first-strand-cdna-due-random-hexamer-mispriming", "reader_count"=>70, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Professor > Associate Professor"=>5, "Librarian"=>1, "Researcher"=>20, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>25, "Student > Postgraduate"=>1, "Other"=>6, "Student > Master"=>7, "Student > Bachelor"=>2}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Professor > Associate Professor"=>5, "Librarian"=>1, "Researcher"=>20, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>25, "Student > Postgraduate"=>1, "Other"=>6, "Student > Master"=>7, "Student > Bachelor"=>2}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Engineering"=>1, "Environmental Science"=>1, "Biochemistry, Genetics and Molecular Biology"=>11, "Agricultural and Biological Sciences"=>40, "Medicine and Dentistry"=>3, "Neuroscience"=>1, "Chemistry"=>2, "Computer Science"=>6, "Immunology and Microbiology"=>3, "Linguistics"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>3}, "Neuroscience"=>{"Neuroscience"=>1}, "Chemistry"=>{"Chemistry"=>2}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>3}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>40}, "Computer Science"=>{"Computer Science"=>6}, "Linguistics"=>{"Linguistics"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>11}, "Unspecified"=>{"Unspecified"=>1}, "Environmental Science"=>{"Environmental Science"=>1}}, "reader_count_by_country"=>{"Netherlands"=>1, "United States"=>4, "Denmark"=>1, "United Kingdom"=>1, "South Africa"=>1, "France"=>1}, "group_count"=>3}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1331823"], "description"=>"<p>For all 4 nucleotides present in RNA the distribution of mismatching nucleotides is shown at selected positions. Mismatch rates are highest for first strand cDNA reads starting with T or A. For position three mismatches are mostly due to RNA-U vs DNA-G mispriming. Per nucleotide mismatch distributions are highly variable for the first seven positions, whereas they are consistent from position 7 onwards.</p>", "links"=>[], "tags"=>["mismatch", "15th", "50th", "positions", "strand"], "article_id"=>888840, "categories"=>["Biological Sciences"], "users"=>["Thomas P. van Gurp", "Lauren M. McIntyre", "Koen J. F. Verhoeven"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085583.g002", "stats"=>{"downloads"=>1, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_mismatch_rate_and_distribution_for_the_first_third_15th_and_50th_positions_of_first_strand_cDNA_/888840", "title"=>"The mismatch rate and distribution for the first, third, 15th and 50th positions of first strand cDNA.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-30 02:56:59"}
  • {"files"=>["https://ndownloader.figshare.com/files/1331824"], "description"=>"<p>Mismatch error patterns observed in ERCC spike reads are correlated between independent data sets for read position 1-6 (panel A) but not for subsequent positions (position 7-20, panel B). For each position the distribution of errors over the 12 mispriming possibilites was determined (4 nucleotides x 3 mispriming options, summing to 100% per position) and plotted between the dandelion RNA-seq ERCC-dataset (see <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085583#pone-0085583-g001\" target=\"_blank\">Figure 1</a>) and ERCC RNA-seq data described in Jiang (2011; GSM517062). The correlation in error distributions between the two data sets shows consistency of specific mismatching errors only in the hexamer binding region (panel A).</p>", "links"=>[], "tags"=>[], "article_id"=>888841, "categories"=>["Biological Sciences"], "users"=>["Thomas P. van Gurp", "Lauren M. McIntyre", "Koen J. F. Verhoeven"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085583.g003", "stats"=>{"downloads"=>1, "page_views"=>20, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Mismatch_error_pattern_correlation_between_two_independent_datasets_/888841", "title"=>"Mismatch error pattern correlation between two independent datasets.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-30 02:56:59"}
  • {"files"=>["https://ndownloader.figshare.com/files/1331822"], "description"=>"<p>Sequence mismatch rates in first (RNA-DNA) and second (DNA-DNA) strand cDNA of reads mapping to ERCC sequences.</p>", "links"=>[], "tags"=>[], "article_id"=>888839, "categories"=>["Biological Sciences"], "users"=>["Thomas P. van Gurp", "Lauren M. McIntyre", "Koen J. F. Verhoeven"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085583.g001", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Read_position_effect_on_sequence_mismatches_/888839", "title"=>"Read position effect on sequence mismatches.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-30 02:56:59"}

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Relative Metric

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