Performance of Ultra-Deep Pyrosequencing in Analysis of HIV-1 pol Gene Variation
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{"title"=>"Performance of Ultra-Deep pyrosequencing in analysis of HIV-1 pol gene variation", "type"=>"journal", "authors"=>[{"first_name"=>"Mattias", "last_name"=>"Mild", "scopus_author_id"=>"56908524200"}, {"first_name"=>"Charlotte", "last_name"=>"Hedskog", "scopus_author_id"=>"36607993800"}, {"first_name"=>"Johanna", "last_name"=>"Jernberg", "scopus_author_id"=>"36608295400"}, {"first_name"=>"Jan", "last_name"=>"Albert", "scopus_author_id"=>"7201985763"}], "year"=>2011, "source"=>"PLoS ONE", "identifiers"=>{"isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "doi"=>"10.1371/journal.pone.0022741", "pmid"=>"21799940", "issn"=>"19326203", "sgr"=>"79960714085", "pui"=>"362208638", "scopus"=>"2-s2.0-79960714085"}, "id"=>"08cb06f3-4865-33d8-8f46-ea96b40fb3bf", "abstract"=>"Ultra-deep pyrosequencing (UDPS) has been used to detect minority variants within HIV-1 populations. Some aspects of the quality and reproducibility of UDPS have been previously evaluated, but comprehensive studies are still needed.", "link"=>"http://www.mendeley.com/research/performance-ultradeep-pyrosequencing-analysis-hiv1-pol-gene-variation", "reader_count"=>36, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>1, "Researcher"=>13, "Student > Doctoral Student"=>4, "Student > Ph. D. Student"=>10, "Student > Master"=>6, "Other"=>1, "Lecturer"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>1, "Researcher"=>13, "Student > Doctoral Student"=>4, "Student > Ph. D. Student"=>10, "Student > Master"=>6, "Other"=>1, "Lecturer"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Environmental Science"=>1, "Biochemistry, Genetics and Molecular Biology"=>4, "Mathematics"=>1, "Agricultural and Biological Sciences"=>24, "Medicine and Dentistry"=>4, "Social Sciences"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>4}, "Social Sciences"=>{"Social Sciences"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>24}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>4}, "Mathematics"=>{"Mathematics"=>1}, "Unspecified"=>{"Unspecified"=>1}, "Environmental Science"=>{"Environmental Science"=>1}}, "reader_count_by_country"=>{"Canada"=>1, "Netherlands"=>1, "United States"=>1, "United Kingdom"=>2, "Switzerland"=>1}, "group_count"=>1}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/753267"], "description"=>"<p>The informative sites of clone 1 and clone 2 (see <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022741#pone-0022741-g001\" target=\"_blank\">Figure 1</a>) are shown in italics in the upper part of the figure. Below are identified recombinant reads presented for the experiment with 100,000 input template molecules (panel A) and 10,000 input template molecules (panel B). The proportion (Prop. %) of each recombinant is also shown.</p>", "links"=>[], "tags"=>["pcr"], "article_id"=>423643, "categories"=>["Genetics", "Infectious Diseases"], "users"=>["Mattias Mild", "Charlotte Hedskog", "Johanna Jernberg", "Jan Albert"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0022741.g005", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Analysis_of_in_vitro_PCR_recombination_/423643", "title"=>"Analysis of <i>in vitro</i> PCR recombination.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-07-25 01:00:43"}
  • {"files"=>["https://ndownloader.figshare.com/files/753380"], "description"=>"<p>Characteristics of the samples and basic information on UDPS.</p>", "links"=>[], "tags"=>["samples"], "article_id"=>423756, "categories"=>["Genetics", "Infectious Diseases"], "users"=>["Mattias Mild", "Charlotte Hedskog", "Johanna Jernberg", "Jan Albert"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0022741.t001", "stats"=>{"downloads"=>1, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Characteristics_of_the_samples_and_basic_information_on_UDPS_/423756", "title"=>"Characteristics of the samples and basic information on UDPS.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2011-07-25 01:02:36"}
  • {"files"=>["https://ndownloader.figshare.com/files/753119"], "description"=>"<p>Templates for UDPS were prepared using original and alternative PCR primers. The number of reads in the second (alternative primers) measurement was weighted by the number of reads in the first measurement. The number of reads per variant was log transformed. The differences in number of reads per variant using the two primer sets are plotted against the average number of reads per variant. Horizontal lines are drawn at the mean difference between the two measurements and at the upper and lower limits of agreement. The 95% confidence intervals are also shown for the mean and the upper and lower limits of agreement.</p>", "links"=>[], "tags"=>["variant", "quantification", "primer"], "article_id"=>423494, "categories"=>["Genetics", "Infectious Diseases"], "users"=>["Mattias Mild", "Charlotte Hedskog", "Johanna Jernberg", "Jan Albert"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0022741.g004", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Bland_Altman_plot_showing_the_agreement_of_variant_quantification_using_two_primer_sets_/423494", "title"=>"Bland-Altman plot showing the agreement of variant quantification using two primer sets.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-07-25 00:58:14"}
  • {"files"=>["https://ndownloader.figshare.com/files/752816"], "description"=>"<p>The clones cover position 3093–3206 in HXB2 and were used for the mixing experiments. The clones differed by 13 informative sites that are highlighted in gray.</p>", "links"=>[], "tags"=>["alignment", "clone"], "article_id"=>423188, "categories"=>["Genetics", "Infectious Diseases"], "users"=>["Mattias Mild", "Charlotte Hedskog", "Johanna Jernberg", "Jan Albert"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0022741.g001", "stats"=>{"downloads"=>0, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Nucleotide_sequence_alignment_of_clone_1_and_clone_2_/423188", "title"=>"Nucleotide sequence alignment of clone 1 and clone 2.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-07-25 00:53:08"}
  • {"files"=>["https://ndownloader.figshare.com/files/753032"], "description"=>"<p>The data from the paired measurements from sample A and sample B were combined and the number of reads in the reverse direction was weighted by the number of reads in the forward direction. The number of reads per variant was log transformed. The differences in number of reads per variant in forward and reverse direction are plotted against the average number of reads per variant. Horizontal lines are drawn at the mean difference between the two measurements and at the upper and lower limits of agreement. The 95% confidence intervals are also shown for the mean and the upper and lower limits of agreement.</p>", "links"=>[], "tags"=>["variant", "quantification"], "article_id"=>423409, "categories"=>["Genetics", "Infectious Diseases"], "users"=>["Mattias Mild", "Charlotte Hedskog", "Johanna Jernberg", "Jan Albert"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0022741.g003", "stats"=>{"downloads"=>0, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Bland_Altman_plot_showing_the_agreement_of_variant_quantification_in_forward_and_reverse_direction_/423409", "title"=>"Bland-Altman plot showing the agreement of variant quantification in forward and reverse direction.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-07-25 00:56:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/752874"], "description"=>"<p>UDPS was performed twice for sample A and sample B. These paired measurements were combined and the number of reads in the second (repeat) measurement was weighted by the number of reads in the first measurement. The number of reads per variant was log transformed. The differences in number of reads per variant in the repeat analyses are plotted against the average number of reads per variant. Horizontal lines are drawn at the mean difference between the two measurements and at the upper and lower limits of agreement. The 95% confidence intervals are also shown for the mean and the upper and lower limits of agreement.</p>", "links"=>[], "tags"=>["repeatability", "variant", "quantification"], "article_id"=>423247, "categories"=>["Genetics", "Infectious Diseases"], "users"=>["Mattias Mild", "Charlotte Hedskog", "Johanna Jernberg", "Jan Albert"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0022741.g002", "stats"=>{"downloads"=>1, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Bland_Altman_plot_showing_the_repeatability_of_variant_quantification_using_UDPS_/423247", "title"=>"Bland-Altman plot showing the repeatability of variant quantification using UDPS.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-07-25 00:54:07"}
  • {"files"=>["https://ndownloader.figshare.com/files/753345"], "description"=>"<p>Limit of detection of repeatedly detected virus variants in samples analyzed using original primers and alternative PCR primers.</p>", "links"=>[], "tags"=>["detection", "variants", "samples", "primers", "pcr"], "article_id"=>423723, "categories"=>["Genetics", "Infectious Diseases"], "users"=>["Mattias Mild", "Charlotte Hedskog", "Johanna Jernberg", "Jan Albert"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0022741.t002", "stats"=>{"downloads"=>1, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Limit_of_detection_of_repeatedly_detected_virus_variants_in_samples_analyzed_using_original_primers_and_alternative_PCR_primers_/423723", "title"=>"Limit of detection of repeatedly detected virus variants in samples analyzed using original primers and alternative PCR primers.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2011-07-25 01:02:03"}
  • {"files"=>["https://ndownloader.figshare.com/files/379054", "https://ndownloader.figshare.com/files/379110"], "description"=>"<div><h3>Introduction</h3><p>Ultra-deep pyrosequencing (UDPS) has been used to detect minority variants within HIV-1 populations. Some aspects of the quality and reproducibility of UDPS have been previously evaluated, but comprehensive studies are still needed.</p> <h3>Principal Finding</h3><p>In this study the UDPS technology (FLX platform) was evaluated by analyzing a 120 base pair fragment of the HIV-1 <em>pol</em> gene from plasma samples from two patients and artificial mixtures of molecular clones. UDPS was performed using an optimized experimental protocol and an in-house data cleaning strategy. Nine samples and mixtures were analyzed and the average number of reads per sample was 19,404 (range 8,858–26,846). The two patient plasma samples were analyzed twice and quantification of viral variants was found to be highly repeatable for variants representing >0.27% of the virus population, whereas some variants representing 0.11–0.27% were detected in only one of the two UDPS runs. Bland-Altman analysis showed that a repeated measurement would have a 95% likelihood to lie approximately within ±0.5 log<sub>10</sub> of the initial estimate. A similar level of agreement was observed for variant frequency estimates in forward vs. reverse sequencing direction, but here the agreement was higher for common variants than for rare variants. UDPS following PCR amplification with alternative primers indicated that some variants may be incorrectly quantified due to primer-related selective amplification. Finally, the <em>in vitro</em> recombination rate during PCR was evaluated using artificial mixtures of clones and was found to be low. The most abundant <em>in vitro</em> recombinant represented 0.25% of all UDPS reads.</p> <h3>Conclusion</h3><p>This study demonstrates that this UDPS protocol results in low experimental noise and high repeatability, which is relevant for future research and clinical use of the UDPS technology. The low rate of <em>in vitro</em> recombination suggests that this UDPS system can be used to study genetic variants and mutational linkage.</p> </div>", "links"=>[], "tags"=>["ultra-deep", "pyrosequencing", "hiv-1"], "article_id"=>134904, "categories"=>["Cancer", "Genetics"], "users"=>["Mattias Mild", "Charlotte Hedskog", "Johanna Jernberg", "Jan Albert"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0022741.s001", "https://dx.doi.org/10.1371/journal.pone.0022741.s002"], "stats"=>{"downloads"=>2, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Performance_of_Ultra_Deep_Pyrosequencing_in_Analysis_of_HIV_1_pol_Gene_Variation/134904", "title"=>"Performance of Ultra-Deep Pyrosequencing in Analysis of HIV-1 <em>pol</em> Gene Variation", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2011-07-25 01:21:44"}

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Relative Metric

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