Improved Methods for Capture, Extraction, and Quantitative Assay of Environmental DNA from Asian Bigheaded Carp (Hypophthalmichthys spp.)
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{"title"=>"Improved methods for capture, extraction, and quantitative assay of environmental DNA from Asian bigheaded carp (hypophthalmichthys spp.)", "type"=>"journal", "authors"=>[{"first_name"=>"Cameron R.", "last_name"=>"Turner", "scopus_author_id"=>"25230535700"}, {"first_name"=>"Derryl J.", "last_name"=>"Miller", "scopus_author_id"=>"55257268600"}, {"first_name"=>"Kathryn J.", "last_name"=>"Coyne", "scopus_author_id"=>"7005117857"}, {"first_name"=>"Joel", "last_name"=>"Corush", "scopus_author_id"=>"35788719300"}], "year"=>2014, "source"=>"PLoS ONE", "identifiers"=>{"scopus"=>"2-s2.0-84916219881", "sgr"=>"84916219881", "issn"=>"19326203", "doi"=>"10.1371/journal.pone.0114329", "pmid"=>"25474207", "isbn"=>"0962-1083", "pui"=>"600684376"}, "id"=>"8a489e72-58ad-30ae-b7a9-af123c42195e", "abstract"=>"Indirect, non-invasive detection of rare aquatic macrofauna using aqueous environmental DNA (eDNA) is a relatively new approach to population and biodiversity monitoring. As such, the sensitivity of monitoring results to different methods of eDNA capture, extraction, and detection is being investigated in many ecosystems and species. One of the first and largest conservation programs with eDNA-based monitoring as a central instrument focuses on Asian bigheaded carp (Hypophthalmichthys spp.), an invasive fish spreading toward the Laurentian Great Lakes. However, the standard eDNA methods of this program have not advanced since their development in 2010. We developed new, quantitative, and more cost-effective methods and tested them against the standard protocols. In laboratory testing, our new quantitative PCR (qPCR) assay for bigheaded carp eDNA was one to two orders of magnitude more sensitive than the existing endpoint PCR assays. When applied to eDNA samples from an experimental pond containing bigheaded carp, the qPCR assay produced a detection probability of 94.8% compared to 4.2% for the endpoint PCR assays. Also, the eDNA capture and extraction method we adapted from aquatic microbiology yielded five times more bigheaded carp eDNA from the experimental pond than the standard method, at a per sample cost over forty times lower. Our new, more sensitive assay provides a quantitative tool for eDNA-based monitoring of bigheaded carp, and the higher-yielding eDNA capture and extraction method we describe can be used for eDNA-based monitoring of any aquatic species.", "link"=>"http://www.mendeley.com/research/improved-methods-capture-extraction-quantitative-assay-environmental-dna-asian-bigheaded-carp-hypoph", "reader_count"=>119, "reader_count_by_academic_status"=>{"Unspecified"=>5, "Professor > Associate Professor"=>2, "Researcher"=>23, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>19, "Student > Postgraduate"=>7, "Student > Master"=>22, "Other"=>12, "Student > Bachelor"=>17, "Lecturer"=>4, "Lecturer > Senior Lecturer"=>1, "Professor"=>4}, "reader_count_by_user_role"=>{"Unspecified"=>5, "Professor > Associate Professor"=>2, "Researcher"=>23, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>19, "Student > Postgraduate"=>7, "Student > Master"=>22, "Other"=>12, "Student > Bachelor"=>17, "Lecturer"=>4, "Lecturer > Senior Lecturer"=>1, "Professor"=>4}, "reader_count_by_subject_area"=>{"Unspecified"=>10, "Engineering"=>1, "Environmental Science"=>22, "Biochemistry, Genetics and Molecular Biology"=>18, "Agricultural and Biological Sciences"=>64, "Medicine and Dentistry"=>1, "Pharmacology, Toxicology and Pharmaceutical Science"=>1, "Psychology"=>1, "Earth and Planetary Sciences"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Psychology"=>{"Psychology"=>1}, "Earth and Planetary Sciences"=>{"Earth and Planetary Sciences"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>64}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>18}, "Unspecified"=>{"Unspecified"=>10}, "Environmental Science"=>{"Environmental Science"=>22}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>1}}, "reader_count_by_country"=>{"Canada"=>1, "Netherlands"=>1, "Hungary"=>1, "United States"=>5, "Brazil"=>2, "Germany"=>1}, "group_count"=>10}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1821950"], "description"=>"<p>Note that the two slightly different versions of the forward and reverse primers reflect three positions (shown in bold text) that vary within bigheaded carp, according to all available GenBank sequences. The “Hm” version of a primer matches all <i>H. molitrix</i> sequences while the “Hn” version matches all <i>H. nobilis</i> sequences in GenBank. Rather than ordering primers with degenerate bases, which would create unnecessary combinations of the two variable positions in the reverse primer, we simply ordered each version separately and combined them at equimolar concentration. The Hm and Hn variants do not provide discrimination between <i>H. molitrix</i> and <i>H. nobilis</i>. Rather, they are used in combination to maximize sensitivity within the genus while avoiding amplification from any non-<i>Hypophthalmichthys</i> species. 6FAM = fluorescein amidite reporter. MGB = minor groove binding moiety. NFQ = non-fluorescent quencher. BHQ1 = black hole quencher. +N = locked nucleic acid (LNA).</p><p>qPCR oligonucleotides developed for this study to produce a genus-specific assay targeting a 100 bp section of the D-loop mtDNA region in bigheaded carp (genus <i>Hypophthalmichthys</i>: <i>H. molitrix</i> and <i>H. nobilis</i>).</p>", "links"=>[], "tags"=>["monitoring", "Asian Bigheaded Carp", "detection", "edna", "dna", "bigheaded", "endpoint PCR assays", "extraction method", "Laurentian Great Lakes"], "article_id"=>1260429, "categories"=>["Biological Sciences"], "users"=>["Cameron R. Turner", "Derryl J. Miller", "Kathryn J. Coyne", "Joel Corush"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0114329.t001", "stats"=>{"downloads"=>2, "page_views"=>14, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_qPCR_oligonucleotides_developed_for_this_study_to_produce_a_genus_specific_assay_targeting_a_100_bp_section_of_the_D_loop_mtDNA_region_in_bigheaded_carp_genus_Hypophthalmichthys_H_molitrix_and_H_nobilis_/1260429", "title"=>"qPCR oligonucleotides developed for this study to produce a genus-specific assay targeting a 100 bp section of the D-loop mtDNA region in bigheaded carp (genus <i>Hypophthalmichthys</i>: <i>H. molitrix</i> and <i>H. nobilis</i>).", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-12-04 03:14:53"}
  • {"files"=>["https://ndownloader.figshare.com/files/1821949"], "description"=>"<p>48 pairs of samples were collected, but following Zuur et al. <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114329#pone.0114329-Zuur1\" target=\"_blank\">[88]</a>, seven pairs with unusually high eDNA concentration (i.e., outliers) were removed prior to statistical analysis. Statistical results were robust to outlier presence or removal, and plots including outliers are provided in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114329#pone.0114329.s001\" target=\"_blank\">Figure S1</a>. PCTE = polycarbonate track-etched filter membrane, GF = glass fiber filter paper, CTAB = cetyl trimethyl ammonium bromide DNA extraction protocol, PowerWater = PowerWater DNA Isolation Kit. (A) Paired data, (B) boxplot, (C) paired differences and the median difference (red point) and 95% confidence interval (red interval) from the Wilcoxon signed-rank test. Note that points in (C) are horizontally ‘jittered’ for better visualization. The PCTE/CTAB method yielded significantly more eDNA than the GF/PowerWater method (paired Student’s t-test, t = 4.1, df = 40, P = 0.00019).</p>", "links"=>[], "tags"=>["monitoring", "Asian Bigheaded Carp", "detection", "edna", "dna", "bigheaded", "endpoint PCR assays", "extraction method", "Laurentian Great Lakes"], "article_id"=>1260428, "categories"=>["Biological Sciences"], "users"=>["Cameron R. Turner", "Derryl J. Miller", "Kathryn J. Coyne", "Joel Corush"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0114329.g002", "stats"=>{"downloads"=>1, "page_views"=>20, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_qPCR_assay_results_comparing_the_amount_of_bigheaded_carp_eDNA_captured_and_recovered_i_e_eDNA_yield_using_two_alternative_capture_extraction_methods_on_paired_2_L_samples_collected_side_by_side_in_the_experimental_pond_at_USGS_CERC_/1260428", "title"=>"qPCR assay results comparing the amount of bigheaded carp eDNA captured and recovered (i.e., eDNA yield) using two alternative capture/extraction methods on paired 2 L samples collected side-by-side in the experimental pond at USGS-CERC.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-12-04 03:14:53"}
  • {"files"=>["https://ndownloader.figshare.com/files/1821944"], "description"=>"<p>The qPCR assay was developed in the present study and the endpoint PCR assays are from a previous study <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114329#pone.0114329-Jerde2\" target=\"_blank\">[40]</a>. Gaps (−) and bases in between oligonucleotides (… n bp…) reflect the actual alignment used to design the qPCR assay. Note that all oligonucleotides are shown on the ‘sense’ strand in 5′ to 3′ orientation, left to right. Thus oligonucleotides that actually bind to the sense strand (i.e., reverse primers and the hydrolysis probe) are shown as reverse-complements of the actual oligonucleotides used in an assay (<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114329#pone-0114329-t001\" target=\"_blank\">Table 1</a>). Degenerate bases are shown in bold text, but oligonucleotides were not synthesized with degenerate bases for the qPCR assay (<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114329#pone-0114329-t001\" target=\"_blank\">Table 1</a>). The forward primer of the Silver Carp endpoint PCR assay was synthesized with degenerate bases, as specified in the study that developed it <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114329#pone.0114329-Jerde2\" target=\"_blank\">[40]</a>.</p>", "links"=>[], "tags"=>["monitoring", "Asian Bigheaded Carp", "detection", "edna", "dna", "bigheaded", "endpoint PCR assays", "extraction method", "Laurentian Great Lakes"], "article_id"=>1260423, "categories"=>["Biological Sciences"], "users"=>["Cameron R. Turner", "Derryl J. Miller", "Kathryn J. Coyne", "Joel Corush"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0114329.g001", "stats"=>{"downloads"=>2, "page_views"=>18, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Alignment_diagram_showing_the_relative_position_of_oligonucleotides_for_the_qPCR_and_endpoint_PCR_assays_/1260423", "title"=>"Alignment diagram showing the relative position of oligonucleotides for the qPCR and endpoint PCR assays.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-12-04 03:14:53"}
  • {"files"=>["https://ndownloader.figshare.com/files/1821966", "https://ndownloader.figshare.com/files/1821967", "https://ndownloader.figshare.com/files/1821968"], "description"=>"<div><p>Indirect, non-invasive detection of rare aquatic macrofauna using aqueous environmental DNA (eDNA) is a relatively new approach to population and biodiversity monitoring. As such, the sensitivity of monitoring results to different methods of eDNA capture, extraction, and detection is being investigated in many ecosystems and species. One of the first and largest conservation programs with eDNA-based monitoring as a central instrument focuses on Asian bigheaded carp (<i>Hypophthalmichthys</i> spp.), an invasive fish spreading toward the Laurentian Great Lakes. However, the standard eDNA methods of this program have not advanced since their development in 2010. We developed new, quantitative, and more cost-effective methods and tested them against the standard protocols. In laboratory testing, our new quantitative PCR (qPCR) assay for bigheaded carp eDNA was one to two orders of magnitude more sensitive than the existing endpoint PCR assays. When applied to eDNA samples from an experimental pond containing bigheaded carp, the qPCR assay produced a detection probability of 94.8% compared to 4.2% for the endpoint PCR assays. Also, the eDNA capture and extraction method we adapted from aquatic microbiology yielded five times more bigheaded carp eDNA from the experimental pond than the standard method, at a per sample cost over forty times lower. Our new, more sensitive assay provides a quantitative tool for eDNA-based monitoring of bigheaded carp, and the higher-yielding eDNA capture and extraction method we describe can be used for eDNA-based monitoring of any aquatic species.</p></div>", "links"=>[], "tags"=>["monitoring", "Asian Bigheaded Carp", "detection", "edna", "dna", "bigheaded", "endpoint PCR assays", "extraction method", "Laurentian Great Lakes"], "article_id"=>1260436, "categories"=>["Biological Sciences"], "users"=>["Cameron R. Turner", "Derryl J. Miller", "Kathryn J. Coyne", "Joel Corush"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0114329.s001", "https://dx.doi.org/10.1371/journal.pone.0114329.s002", "https://dx.doi.org/10.1371/journal.pone.0114329.s003"], "stats"=>{"downloads"=>8, "page_views"=>21, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Improved_Methods_for_Capture_Extraction_and_Quantitative_Assay_of_Environmental_DNA_from_Asian_Bigheaded_Carp_Hypophthalmichthys_spp_/1260436", "title"=>"Improved Methods for Capture, Extraction, and Quantitative Assay of Environmental DNA from Asian Bigheaded Carp (<i>Hypophthalmichthys</i> spp.)", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2014-12-04 03:14:53"}
  • {"files"=>["https://ndownloader.figshare.com/files/1821952"], "description"=>"<p>PCTE = polycarbonate track-etched filter membrane. GF = glass fiber filter paper. CTAB = cetyl trimethyl ammonium bromide DNA extraction protocol. PowerWater = PowerWater DNA Isolation Kit, MO BIO Laboratories.</p><p>Detection probabilities of different methods for eDNA-based detection of bigheaded carp presence in an experimental pond containing six bigheaded carp.</p>", "links"=>[], "tags"=>["monitoring", "Asian Bigheaded Carp", "detection", "edna", "dna", "bigheaded", "endpoint PCR assays", "extraction method", "Laurentian Great Lakes"], "article_id"=>1260431, "categories"=>["Biological Sciences"], "users"=>["Cameron R. Turner", "Derryl J. Miller", "Kathryn J. Coyne", "Joel Corush"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0114329.t002", "stats"=>{"downloads"=>0, "page_views"=>20, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Detection_probabilities_of_different_methods_for_eDNA_based_detection_of_bigheaded_carp_presence_in_an_experimental_pond_containing_six_bigheaded_carp_/1260431", "title"=>"Detection probabilities of different methods for eDNA-based detection of bigheaded carp presence in an experimental pond containing six bigheaded carp.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-12-04 03:14:53"}
  • {"files"=>["https://ndownloader.figshare.com/files/1821953"], "description"=>"<p>*The latest Cq observed was in cycle 41.</p><p>Comparison of existing and new protocols for eDNA-based monitoring of bigheaded carp.</p>", "links"=>[], "tags"=>["monitoring", "Asian Bigheaded Carp", "detection", "edna", "dna", "bigheaded", "endpoint PCR assays", "extraction method", "Laurentian Great Lakes"], "article_id"=>1260432, "categories"=>["Biological Sciences"], "users"=>["Cameron R. Turner", "Derryl J. Miller", "Kathryn J. Coyne", "Joel Corush"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0114329.t003", "stats"=>{"downloads"=>0, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Comparison_of_existing_and_new_protocols_for_eDNA_based_monitoring_of_bigheaded_carp_/1260432", "title"=>"Comparison of existing and new protocols for eDNA-based monitoring of bigheaded carp.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-12-04 03:14:53"}

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Relative Metric

{"start_date"=>"2014-01-01T00:00:00Z", "end_date"=>"2014-12-31T00:00:00Z", "subject_areas"=>[{"subject_area"=>"/Biology and life sciences/Molecular biology", "average_usage"=>[292, 461]}, {"subject_area"=>"/Earth sciences/Marine and aquatic sciences", "average_usage"=>[343]}]}
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