Low-Cost Ultra-Wide Genotyping Using Roche/454 Pyrosequencing for Surveillance of HIV Drug Resistance
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{"title"=>"Low-cost ultra-wide genotyping using roche/454 pyrosequencing for surveillance of HIV drug resistance", "type"=>"journal", "authors"=>[{"first_name"=>"Dawn M.", "last_name"=>"Dudley", "scopus_author_id"=>"26532672700"}, {"first_name"=>"Emily N.", "last_name"=>"Chin", "scopus_author_id"=>"57196833237"}, {"first_name"=>"Benjamin N.", "last_name"=>"Bimber", "scopus_author_id"=>"14053443700"}, {"first_name"=>"Sabri S.", "last_name"=>"Sanabani", "scopus_author_id"=>"8290541600"}, {"first_name"=>"Leandro F.", "last_name"=>"Tarosso", "scopus_author_id"=>"24825390600"}, {"first_name"=>"Priscilla R.", "last_name"=>"Costa", "scopus_author_id"=>"16240934500"}, {"first_name"=>"Mariana M.", "last_name"=>"Sauer", "scopus_author_id"=>"7202098162"}, {"first_name"=>"Esper G.", "last_name"=>"Kallas", "scopus_author_id"=>"35499569700"}, {"first_name"=>"David H.", "last_name"=>"O'Connor", "scopus_author_id"=>"7401496996"}], "year"=>2012, "source"=>"PLoS ONE", "identifiers"=>{"sgr"=>"84860521943", "doi"=>"10.1371/journal.pone.0036494", "issn"=>"19326203", "pui"=>"364730708", "isbn"=>"1932-6203", "pmid"=>"22574170", "scopus"=>"2-s2.0-84860521943"}, "id"=>"7cdc1aca-fb33-3d65-8b8e-2cfdbcc4d07a", "abstract"=>"BACKGROUND: Great efforts have been made to increase accessibility of HIV antiretroviral therapy (ART) in low and middle-income countries. The threat of wide-scale emergence of drug resistance could severely hamper ART scale-up efforts. Population-based surveillance of transmitted HIV drug resistance ensures the use of appropriate first-line regimens to maximize efficacy of ART programs where drug options are limited. However, traditional HIV genotyping is extremely expensive, providing a cost barrier to wide-scale and frequent HIV drug resistance surveillance.\\n\\nMETHODS/RESULTS: We have developed a low-cost laboratory-scale next-generation sequencing-based genotyping method to monitor drug resistance. We designed primers specifically to amplify protease and reverse transcriptase from Brazilian HIV subtypes and developed a multiplexing scheme using multiplex identifier tags to minimize cost while providing more robust data than traditional genotyping techniques. Using this approach, we characterized drug resistance from plasma in 81 HIV infected individuals collected in São Paulo, Brazil. We describe the complexities of analyzing next-generation sequencing data and present a simplified open-source workflow to analyze drug resistance data. From this data, we identified drug resistance mutations in 20% of treatment naïve individuals in our cohort, which is similar to frequencies identified using traditional genotyping in Brazilian patient samples.\\n\\nCONCLUSION: The developed ultra-wide sequencing approach described here allows multiplexing of at least 48 patient samples per sequencing run, 4 times more than the current genotyping method. This method is also 4-fold more sensitive (5% minimal detection frequency vs. 20%) at a cost 3-5× less than the traditional Sanger-based genotyping method. Lastly, by using a benchtop next-generation sequencer (Roche/454 GS Junior), this approach can be more easily implemented in low-resource settings. This data provides proof-of-concept that next-generation HIV drug resistance genotyping is a feasible and low-cost alternative to current genotyping methods and may be particularly beneficial for in-country surveillance of transmitted drug resistance.", "link"=>"http://www.mendeley.com/research/lowcost-ultrawide-genotyping-using-roche454-pyrosequencing-surveillance-hiv-drug-resistance", "reader_count"=>81, "reader_count_by_academic_status"=>{"Unspecified"=>2, "Professor > Associate Professor"=>6, "Researcher"=>20, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>18, "Student > Postgraduate"=>5, "Student > Master"=>15, "Other"=>5, "Student > Bachelor"=>5, "Professor"=>2}, "reader_count_by_user_role"=>{"Unspecified"=>2, "Professor > Associate Professor"=>6, "Researcher"=>20, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>18, "Student > Postgraduate"=>5, "Student > Master"=>15, "Other"=>5, "Student > Bachelor"=>5, "Professor"=>2}, "reader_count_by_subject_area"=>{"Unspecified"=>5, "Agricultural and Biological Sciences"=>37, "Arts and Humanities"=>1, "Computer Science"=>2, "Economics, Econometrics and Finance"=>1, "Engineering"=>2, "Environmental Science"=>1, "Biochemistry, Genetics and Molecular Biology"=>5, "Mathematics"=>1, "Medicine and Dentistry"=>16, "Pharmacology, Toxicology and Pharmaceutical Science"=>1, "Social Sciences"=>2, "Immunology and Microbiology"=>7}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>16}, "Social Sciences"=>{"Social Sciences"=>2}, "Mathematics"=>{"Mathematics"=>1}, "Unspecified"=>{"Unspecified"=>5}, "Environmental Science"=>{"Environmental Science"=>1}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>1}, "Arts and Humanities"=>{"Arts and Humanities"=>1}, "Engineering"=>{"Engineering"=>2}, "Economics, Econometrics and Finance"=>{"Economics, Econometrics and Finance"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>7}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>37}, "Computer Science"=>{"Computer Science"=>2}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>5}}, "reader_count_by_country"=>{"Belgium"=>1, "United States"=>3, "Brazil"=>1, "United Kingdom"=>1, "Chile"=>1, "Switzerland"=>1, "Germany"=>1}, "group_count"=>2}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/642581"], "description"=>"<p>The number of sequences representing each nucleotide position (coverage) from three patient samples sequenced in the same GS Junior run is shown after alignment to the NC_001802 HXB2 HIV reference sequence. Also shown is the number of sequences that align to each nucleotide position after eliminating sequences with low quality scores (adjusted coverage) at each nucleotide position for each patient sample. Pyrosequencing was performed on three overlapping amplicons with nucleotide positions for each amplicon represented at the top of the graph.</p>", "links"=>[], "tags"=>["adjusted"], "article_id"=>313069, "categories"=>["Virology", "Biological Sciences", "Infectious Diseases"], "users"=>["Dawn M. Dudley", "Emily N. Chin", "Benjamin N. Bimber", "Sabri S. Sanabani", "Leandro F. Tarosso", "Priscilla R. Costa", "Mariana M. Sauer", "Esper G. Kallas", "David H. O.’Connor"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036494.g003", "stats"=>{"downloads"=>1, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Sequence_coverage_and_adjusted_sequence_coverage_of_three_representative_patient_samples_/313069", "title"=>"Sequence coverage and adjusted sequence coverage of three representative patient samples.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-05-04 00:51:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/642764"], "description"=>"1<p>Codons representing the mutated amino acid are shown in boldface.</p>", "links"=>[], "tags"=>["mutations", "located", "adjacent", "homopolymers", "hxb2", "hiv", "subtype"], "article_id"=>313246, "categories"=>["Virology", "Biological Sciences", "Infectious Diseases"], "users"=>["Dawn M. Dudley", "Emily N. Chin", "Benjamin N. Bimber", "Sabri S. Sanabani", "Leandro F. Tarosso", "Priscilla R. Costa", "Mariana M. Sauer", "Esper G. Kallas", "David H. O.’Connor"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036494.t004", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Drug_resistance_mutations_located_within_or_adjacent_to_homopolymers_in_the_HXB2_HIV_subtype_B_reference_sequence_/313246", "title"=>"Drug resistance mutations located within or adjacent to homopolymers in the HXB2 HIV subtype B reference sequence.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-05-04 00:54:06"}
  • {"files"=>["https://ndownloader.figshare.com/files/642406"], "description"=>"<p>A.) Plasma is isolated from 48 patient samples using centrifugation. B.) Viral RNA is extracted from ∼1 ml of plasma from each sample. C.) One-step RT-PCR is used to reverse-transcribe and PCR-amplify 3 amplicons spanning the HIV pol gene from each sample as shown. Each sample is amplified with primers containing a unique multiplex identifier (MID) tag (1–48). D.) PCR products are gel purified and purified further using size exclusion magnetic beads. E.) Purified samples are quantitated and pooled together at equimolar ratios for a total of 144 amplicons/pool. F.) Each pool is subjected to emPCR followed by pyrosequencing on the Roche/454 GS Junior.</p>", "links"=>[], "tags"=>["ultra-wide", "hiv", "genotyping"], "article_id"=>312891, "categories"=>["Virology", "Biological Sciences", "Infectious Diseases"], "users"=>["Dawn M. Dudley", "Emily N. Chin", "Benjamin N. Bimber", "Sabri S. Sanabani", "Leandro F. Tarosso", "Priscilla R. Costa", "Mariana M. Sauer", "Esper G. Kallas", "David H. O.’Connor"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036494.g001", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Schematic_representation_of_the_sample_preparation_for_ultra_wide_HIV_drug_resistance_genotyping_using_Roche_454_pyrosequencing_/312891", "title"=>"Schematic representation of the sample preparation for ultra-wide HIV drug resistance genotyping using Roche/454 pyrosequencing.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-05-04 00:48:11"}
  • {"files"=>["https://ndownloader.figshare.com/files/642639"], "description"=>"<p>The statistics of the two GS Junior runs used to assess the patient samples in this study.</p>", "links"=>[], "tags"=>["gs", "runs", "samples"], "article_id"=>313126, "categories"=>["Virology", "Biological Sciences", "Infectious Diseases"], "users"=>["Dawn M. Dudley", "Emily N. Chin", "Benjamin N. Bimber", "Sabri S. Sanabani", "Leandro F. Tarosso", "Priscilla R. Costa", "Mariana M. Sauer", "Esper G. Kallas", "David H. O.’Connor"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036494.t002", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_statistics_of_the_two_GS_Junior_runs_used_to_assess_the_patient_samples_in_this_study_/313126", "title"=>"The statistics of the two GS Junior runs used to assess the patient samples in this study.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-05-04 00:52:06"}
  • {"files"=>["https://ndownloader.figshare.com/files/331397", "https://ndownloader.figshare.com/files/331438", "https://ndownloader.figshare.com/files/331456"], "description"=>"<div><h3>Background</h3><p>Great efforts have been made to increase accessibility of HIV antiretroviral therapy (ART) in low and middle-income countries. The threat of wide-scale emergence of drug resistance could severely hamper ART scale-up efforts. Population-based surveillance of transmitted HIV drug resistance ensures the use of appropriate first-line regimens to maximize efficacy of ART programs where drug options are limited. However, traditional HIV genotyping is extremely expensive, providing a cost barrier to wide-scale and frequent HIV drug resistance surveillance.</p> <h3>Methods/Results</h3><p>We have developed a low-cost laboratory-scale next-generation sequencing-based genotyping method to monitor drug resistance. We designed primers specifically to amplify protease and reverse transcriptase from Brazilian HIV subtypes and developed a multiplexing scheme using multiplex identifier tags to minimize cost while providing more robust data than traditional genotyping techniques. Using this approach, we characterized drug resistance from plasma in 81 HIV infected individuals collected in São Paulo, Brazil. We describe the complexities of analyzing next-generation sequencing data and present a simplified open-source workflow to analyze drug resistance data. From this data, we identified drug resistance mutations in 20% of treatment naïve individuals in our cohort, which is similar to frequencies identified using traditional genotyping in Brazilian patient samples.</p> <h3>Conclusion</h3><p>The developed ultra-wide sequencing approach described here allows multiplexing of at least 48 patient samples per sequencing run, 4 times more than the current genotyping method. This method is also 4-fold more sensitive (5% minimal detection frequency vs. 20%) at a cost 3–5× less than the traditional Sanger-based genotyping method. Lastly, by using a benchtop next-generation sequencer (Roche/454 GS Junior), this approach can be more easily implemented in low-resource settings. This data provides proof-of-concept that next-generation HIV drug resistance genotyping is a feasible and low-cost alternative to current genotyping methods and may be particularly beneficial for in-country surveillance of transmitted drug resistance.</p> </div>", "links"=>[], "tags"=>["low-cost", "ultra-wide", "genotyping", "pyrosequencing", "hiv"], "article_id"=>125423, "categories"=>["Biological Sciences", "Cancer"], "users"=>["Dawn M. Dudley", "Emily N. Chin", "Benjamin N. Bimber", "Sabri S. Sanabani", "Leandro F. Tarosso", "Priscilla R. Costa", "Mariana M. Sauer", "Esper G. Kallas", "David H. O.’Connor"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0036494.s001", "https://dx.doi.org/10.1371/journal.pone.0036494.s002", "https://dx.doi.org/10.1371/journal.pone.0036494.s003"], "stats"=>{"downloads"=>3, "page_views"=>22, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Low_Cost_Ultra_Wide_Genotyping_Using_Roche_454_Pyrosequencing_for_Surveillance_of_HIV_Drug_Resistance/125423", "title"=>"Low-Cost Ultra-Wide Genotyping Using Roche/454 Pyrosequencing for Surveillance of HIV Drug Resistance", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2012-05-04 01:30:23"}
  • {"files"=>["https://ndownloader.figshare.com/files/642680"], "description"=>"<p>Drug resistance mutations identified in the patient samples sequenced using the Roche/454 GS Junior HIV drug resistance genotyping method.</p>", "links"=>[], "tags"=>["mutations", "samples", "sequenced", "gs", "hiv", "genotyping"], "article_id"=>313163, "categories"=>["Virology", "Biological Sciences", "Infectious Diseases"], "users"=>["Dawn M. Dudley", "Emily N. Chin", "Benjamin N. Bimber", "Sabri S. Sanabani", "Leandro F. Tarosso", "Priscilla R. Costa", "Mariana M. Sauer", "Esper G. Kallas", "David H. O.’Connor"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036494.t003", "stats"=>{"downloads"=>1, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Drug_resistance_mutations_identified_in_the_patient_samples_sequenced_using_the_Roche_454_GS_Junior_HIV_drug_resistance_genotyping_method_/313163", "title"=>"Drug resistance mutations identified in the patient samples sequenced using the Roche/454 GS Junior HIV drug resistance genotyping method.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-05-04 00:52:43"}
  • {"files"=>["https://ndownloader.figshare.com/files/642721"], "description"=>"1<p>Plasmid 1 and 2 refer to data from two independent experiments used to sequence the same HXBn plasmid. Virus refers to the data from sequencing a viral stock derived from the HXBn plasmid.</p>", "links"=>[], "tags"=>["mutations", "hxb2", "plasmid", "viral"], "article_id"=>313207, "categories"=>["Virology", "Biological Sciences", "Infectious Diseases"], "users"=>["Dawn M. Dudley", "Emily N. Chin", "Benjamin N. Bimber", "Sabri S. Sanabani", "Leandro F. Tarosso", "Priscilla R. Costa", "Mariana M. Sauer", "Esper G. Kallas", "David H. O.’Connor"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036494.t001", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_8220_False_8221_drug_resistance_mutations_found_in_the_HXB2_plasmid_or_HXB2_viral_stock_/313207", "title"=>"“False” drug resistance mutations found in the HXB2 plasmid or HXB2 viral stock.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-05-04 00:53:27"}
  • {"files"=>["https://ndownloader.figshare.com/files/642502"], "description"=>"<p>The number of sequences from the clonal viral stock (red) or HIV plasmid (blue and black) that aligned to each nucleotide position of the NC_001802 HXB2 HIV reference sequence is shown across the <i>pol</i> gene. The HXB2 vRNA clonal viral stock was sequenced under one GS Junior sequencing run, while two independent PCR amplifications were used to sequence the plasmid under two different MID tags in a separate GS Junior sequencing run. Pyrosequencing was performed on three overlapping amplicons with nucleotide positions for each amplicon represented at the top of the graph.</p>", "links"=>[], "tags"=>["amplicons", "clonal", "hxb2", "viral"], "article_id"=>312978, "categories"=>["Virology", "Biological Sciences", "Infectious Diseases"], "users"=>["Dawn M. Dudley", "Emily N. Chin", "Benjamin N. Bimber", "Sabri S. Sanabani", "Leandro F. Tarosso", "Priscilla R. Costa", "Mariana M. Sauer", "Esper G. Kallas", "David H. O.’Connor"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036494.g002", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Sequence_coverage_of_three_amplicons_from_a_clonal_HXB2_viral_stock_and_HXB2_plasmid_/312978", "title"=>"Sequence coverage of three amplicons from a clonal HXB2 viral stock and HXB2 plasmid.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-05-04 00:49:38"}

PMC Usage Stats | Further Information

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Relative Metric

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