Gene Coverage Count and Classification (GC3): a locus sequence coverage assessment tool using short-read whole genome sequencing data, and its application to identify and classify histidine-rich protein 2 and 3 deletions in Plasmodium falciparum
Abstract Background The ability of malaria rapid diagnostic tests (RDTs) to effectively detect active infections is being compromised by the presence of malaria strains with genomic deletions at the hrp2 and hrp3 loci, encoding the antigens most commonly targeted in diagnostics for Plasmodium falcip...
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| Format: | Article |
| Language: | English |
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BMC
2022-11-01
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| Series: | Malaria Journal |
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| Online Access: | https://doi.org/10.1186/s12936-022-04376-3 |
| _version_ | 1811315288137793536 |
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| author | Thomas C. Stabler Ankit Dwivedi Biraj Shrestha Sudhaunshu Joshi Tobias Schindler Amed Ouattara Guillermo A. García Claudia Daubenberger Joana C. Silva |
| author_facet | Thomas C. Stabler Ankit Dwivedi Biraj Shrestha Sudhaunshu Joshi Tobias Schindler Amed Ouattara Guillermo A. García Claudia Daubenberger Joana C. Silva |
| author_sort | Thomas C. Stabler |
| collection | DOAJ |
| description | Abstract Background The ability of malaria rapid diagnostic tests (RDTs) to effectively detect active infections is being compromised by the presence of malaria strains with genomic deletions at the hrp2 and hrp3 loci, encoding the antigens most commonly targeted in diagnostics for Plasmodium falciparum detection. The presence of such deletions can be determined in publically available P. falciparum whole genome sequencing (WGS) datasets. A computational approach was developed and validated, termed Gene Coverage Count and Classification (GC3), to analyse genome-wide sequence coverage data and provide informative outputs to assess presence and coverage profile of a target locus in WGS data. GC3 was applied to detect deletions at hrp2 and hrp3 (hrp2/3) and flanking genes in different geographic regions and across time points. Methods GC3 uses Python and R scripts to extract locus read coverage metrics from mapped WGS data according to user-defined parameters and generates relevant tables and figures. GC3 was tested using WGS data for laboratory reference strains with known hrp2/3 genotypes, and its results compared to those of a hrp2/3-specific qPCR assay. Samples with at least 25% of coding region positions with zero coverage were classified as having a deletion. Publicly available sequence data was analysed and compared with published deletion frequency estimates. Results GC3 results matched the expected coverage of known laboratory reference strains. Agreement between GC3 and a hrp2/3-specific qPCR assay reported for 19/19 (100%) hrp2 deletions and 18/19 (94.7%) hrp3 deletions. Among Cambodian (n = 127) and Brazilian (n = 20) WGS datasets, which had not been previously analysed for hrp2/3 deletions, GC3 identified hrp2 deletions in three and four samples, and hrp3 deletions in 10 and 15 samples, respectively. Plots of hrp2/3 coding regions, grouped by year of sample collection, showed a decrease in median standardized coverage among Malawian samples (n = 150) suggesting the importance of a careful, properly controlled follow up to determine if an increase in frequency of deletions has occurred between 2007–2008 and 2014–2015. Among Malian (n = 90) samples, median standardized coverage was lower in 2002 than 2010, indicating widespread deletions present at the gene locus in 2002. Conclusions The GC3 tool accurately classified hrp2/3 deletions and provided informative tables and figures to analyse targeted gene coverage. GC3 is an appropriate tool when performing preliminary and exploratory assessment of locus coverage data. |
| first_indexed | 2024-04-13T11:28:47Z |
| format | Article |
| id | doaj.art-7f04ab0869a14203a0550948bbf8e570 |
| institution | Directory Open Access Journal |
| issn | 1475-2875 |
| language | English |
| last_indexed | 2024-04-13T11:28:47Z |
| publishDate | 2022-11-01 |
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| spelling | doaj.art-7f04ab0869a14203a0550948bbf8e5702022-12-22T02:48:38ZengBMCMalaria Journal1475-28752022-11-0121111710.1186/s12936-022-04376-3Gene Coverage Count and Classification (GC3): a locus sequence coverage assessment tool using short-read whole genome sequencing data, and its application to identify and classify histidine-rich protein 2 and 3 deletions in Plasmodium falciparumThomas C. Stabler0Ankit Dwivedi1Biraj Shrestha2Sudhaunshu Joshi3Tobias Schindler4Amed Ouattara5Guillermo A. García6Claudia Daubenberger7Joana C. Silva8Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health InstituteInstitute for Genome Sciences, University of Maryland School of MedicineMalaria Research Program, Center for Vaccine Development and Global Health, University of Maryland School of MedicineMalaria Research Program, Center for Vaccine Development and Global Health, University of Maryland School of MedicineDepartment of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health InstituteMalaria Research Program, Center for Vaccine Development and Global Health, University of Maryland School of MedicineMedical Care Development InternationalDepartment of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health InstituteInstitute for Genome Sciences, University of Maryland School of MedicineAbstract Background The ability of malaria rapid diagnostic tests (RDTs) to effectively detect active infections is being compromised by the presence of malaria strains with genomic deletions at the hrp2 and hrp3 loci, encoding the antigens most commonly targeted in diagnostics for Plasmodium falciparum detection. The presence of such deletions can be determined in publically available P. falciparum whole genome sequencing (WGS) datasets. A computational approach was developed and validated, termed Gene Coverage Count and Classification (GC3), to analyse genome-wide sequence coverage data and provide informative outputs to assess presence and coverage profile of a target locus in WGS data. GC3 was applied to detect deletions at hrp2 and hrp3 (hrp2/3) and flanking genes in different geographic regions and across time points. Methods GC3 uses Python and R scripts to extract locus read coverage metrics from mapped WGS data according to user-defined parameters and generates relevant tables and figures. GC3 was tested using WGS data for laboratory reference strains with known hrp2/3 genotypes, and its results compared to those of a hrp2/3-specific qPCR assay. Samples with at least 25% of coding region positions with zero coverage were classified as having a deletion. Publicly available sequence data was analysed and compared with published deletion frequency estimates. Results GC3 results matched the expected coverage of known laboratory reference strains. Agreement between GC3 and a hrp2/3-specific qPCR assay reported for 19/19 (100%) hrp2 deletions and 18/19 (94.7%) hrp3 deletions. Among Cambodian (n = 127) and Brazilian (n = 20) WGS datasets, which had not been previously analysed for hrp2/3 deletions, GC3 identified hrp2 deletions in three and four samples, and hrp3 deletions in 10 and 15 samples, respectively. Plots of hrp2/3 coding regions, grouped by year of sample collection, showed a decrease in median standardized coverage among Malawian samples (n = 150) suggesting the importance of a careful, properly controlled follow up to determine if an increase in frequency of deletions has occurred between 2007–2008 and 2014–2015. Among Malian (n = 90) samples, median standardized coverage was lower in 2002 than 2010, indicating widespread deletions present at the gene locus in 2002. Conclusions The GC3 tool accurately classified hrp2/3 deletions and provided informative tables and figures to analyse targeted gene coverage. GC3 is an appropriate tool when performing preliminary and exploratory assessment of locus coverage data.https://doi.org/10.1186/s12936-022-04376-3MalariaRapid Diagnostic Testhrp2hrp3DeletionGene coverage |
| spellingShingle | Thomas C. Stabler Ankit Dwivedi Biraj Shrestha Sudhaunshu Joshi Tobias Schindler Amed Ouattara Guillermo A. García Claudia Daubenberger Joana C. Silva Gene Coverage Count and Classification (GC3): a locus sequence coverage assessment tool using short-read whole genome sequencing data, and its application to identify and classify histidine-rich protein 2 and 3 deletions in Plasmodium falciparum Malaria Journal Malaria Rapid Diagnostic Test hrp2 hrp3 Deletion Gene coverage |
| title | Gene Coverage Count and Classification (GC3): a locus sequence coverage assessment tool using short-read whole genome sequencing data, and its application to identify and classify histidine-rich protein 2 and 3 deletions in Plasmodium falciparum |
| title_full | Gene Coverage Count and Classification (GC3): a locus sequence coverage assessment tool using short-read whole genome sequencing data, and its application to identify and classify histidine-rich protein 2 and 3 deletions in Plasmodium falciparum |
| title_fullStr | Gene Coverage Count and Classification (GC3): a locus sequence coverage assessment tool using short-read whole genome sequencing data, and its application to identify and classify histidine-rich protein 2 and 3 deletions in Plasmodium falciparum |
| title_full_unstemmed | Gene Coverage Count and Classification (GC3): a locus sequence coverage assessment tool using short-read whole genome sequencing data, and its application to identify and classify histidine-rich protein 2 and 3 deletions in Plasmodium falciparum |
| title_short | Gene Coverage Count and Classification (GC3): a locus sequence coverage assessment tool using short-read whole genome sequencing data, and its application to identify and classify histidine-rich protein 2 and 3 deletions in Plasmodium falciparum |
| title_sort | gene coverage count and classification gc3 a locus sequence coverage assessment tool using short read whole genome sequencing data and its application to identify and classify histidine rich protein 2 and 3 deletions in plasmodium falciparum |
| topic | Malaria Rapid Diagnostic Test hrp2 hrp3 Deletion Gene coverage |
| url | https://doi.org/10.1186/s12936-022-04376-3 |
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