Work

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

Year: 2022

Type: preprint

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 (Pf) detection. The frequency of such deletions can be determined in publically available Pf whole genome sequencing (WGS) datasets. A computational approach was developed and validated, termed Gene Coverage Count and Classification (GC3), to analyze 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 genotype, 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 analyzed 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 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 analyzed 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 an increase in frequency of deletions between 2007-08 and 2014-15. 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 classifies hrp2/3 deletions and provided informative tables and figures to analyze targeted gene coverage. GC3 is an appropriate tool when performing preliminary and exploratory assessment of locus coverage data.

Source: Research Square

DOI: 10.21203/rs.3.rs-1862557/v1