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Selection and exploitation of prevalent, tandemly repeated genomic targets for improved real-time PCR-based detection of Wuchereria bancrofti and Plasmodium falciparum in mosquitoes

Citation

Zulch, Michael et al. (2020), Selection and exploitation of prevalent, tandemly repeated genomic targets for improved real-time PCR-based detection of Wuchereria bancrofti and Plasmodium falciparum in mosquitoes, Dryad, Dataset, https://doi.org/10.5061/dryad.d7wm37pxz

Abstract

Background

Optimization of polymerase chain reaction (PCR)-based diagnostics requires the careful selection of molecular targets that are both highly repetitive and pathogen-specific.  Advances in both next-generation sequencing (NGS) technologies and bioinformatics-based analysis tools are facilitating this selection process, informing target choices and reducing labor.  Once developed, such assays provide disease control and elimination programs with an additional set of tools capable of evaluating and monitoring intervention successes.  The importance of such tools is heightened as intervention efforts approach their endpoints, as accurate and complete information is an essential component of the informed decision-making process.  As global efforts for the control and elimination of both lymphatic filariasis and malaria continue to make significant gains, the benefits of diagnostics with improved analytical and clinical/field-based sensitivities and specificities will become increasingly apparent.

Methodology/Principal Findings       

Coupling Illumina-based NGS with informatics approaches, we have successfully identified the tandemly repeated elements in both the Wuchereria bancrofti and Plasmodium falciparum genomes of putatively greatest copy number.  Utilizing these sequences as quantitative real-time PCR (qPCR)-based targets, we have developed assays capable of exploiting the most abundant tandem repeats for both organisms.  For the detection of P. falciparum, analysis and development resulted in an assay with improved analytical and field-based sensitivity vs. an established ribosomal sequence-targeting assay.  Surprisingly, analysis of the W. bancrofti genome predicted a ribosomal sequence to be the genome’s most abundant tandem repeat.  While resulting cycle quantification values comparing a qPCR assay targeting this ribosomal sequence and a commonly targeted repetitive DNA sequence from the literature supported our finding that this ribosomal sequence was the most prevalent tandemly repeated target in the W. bancrofti genome, the resulting assay did not significantly improve detection sensitivity in conjunction with field sample testing. 

Conclusions/Significance

Examination of pathogen genomes facilitates the development of PCR-based diagnostics targeting the most abundant and specific genomic elements.  While in some instances currently available tools may deliver equal or superior performance, systematic analysis of potential targets provides confidence that the selected assays represent the most advantageous options available and that informed assay selection is occurring in the context of a particular study’s objectives.

Methods

This dataset is the output from RepeatExplorer and TAREAN-based analyses of the genomes of Plasmodium falciparum and Wuchereria bancrofti.

Usage Notes

This dataset is full and complete.  An understanding of the output of the RepeatExplorer and TAREAN analysis suites is required to interpret this data.