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Improved PCR-Based Detection of Soil Transmitted Helminth Infections Using a Next-Generation Sequencing Approach to Assay Design

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      There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

      Abstract

      Background

      The soil transmitted helminths are a group of parasitic worms responsible for extensive morbidity in many of the world’s most economically depressed locations. With growing emphasis on disease mapping and eradication, the availability of accurate and cost-effective diagnostic measures is of paramount importance to global control and elimination efforts. While real-time PCR-based molecular detection assays have shown great promise, to date, these assays have utilized sub-optimal targets. By performing next-generation sequencing-based repeat analyses, we have identified high copy-number, non-coding DNA sequences from a series of soil transmitted pathogens. We have used these repetitive DNA elements as targets in the development of novel, multi-parallel, PCR-based diagnostic assays.

      Methodology/Principal Findings

      Utilizing next-generation sequencing and the Galaxy-based RepeatExplorer web server, we performed repeat DNA analysis on five species of soil transmitted helminths ( Necator americanus, Ancylostoma duodenale, Trichuris trichiura, Ascaris lumbricoides, and Strongyloides stercoralis). Employing high copy-number, non-coding repeat DNA sequences as targets, novel real-time PCR assays were designed, and assays were tested against established molecular detection methods. Each assay provided consistent detection of genomic DNA at quantities of 2 fg or less, demonstrated species-specificity, and showed an improved limit of detection over the existing, proven PCR-based assay.

      Conclusions/Significance

      The utilization of next-generation sequencing-based repeat DNA analysis methodologies for the identification of molecular diagnostic targets has the ability to improve assay species-specificity and limits of detection. By exploiting such high copy-number repeat sequences, the assays described here will facilitate soil transmitted helminth diagnostic efforts. We recommend similar analyses when designing PCR-based diagnostic tests for the detection of other eukaryotic pathogens.

      Author Summary

      With a growing emphasis on the mapping and elimination of soil transmitted helminth (STH) infections, the need for optimal and specific diagnostic methods is increasing. While PCR-based diagnostic methods for the detection of these parasitic organisms exist, these assays make use of sub-optimal target sequences. By designing assays that target non-coding, high copy-number repetitive sequences, both the limit of detection and species-specificity of detection improve. Using next-generation sequencing technology, we have identified high copy-number repeats for a series of STH species responsible for the greatest burden of disease. Using these repetitive sequences as targets in the design of novel real-time PCR assays, we have improved both the limits of detection and species-specificity of detection, and we have demonstrated this improved detection by testing these assays against an established PCR-based diagnostic methodology. Accordingly, these assays should facilitate mapping and monitoring efforts, and the generalized application of this approach to assay design should improve detection efforts for other eukaryotic pathogens.

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      Most cited references 59

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      Genome sequence of the nematode C. elegans: a platform for investigating biology.

       James Mussell (1998)
      The 97-megabase genomic sequence of the nematode Caenorhabditis elegans reveals over 19,000 genes. More than 40 percent of the predicted protein products find significant matches in other organisms. There is a variety of repeated sequences, both local and dispersed. The distinctive distribution of some repeats and highly conserved genes provides evidence for a regional organization of the chromosomes.
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        Repetitive DNA and next-generation sequencing: computational challenges and solutions.

        Repetitive DNA sequences are abundant in a broad range of species, from bacteria to mammals, and they cover nearly half of the human genome. Repeats have always presented technical challenges for sequence alignment and assembly programs. Next-generation sequencing projects, with their short read lengths and high data volumes, have made these challenges more difficult. From a computational perspective, repeats create ambiguities in alignment and assembly, which, in turn, can produce biases and errors when interpreting results. Simply ignoring repeats is not an option, as this creates problems of its own and may mean that important biological phenomena are missed. We discuss the computational problems surrounding repeats and describe strategies used by current bioinformatics systems to solve them.
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          RepeatExplorer: a Galaxy-based web server for genome-wide characterization of eukaryotic repetitive elements from next-generation sequence reads.

          Repetitive DNA makes up large portions of plant and animal nuclear genomes, yet it remains the least-characterized genome component in most species studied so far. Although the recent availability of high-throughput sequencing data provides necessary resources for in-depth investigation of genomic repeats, its utility is hampered by the lack of specialized bioinformatics tools and appropriate computational resources that would enable large-scale repeat analysis to be run by biologically oriented researchers. Here we present RepeatExplorer, a collection of software tools for characterization of repetitive elements, which is accessible via web interface. A key component of the server is the computational pipeline using a graph-based sequence clustering algorithm to facilitate de novo repeat identification without the need for reference databases of known elements. Because the algorithm uses short sequences randomly sampled from the genome as input, it is ideal for analyzing next-generation sequence reads. Additional tools are provided to aid in classification of identified repeats, investigate phylogenetic relationships of retroelements and perform comparative analysis of repeat composition between multiple species. The server allows to analyze several million sequence reads, which typically results in identification of most high and medium copy repeats in higher plant genomes.
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            Author and article information

            Affiliations
            [1 ]Department of Biological Sciences, Smith College, Northampton, Massachusetts, United States of America
            [2 ]Molecular and Cellular Biology Program, University of Massachusetts Amherst, Amherst, Massachusetts, United States of America
            [3 ]QIMR Berghofer Medical Research Institute, Brisbane, Australia
            Johns Hopkins Bloomberg School of Public Health, UNITED STATES
            Author notes

            The authors have declared that no competing interests exist.

            Conceived and designed the experiments: NP MP SL JSM SAW. Performed the experiments: NP MP JRG LAB SL. Analyzed the data: NP MP JRG JSM SL SAW. Contributed reagents/materials/analysis tools: LAB JSM SAW. Wrote the paper: NP MP SAW. Contributed to the editing of the manuscript: JRG LAB SL JSM.

            Contributors
            Role: Editor
            Journal
            PLoS Negl Trop Dis
            PLoS Negl Trop Dis
            plos
            plosntds
            PLoS Neglected Tropical Diseases
            Public Library of Science (San Francisco, CA USA )
            1935-2727
            1935-2735
            30 March 2016
            March 2016
            : 10
            : 3
            27027771 4814118 10.1371/journal.pntd.0004578 PNTD-D-15-01933
            © 2016 Pilotte et al

            This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

            Counts
            Figures: 3, Tables: 3, Pages: 18
            Product
            Funding
            This work was funded through an award to SAW from the Bill and Melinda Gates Foundation ( www.gatesfoundation.org) (grant #OPP1053230). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
            Categories
            Research Article
            Biology and Life Sciences
            Genetics
            Genomics
            Repeated Sequences
            Biology and Life Sciences
            Molecular Biology
            Molecular Biology Techniques
            Artificial Gene Amplification and Extension
            Polymerase Chain Reaction
            Research and Analysis Methods
            Molecular Biology Techniques
            Artificial Gene Amplification and Extension
            Polymerase Chain Reaction
            Biology and life sciences
            Molecular biology
            Molecular biology techniques
            Sequencing techniques
            Sequence analysis
            DNA sequence analysis
            Research and analysis methods
            Molecular biology techniques
            Sequencing techniques
            Sequence analysis
            DNA sequence analysis
            Biology and Life Sciences
            Organisms
            Animals
            Invertebrates
            Nematoda
            Ascaris
            Ascaris Lumbricoides
            Biology and Life Sciences
            Organisms
            Animals
            Invertebrates
            Nematoda
            Strongyloides
            Strongyloides Stercoralis
            Biology and Life Sciences
            Organisms
            Animals
            Invertebrates
            Nematoda
            Necator
            Necator Americanus
            Biology and Life Sciences
            Organisms
            Animals
            Invertebrates
            Nematoda
            Trichuris
            Biology and Life Sciences
            Organisms
            Animals
            Invertebrates
            Nematoda
            Ancylostoma
            Custom metadata
            All raw sequence read files are available from the Sequence Read Archive database ( http://www.ncbi.nlm.nih.gov/sra). The accession number for the BioProject is PRJNA304165. The accession numbers for individual BioSamples are SAMN04296119, SAMN04296117, SAMN04296116, SAMN04296070, and SAMN04296069.

            Infectious disease & Microbiology

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