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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 (1999)
          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

                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
                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.

                Article
                PNTD-D-15-01933
                10.1371/journal.pntd.0004578
                4814118
                27027771
                © 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.

                Page count
                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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