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

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.

Fig 1. Workflow for repeat analysis.

Output data from a next-generation sequencing run are uploaded to the RepeatExplorer Galaxy-based platform. During the QC and manipulation phase, the FASTQ Groomer tool is used to convert sequence reads into Sanger format. The FASTQ: READ QC tool is then used to verify the quality of the reads before removing unnecessary sequence (i.e. adapter sequences, etc.) from the ends of each read using the FASTQ Trimmer tool. The QC analysis is then repeated, and the FASTQ to FASTA converter tool is used to convert each read into FASTA format. Using these DNA sequence reads as input, sequences undergo clustering, during which an “all-to-all” sequence comparison is performed, and similar sequences are grouped together into clusters. Clusters containing the most highly repetitive sequences are then selected as putative diagnostic targets to be used for primer and probe-based real-time PCR assay design.

Fig 2. Illustrative output from RepeatExplorer analysis of Necator americanus.

During “clustering” each nucleotide within a cluster is assigned a number. That number corresponds to how many individual next-generation sequencing reads that particular nucleotide appeared in. Using this output, a stretch of the most abundant nucleotides (depicted in green within the larger cluster’s sequence) is selected, and the corresponding nucleotides (highlighted in yellow) are selected as the candidate sequence from which the primers and probe are designed.

Fig 3. Comparative probe testing.

For each novel probe design, FAM-TAMRA and double quenched FAM-ZEN-IOWA BLACK probes were synthesized. Comparative testing revealed that double quenched probes outperformed traditional probes, as evidenced by lower Ct values and greater ΔRn values. The plot above demonstrates these findings with the amplification of three concentrations of N. americanus template DNA using both double quenched (yellow) and traditional (blue) probe designs.