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

Pilotte N, Papaiakovou M, Grant JR, Bierwert LA, Llewellyn S, McCarthy JS, Williams SA - PLoS Negl Trop Dis (2016)

Bottom Line: 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.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.We recommend similar analyses when designing PCR-based diagnostic tests for the detection of other eukaryotic pathogens.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Smith College, Northampton, Massachusetts, United States of America.

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4814118&req=5

pntd.0004578.g001: 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.

Mentions: For each parasite analyzed, raw sequencing reads were uploaded to the Galaxy-based RepeatExplorer web server [39]. Reads were processed according to the workflow in Fig 1, enabling the identification of high copy-number repeat DNA sequences for each organism. Promising repeat families were further analyzed using the Nucleotide BLAST tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi) available from the National Center for Biotechnology Information (NCBI). Results from each organism were screened for repetitive DNA elements found to have high degrees of homology with elements of the human genome, common bacteria of the human microbiome, or other parasitic organisms likely to be found within the human gut. Had such sequences been identified as among the most repetitive, they would have been eliminated from further consideration as they would be expected to cause species-specificity challenges during downstream PCR assay development. However, no such conserved highly repetitive elements were identified. Following screening, sequences from each organism, putatively determined to be among the most highly repetitive, were utilized for further assay development (Fig 2).


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

Pilotte N, Papaiakovou M, Grant JR, Bierwert LA, Llewellyn S, McCarthy JS, Williams SA - PLoS Negl Trop Dis (2016)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4814118&req=5

pntd.0004578.g001: 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.
Mentions: For each parasite analyzed, raw sequencing reads were uploaded to the Galaxy-based RepeatExplorer web server [39]. Reads were processed according to the workflow in Fig 1, enabling the identification of high copy-number repeat DNA sequences for each organism. Promising repeat families were further analyzed using the Nucleotide BLAST tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi) available from the National Center for Biotechnology Information (NCBI). Results from each organism were screened for repetitive DNA elements found to have high degrees of homology with elements of the human genome, common bacteria of the human microbiome, or other parasitic organisms likely to be found within the human gut. Had such sequences been identified as among the most repetitive, they would have been eliminated from further consideration as they would be expected to cause species-specificity challenges during downstream PCR assay development. However, no such conserved highly repetitive elements were identified. Following screening, sequences from each organism, putatively determined to be among the most highly repetitive, were utilized for further assay development (Fig 2).

Bottom Line: 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.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.We recommend similar analyses when designing PCR-based diagnostic tests for the detection of other eukaryotic pathogens.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Smith College, Northampton, Massachusetts, United States of America.

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.

Show MeSH
Related in: MedlinePlus