Detection of 16S rRNA and KPC Genes from Complex Matrix Utilizing a Molecular Inversion Probe Assay for Next-Generation Sequencing

Abstract Targeted sequencing promises to bring next-generation sequencing (NGS) into routine clinical use for infectious disease diagnostics. In this context, upfront processing techniques, including pathogen signature enrichment, must amplify multiple targets of interest for NGS to be relevant when...

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Autores principales:	Christopher P. Stefan, Adrienne T. Hall, Timothy D. Minogue
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2018
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/5d99595fcf2844c9992d0e93e5cbd52a
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spelling	oai:doaj.org-article:5d99595fcf2844c9992d0e93e5cbd52a2021-12-02T15:08:03ZDetection of 16S rRNA and KPC Genes from Complex Matrix Utilizing a Molecular Inversion Probe Assay for Next-Generation Sequencing10.1038/s41598-018-19501-z2045-2322https://doaj.org/article/5d99595fcf2844c9992d0e93e5cbd52a2018-02-01T00:00:00Zhttps://doi.org/10.1038/s41598-018-19501-zhttps://doaj.org/toc/2045-2322Abstract Targeted sequencing promises to bring next-generation sequencing (NGS) into routine clinical use for infectious disease diagnostics. In this context, upfront processing techniques, including pathogen signature enrichment, must amplify multiple targets of interest for NGS to be relevant when applied to patient samples with limited volumes. Here, we demonstrate an optimized molecular inversion probe (MIP) assay targeting multiple variable regions within the 16S ribosomal gene for the identification of biothreat and ESKAPE pathogens in a process that significantly reduces complexity, labor, and processing time. Probes targeting the Klebsiella pneumoniae carbapenemase (KPC) antibiotic resistance (AR) gene were also included to demonstrate the ability to concurrently identify etiologic agent and ascertain valuable secondary genetic information. Our assay captured gene sequences in 100% of mock clinical samples prepared from flagged positive blood culture bottles. Using a simplified processing and adjudication method for mapped sequencing reads, genus and species level concordance was 100% and 80%, respectively. In addition, sensitivity and specificity for KPC gene detection was 100%. Our MIP assay produced sequenceable amplicons for the identification of etiologic agents and the detection of AR genes directly from blood culture bottles in a simplified single tube assay.Christopher P. StefanAdrienne T. HallTimothy D. MinogueNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 8, Iss 1, Pp 1-11 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Christopher P. Stefan Adrienne T. Hall Timothy D. Minogue Detection of 16S rRNA and KPC Genes from Complex Matrix Utilizing a Molecular Inversion Probe Assay for Next-Generation Sequencing
description	Abstract Targeted sequencing promises to bring next-generation sequencing (NGS) into routine clinical use for infectious disease diagnostics. In this context, upfront processing techniques, including pathogen signature enrichment, must amplify multiple targets of interest for NGS to be relevant when applied to patient samples with limited volumes. Here, we demonstrate an optimized molecular inversion probe (MIP) assay targeting multiple variable regions within the 16S ribosomal gene for the identification of biothreat and ESKAPE pathogens in a process that significantly reduces complexity, labor, and processing time. Probes targeting the Klebsiella pneumoniae carbapenemase (KPC) antibiotic resistance (AR) gene were also included to demonstrate the ability to concurrently identify etiologic agent and ascertain valuable secondary genetic information. Our assay captured gene sequences in 100% of mock clinical samples prepared from flagged positive blood culture bottles. Using a simplified processing and adjudication method for mapped sequencing reads, genus and species level concordance was 100% and 80%, respectively. In addition, sensitivity and specificity for KPC gene detection was 100%. Our MIP assay produced sequenceable amplicons for the identification of etiologic agents and the detection of AR genes directly from blood culture bottles in a simplified single tube assay.
format	article
author	Christopher P. Stefan Adrienne T. Hall Timothy D. Minogue
author_facet	Christopher P. Stefan Adrienne T. Hall Timothy D. Minogue
author_sort	Christopher P. Stefan
title	Detection of 16S rRNA and KPC Genes from Complex Matrix Utilizing a Molecular Inversion Probe Assay for Next-Generation Sequencing
title_short	Detection of 16S rRNA and KPC Genes from Complex Matrix Utilizing a Molecular Inversion Probe Assay for Next-Generation Sequencing
title_full	Detection of 16S rRNA and KPC Genes from Complex Matrix Utilizing a Molecular Inversion Probe Assay for Next-Generation Sequencing
title_fullStr	Detection of 16S rRNA and KPC Genes from Complex Matrix Utilizing a Molecular Inversion Probe Assay for Next-Generation Sequencing
title_full_unstemmed	Detection of 16S rRNA and KPC Genes from Complex Matrix Utilizing a Molecular Inversion Probe Assay for Next-Generation Sequencing
title_sort	detection of 16s rrna and kpc genes from complex matrix utilizing a molecular inversion probe assay for next-generation sequencing
publisher	Nature Portfolio
publishDate	2018
url	https://doaj.org/article/5d99595fcf2844c9992d0e93e5cbd52a
work_keys_str_mv	AT christopherpstefan detectionof16srrnaandkpcgenesfromcomplexmatrixutilizingamolecularinversionprobeassayfornextgenerationsequencing AT adriennethall detectionof16srrnaandkpcgenesfromcomplexmatrixutilizingamolecularinversionprobeassayfornextgenerationsequencing AT timothydminogue detectionof16srrnaandkpcgenesfromcomplexmatrixutilizingamolecularinversionprobeassayfornextgenerationsequencing
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Detection of 16S rRNA and KPC Genes from Complex Matrix Utilizing a Molecular Inversion Probe Assay for Next-Generation Sequencing

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