Restriction enzyme digestion of host DNA enhances universal detection of parasitic pathogens in blood via targeted amplicon deep sequencing
Flaherty, Briana R., Talundzic, Eldin, Barratt, Joel, Kines, Kristine J., Olsen, Christian, Lane, Meredith, Sheth, Mili, and Bradbury, Richard S. (2018) Restriction enzyme digestion of host DNA enhances universal detection of parasitic pathogens in blood via targeted amplicon deep sequencing. Microbiome, 6 (1). 164.
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Abstract
Background: Targeted amplicon deep sequencing (TADS) of the 16S rRNA gene is commonly used to explore and characterize bacterial microbiomes. Meanwhile, attempts to apply TADS to the detection and characterization of entire parasitic communities have been hampered since conserved regions of many conserved parasite genes, such as the 18S rRNA gene, are also conserved in their eukaryotic hosts. As a result, targeted amplification of 18S rRNA from clinical samples using universal primers frequently results in competitive priming and preferential amplification of host DNA. Here, we describe a novel method that employs a single pair of universal primers to capture all blood-borne parasites while reducing host 18S rRNA template and enhancing the amplification of parasite 18S rRNA for TADS. This was achieved using restriction enzymes to digest the 18S rRNA gene at cut sites present only in the host sequence prior to PCR amplification. Results: This method was validated against 16 species of blood-borne helminths and protozoa. Enzyme digestion prior to PCR enrichment and Illumina amplicon deep sequencing led to a substantial reduction in human reads and a corresponding 5- to 10-fold increase in parasite reads relative to undigested samples. This method allowed for discrimination of all common parasitic agents found in human blood, even in cases of multi-parasite infection, and markedly reduced the limit of detection in digested versus undigested samples. Conclusions: The results herein provide a novel methodology for the reduction of host DNA prior to TADS and establish the validity of a next-generation sequencing-based platform for universal parasite detection.
| Item ID: | 81857 |
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| Item Type: | Article (Research - C1) |
| ISSN: | 2049-2618 |
| Keywords: | Amplicon sequencing, Blood microbiota, Molecular parasitology, Parasite biodiversity |
| Copyright Information: | © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
| Date Deposited: | 20 Jun 2024 02:49 |
| FoR Codes: | 32 BIOMEDICAL AND CLINICAL SCIENCES > 3207 Medical microbiology > 320704 Medical parasitology @ 80% 31 BIOLOGICAL SCIENCES > 3102 Bioinformatics and computational biology > 310201 Bioinformatic methods development @ 20% |
| SEO Codes: | 28 EXPANDING KNOWLEDGE > 2801 Expanding knowledge > 280103 Expanding knowledge in the biomedical and clinical sciences @ 100% |
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