Restriction enzyme digestion of host DNA enhances universal detection of parasitic pathogens in blood via targeted amplicon deep sequencing
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...
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| Format: | Article |
| Language: | English |
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BMC
2018-09-01
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| Series: | Microbiome |
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| Online Access: | http://link.springer.com/article/10.1186/s40168-018-0540-2 |
| _version_ | 1811220671502483456 |
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| author | Briana R. Flaherty Eldin Talundzic Joel Barratt Kristine J. Kines Christian Olsen Meredith Lane Mili Sheth Richard S. Bradbury |
| author_facet | Briana R. Flaherty Eldin Talundzic Joel Barratt Kristine J. Kines Christian Olsen Meredith Lane Mili Sheth Richard S. Bradbury |
| author_sort | Briana R. Flaherty |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-12T07:46:38Z |
| format | Article |
| id | doaj.art-6d744872b310419692c824948f3343c0 |
| institution | Directory Open Access Journal |
| issn | 2049-2618 |
| language | English |
| last_indexed | 2024-04-12T07:46:38Z |
| publishDate | 2018-09-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbiome |
| spelling | doaj.art-6d744872b310419692c824948f3343c02022-12-22T03:41:42ZengBMCMicrobiome2049-26182018-09-016111310.1186/s40168-018-0540-2Restriction enzyme digestion of host DNA enhances universal detection of parasitic pathogens in blood via targeted amplicon deep sequencingBriana R. Flaherty0Eldin Talundzic1Joel Barratt2Kristine J. Kines3Christian Olsen4Meredith Lane5Mili Sheth6Richard S. Bradbury7Parasitic Diseases Branch, Division of Parasitic Diseases and Malaria, Centers for Disease Control and PreventionMalaria Branch, Division of Parasitic Diseases and Malaria, Centers for Disease Control and PreventionParasitic Diseases Branch, Division of Parasitic Diseases and Malaria, Centers for Disease Control and PreventionParasitic Diseases Branch, Division of Parasitic Diseases and Malaria, Centers for Disease Control and PreventionPacific BiosciencesParasitic Diseases Branch, Division of Parasitic Diseases and Malaria, Centers for Disease Control and PreventionBiotechnology Core Facility, Centers for Disease Control and PreventionParasitic Diseases Branch, Division of Parasitic Diseases and Malaria, Centers for Disease Control and PreventionAbstract 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.http://link.springer.com/article/10.1186/s40168-018-0540-2Molecular parasitologyAmplicon sequencingBlood microbiotaParasite biodiversity |
| spellingShingle | Briana R. Flaherty Eldin Talundzic Joel Barratt Kristine J. Kines Christian Olsen Meredith Lane Mili Sheth Richard S. Bradbury Restriction enzyme digestion of host DNA enhances universal detection of parasitic pathogens in blood via targeted amplicon deep sequencing Microbiome Molecular parasitology Amplicon sequencing Blood microbiota Parasite biodiversity |
| title | Restriction enzyme digestion of host DNA enhances universal detection of parasitic pathogens in blood via targeted amplicon deep sequencing |
| title_full | Restriction enzyme digestion of host DNA enhances universal detection of parasitic pathogens in blood via targeted amplicon deep sequencing |
| title_fullStr | Restriction enzyme digestion of host DNA enhances universal detection of parasitic pathogens in blood via targeted amplicon deep sequencing |
| title_full_unstemmed | Restriction enzyme digestion of host DNA enhances universal detection of parasitic pathogens in blood via targeted amplicon deep sequencing |
| title_short | Restriction enzyme digestion of host DNA enhances universal detection of parasitic pathogens in blood via targeted amplicon deep sequencing |
| title_sort | restriction enzyme digestion of host dna enhances universal detection of parasitic pathogens in blood via targeted amplicon deep sequencing |
| topic | Molecular parasitology Amplicon sequencing Blood microbiota Parasite biodiversity |
| url | http://link.springer.com/article/10.1186/s40168-018-0540-2 |
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