Development and Optimization of an Unbiased, Metagenomics-Based Pathogen Detection Workflow for Infectious Disease and Biosurveillance Applications
Rapid, specific, and sensitive identification of microbial pathogens is critical to infectious disease diagnosis and surveillance. Classical culture-based methods can be applied to a broad range of pathogens but have long turnaround times. Molecular methods, such as PCR, are time-effective but are n...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-02-01
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| Series: | Tropical Medicine and Infectious Disease |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2414-6366/8/2/121 |
| _version_ | 1797617951836209152 |
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| author | Kyle Parker Hillary Wood Joseph A. Russell David Yarmosh Alan Shteyman John Bagnoli Brittany Knight Jacob R. Aspinwall Jonathan Jacobs Kristine Werking Richard Winegar |
| author_facet | Kyle Parker Hillary Wood Joseph A. Russell David Yarmosh Alan Shteyman John Bagnoli Brittany Knight Jacob R. Aspinwall Jonathan Jacobs Kristine Werking Richard Winegar |
| author_sort | Kyle Parker |
| collection | DOAJ |
| description | Rapid, specific, and sensitive identification of microbial pathogens is critical to infectious disease diagnosis and surveillance. Classical culture-based methods can be applied to a broad range of pathogens but have long turnaround times. Molecular methods, such as PCR, are time-effective but are not comprehensive and may not detect novel strains. Metagenomic shotgun next-generation sequencing (NGS) promises specific identification and characterization of any pathogen (viruses, bacteria, fungi, and protozoa) in a less biased way. Despite its great potential, NGS has yet to be widely adopted by clinical microbiology laboratories due in part to the absence of standardized workflows. Here, we describe a sample-to-answer workflow called PanGIA (Pan-Genomics for Infectious Agents) that includes simplified, standardized wet-lab procedures and data analysis with an easy-to-use bioinformatics tool. PanGIA is an end-to-end, multi-use workflow that can be used for pathogen detection and related applications, such as biosurveillance and biothreat detection. We performed a comprehensive survey and assessment of current, commercially available wet-lab technologies and open-source bioinformatics tools for each workflow component. The workflow includes total nucleic acid extraction from clinical human whole blood and environmental microbial forensic swabs as sample inputs, host nucleic acid depletion, dual DNA and RNA library preparation, shotgun sequencing on an Illumina MiSeq, and sequencing data analysis. The PanGIA workflow can be completed within 24 h and is currently compatible with bacteria and viruses. Here, we present data from the development and application of the clinical and environmental workflows, enabling the specific detection of pathogens associated with bloodstream infections and environmental biosurveillance, without the need for targeted assay development. |
| first_indexed | 2024-03-11T08:03:16Z |
| format | Article |
| id | doaj.art-d6014297542043ab9c242f773d97872c |
| institution | Directory Open Access Journal |
| issn | 2414-6366 |
| language | English |
| last_indexed | 2024-03-11T08:03:16Z |
| publishDate | 2023-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Tropical Medicine and Infectious Disease |
| spelling | doaj.art-d6014297542043ab9c242f773d97872c2023-11-16T23:40:13ZengMDPI AGTropical Medicine and Infectious Disease2414-63662023-02-018212110.3390/tropicalmed8020121Development and Optimization of an Unbiased, Metagenomics-Based Pathogen Detection Workflow for Infectious Disease and Biosurveillance ApplicationsKyle Parker0Hillary Wood1Joseph A. Russell2David Yarmosh3Alan Shteyman4John Bagnoli5Brittany Knight6Jacob R. Aspinwall7Jonathan Jacobs8Kristine Werking9Richard Winegar10MRIGlobal, 425 Dr. Martin Luther King Jr. Blvd, Kansas City, MO 64110, USAMRIGlobal, 425 Dr. Martin Luther King Jr. Blvd, Kansas City, MO 64110, USAMRIGlobal, 65 West Watkins Mill Road, Gaithersburg, MD 20850, USAMRIGlobal, 65 West Watkins Mill Road, Gaithersburg, MD 20850, USAMRIGlobal, 65 West Watkins Mill Road, Gaithersburg, MD 20850, USAMRIGlobal, 65 West Watkins Mill Road, Gaithersburg, MD 20850, USAMRIGlobal, 425 Dr. Martin Luther King Jr. Blvd, Kansas City, MO 64110, USAMRIGlobal, 425 Dr. Martin Luther King Jr. Blvd, Kansas City, MO 64110, USAMRIGlobal, 65 West Watkins Mill Road, Gaithersburg, MD 20850, USAMRIGlobal, 425 Dr. Martin Luther King Jr. Blvd, Kansas City, MO 64110, USAMRIGlobal, 425 Dr. Martin Luther King Jr. Blvd, Kansas City, MO 64110, USARapid, specific, and sensitive identification of microbial pathogens is critical to infectious disease diagnosis and surveillance. Classical culture-based methods can be applied to a broad range of pathogens but have long turnaround times. Molecular methods, such as PCR, are time-effective but are not comprehensive and may not detect novel strains. Metagenomic shotgun next-generation sequencing (NGS) promises specific identification and characterization of any pathogen (viruses, bacteria, fungi, and protozoa) in a less biased way. Despite its great potential, NGS has yet to be widely adopted by clinical microbiology laboratories due in part to the absence of standardized workflows. Here, we describe a sample-to-answer workflow called PanGIA (Pan-Genomics for Infectious Agents) that includes simplified, standardized wet-lab procedures and data analysis with an easy-to-use bioinformatics tool. PanGIA is an end-to-end, multi-use workflow that can be used for pathogen detection and related applications, such as biosurveillance and biothreat detection. We performed a comprehensive survey and assessment of current, commercially available wet-lab technologies and open-source bioinformatics tools for each workflow component. The workflow includes total nucleic acid extraction from clinical human whole blood and environmental microbial forensic swabs as sample inputs, host nucleic acid depletion, dual DNA and RNA library preparation, shotgun sequencing on an Illumina MiSeq, and sequencing data analysis. The PanGIA workflow can be completed within 24 h and is currently compatible with bacteria and viruses. Here, we present data from the development and application of the clinical and environmental workflows, enabling the specific detection of pathogens associated with bloodstream infections and environmental biosurveillance, without the need for targeted assay development.https://www.mdpi.com/2414-6366/8/2/121biosurveillancehost depletioninfectious diseasenext-generation sequencing |
| spellingShingle | Kyle Parker Hillary Wood Joseph A. Russell David Yarmosh Alan Shteyman John Bagnoli Brittany Knight Jacob R. Aspinwall Jonathan Jacobs Kristine Werking Richard Winegar Development and Optimization of an Unbiased, Metagenomics-Based Pathogen Detection Workflow for Infectious Disease and Biosurveillance Applications Tropical Medicine and Infectious Disease biosurveillance host depletion infectious disease next-generation sequencing |
| title | Development and Optimization of an Unbiased, Metagenomics-Based Pathogen Detection Workflow for Infectious Disease and Biosurveillance Applications |
| title_full | Development and Optimization of an Unbiased, Metagenomics-Based Pathogen Detection Workflow for Infectious Disease and Biosurveillance Applications |
| title_fullStr | Development and Optimization of an Unbiased, Metagenomics-Based Pathogen Detection Workflow for Infectious Disease and Biosurveillance Applications |
| title_full_unstemmed | Development and Optimization of an Unbiased, Metagenomics-Based Pathogen Detection Workflow for Infectious Disease and Biosurveillance Applications |
| title_short | Development and Optimization of an Unbiased, Metagenomics-Based Pathogen Detection Workflow for Infectious Disease and Biosurveillance Applications |
| title_sort | development and optimization of an unbiased metagenomics based pathogen detection workflow for infectious disease and biosurveillance applications |
| topic | biosurveillance host depletion infectious disease next-generation sequencing |
| url | https://www.mdpi.com/2414-6366/8/2/121 |
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