Towards a Rapid-Turnaround Low-Depth Unbiased Metagenomics Sequencing Workflow on the Illumina Platforms
Unbiased metagenomic sequencing is conceptually well-suited for first-line diagnosis as all known and unknown infectious entities can be detected, but costs, turnaround time and human background reads in complex biofluids, such as plasma, hinder widespread deployment. Separate preparations of DNA an...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-04-01
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| Series: | Bioengineering |
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| Online Access: | https://www.mdpi.com/2306-5354/10/5/520 |
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| author | Winston Lian Chye Koh Si En Poh Chun Kiat Lee Tim Hon Man Chan Gabriel Yan Kiat Whye Kong Lalita Lau Wai Yip Thomas Lee Clark Cheng Shawn Hoon Yiqi Seow |
| author_facet | Winston Lian Chye Koh Si En Poh Chun Kiat Lee Tim Hon Man Chan Gabriel Yan Kiat Whye Kong Lalita Lau Wai Yip Thomas Lee Clark Cheng Shawn Hoon Yiqi Seow |
| author_sort | Winston Lian Chye Koh |
| collection | DOAJ |
| description | Unbiased metagenomic sequencing is conceptually well-suited for first-line diagnosis as all known and unknown infectious entities can be detected, but costs, turnaround time and human background reads in complex biofluids, such as plasma, hinder widespread deployment. Separate preparations of DNA and RNA also increases costs. In this study, we developed a rapid unbiased metagenomics next-generation sequencing (mNGS) workflow with a human background depletion method (HostEL) and a combined DNA/RNA library preparation kit (AmpRE) to address this issue. We enriched and detected bacterial and fungal standards spiked in plasma at physiological levels with low-depth sequencing (<1 million reads) for analytical validation. Clinical validation also showed 93% of plasma samples agreed with the clinical diagnostic test results when the diagnostic qPCR had a Ct < 33. The effect of different sequencing times was evaluated with the 19 h iSeq 100 paired end run, a more clinically palatable simulated iSeq 100 truncated run and the rapid 7 h MiniSeq platform. Our results demonstrate the ability to detect both DNA and RNA pathogens with low-depth sequencing and that iSeq 100 and MiniSeq platforms are compatible with unbiased low-depth metagenomics identification with the HostEL and AmpRE workflow. |
| first_indexed | 2024-03-11T03:56:53Z |
| format | Article |
| id | doaj.art-9ba1cc59fa104afd9824f00cf9a83fa1 |
| institution | Directory Open Access Journal |
| issn | 2306-5354 |
| language | English |
| last_indexed | 2024-03-11T03:56:53Z |
| publishDate | 2023-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Bioengineering |
| spelling | doaj.art-9ba1cc59fa104afd9824f00cf9a83fa12023-11-18T00:30:34ZengMDPI AGBioengineering2306-53542023-04-0110552010.3390/bioengineering10050520Towards a Rapid-Turnaround Low-Depth Unbiased Metagenomics Sequencing Workflow on the Illumina PlatformsWinston Lian Chye Koh0Si En Poh1Chun Kiat Lee2Tim Hon Man Chan3Gabriel Yan4Kiat Whye Kong5Lalita Lau6Wai Yip Thomas Lee7Clark Cheng8Shawn Hoon9Yiqi Seow10Bioinformatic Institute, A*STAR (Agency for Science, Technology and Research), Singapore 138632, SingaporeInstitute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore 138673, SingaporeDepartment of Laboratory Medicine, National University Hospital, Singapore 119228, SingaporeDepartment of Laboratory Medicine, National University Hospital, Singapore 119228, SingaporeDepartment of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119228, SingaporeInstitute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore 138673, SingaporeInstitute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore 138673, SingaporePaths Diagnostics Pte Limited, Singapore 349317, SingaporePaths Diagnostics Pte Limited, Singapore 349317, SingaporeInstitute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore 138673, SingaporeInstitute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore 138673, SingaporeUnbiased metagenomic sequencing is conceptually well-suited for first-line diagnosis as all known and unknown infectious entities can be detected, but costs, turnaround time and human background reads in complex biofluids, such as plasma, hinder widespread deployment. Separate preparations of DNA and RNA also increases costs. In this study, we developed a rapid unbiased metagenomics next-generation sequencing (mNGS) workflow with a human background depletion method (HostEL) and a combined DNA/RNA library preparation kit (AmpRE) to address this issue. We enriched and detected bacterial and fungal standards spiked in plasma at physiological levels with low-depth sequencing (<1 million reads) for analytical validation. Clinical validation also showed 93% of plasma samples agreed with the clinical diagnostic test results when the diagnostic qPCR had a Ct < 33. The effect of different sequencing times was evaluated with the 19 h iSeq 100 paired end run, a more clinically palatable simulated iSeq 100 truncated run and the rapid 7 h MiniSeq platform. Our results demonstrate the ability to detect both DNA and RNA pathogens with low-depth sequencing and that iSeq 100 and MiniSeq platforms are compatible with unbiased low-depth metagenomics identification with the HostEL and AmpRE workflow.https://www.mdpi.com/2306-5354/10/5/520metagenomic sequencinghost depletionDNA/RNA library preparationliquid biopsyinfectious disease |
| spellingShingle | Winston Lian Chye Koh Si En Poh Chun Kiat Lee Tim Hon Man Chan Gabriel Yan Kiat Whye Kong Lalita Lau Wai Yip Thomas Lee Clark Cheng Shawn Hoon Yiqi Seow Towards a Rapid-Turnaround Low-Depth Unbiased Metagenomics Sequencing Workflow on the Illumina Platforms Bioengineering metagenomic sequencing host depletion DNA/RNA library preparation liquid biopsy infectious disease |
| title | Towards a Rapid-Turnaround Low-Depth Unbiased Metagenomics Sequencing Workflow on the Illumina Platforms |
| title_full | Towards a Rapid-Turnaround Low-Depth Unbiased Metagenomics Sequencing Workflow on the Illumina Platforms |
| title_fullStr | Towards a Rapid-Turnaround Low-Depth Unbiased Metagenomics Sequencing Workflow on the Illumina Platforms |
| title_full_unstemmed | Towards a Rapid-Turnaround Low-Depth Unbiased Metagenomics Sequencing Workflow on the Illumina Platforms |
| title_short | Towards a Rapid-Turnaround Low-Depth Unbiased Metagenomics Sequencing Workflow on the Illumina Platforms |
| title_sort | towards a rapid turnaround low depth unbiased metagenomics sequencing workflow on the illumina platforms |
| topic | metagenomic sequencing host depletion DNA/RNA library preparation liquid biopsy infectious disease |
| url | https://www.mdpi.com/2306-5354/10/5/520 |
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