Benchmarking microbial DNA enrichment protocols from human intestinal biopsies
Shotgun metagenomic sequencing is a powerful tool for studying bacterial communities in their natural habitats or sites of infection, without the need for cultivation. However, low microbial signals in metagenomic sequencing can be overwhelmed by host DNA contamination, resulting in decreased sensit...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-04-01
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| Series: | Frontiers in Genetics |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fgene.2023.1184473/full |
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| author | Dmitrij Marchukov Jiaqi Li Jiaqi Li Pascal Juillerat Pascal Juillerat Pascal Juillerat Benjamin Misselwitz Benjamin Misselwitz Bahtiyar Yilmaz Bahtiyar Yilmaz |
| author_facet | Dmitrij Marchukov Jiaqi Li Jiaqi Li Pascal Juillerat Pascal Juillerat Pascal Juillerat Benjamin Misselwitz Benjamin Misselwitz Bahtiyar Yilmaz Bahtiyar Yilmaz |
| author_sort | Dmitrij Marchukov |
| collection | DOAJ |
| description | Shotgun metagenomic sequencing is a powerful tool for studying bacterial communities in their natural habitats or sites of infection, without the need for cultivation. However, low microbial signals in metagenomic sequencing can be overwhelmed by host DNA contamination, resulting in decreased sensitivity for microbial read detection. Several commercial kits and other methods have been developed to enrich bacterial sequences; however, these assays have not been tested extensively for human intestinal tissues yet. Therefore, the objective of this study was to assess the effectiveness of various wet-lab and software-based approaches for depleting host DNA from microbiome samples. Four different microbiome DNA enrichment methods, namely the NEBNext Microbiome DNA Enrichment kit, Molzym Ultra-Deep Microbiome Prep, QIAamp DNA Microbiome kit, and Zymo HostZERO microbial DNA kit, were evaluated, along with a software-controlled adaptive sampling (AS) approach by Oxford Nanopore Technologies (ONT) providing microbial signal enrichment by aborting unwanted host DNA sequencing. The NEBNext and QIAamp kits proved to be effective in shotgun metagenomic sequencing studies, as they efficiently reduced host DNA contamination, resulting in 24% and 28% bacterial DNA sequences, respectively, compared to <1% in the AllPrep controls. Additional optimization steps using further detergents and bead-beating steps improved the efficacy of less efficient protocols but not of the QIAamp kit. In contrast, ONT AS increased the overall number of bacterial reads resulting in a better bacterial metagenomic assembly with more bacterial contigs with greater completeness compared to non-AS approaches. Additionally, AS also allowed for the recovery of antimicrobial resistance markers and the identification of plasmids, demonstrating the potential utility of AS for targeted sequencing of microbial signals in complex samples with high amounts of host DNA. However, ONT AS resulted in relevant shifts in the observed bacterial abundance, including 2 to 5 times more Escherichia coli reads. Furthermore, a modest enrichment of Bacteroides fragilis and Bacteroides thetaiotaomicron was also observed with AS. Overall, this study provides insight into the efficacy and limitations of various methods for reducing host DNA contamination in human intestinal samples to improve the utility of metagenomic sequencing. |
| first_indexed | 2024-03-13T10:51:53Z |
| format | Article |
| id | doaj.art-0946029260034afa9d43d19bc59b626d |
| institution | Directory Open Access Journal |
| issn | 1664-8021 |
| language | English |
| last_indexed | 2024-03-13T10:51:53Z |
| publishDate | 2023-04-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Genetics |
| spelling | doaj.art-0946029260034afa9d43d19bc59b626d2023-05-17T09:13:56ZengFrontiers Media S.A.Frontiers in Genetics1664-80212023-04-011410.3389/fgene.2023.11844731184473Benchmarking microbial DNA enrichment protocols from human intestinal biopsiesDmitrij Marchukov0Jiaqi Li1Jiaqi Li2Pascal Juillerat3Pascal Juillerat4Pascal Juillerat5Benjamin Misselwitz6Benjamin Misselwitz7Bahtiyar Yilmaz8Bahtiyar Yilmaz9University Hospital Zürich, University of Zürich, Zürich, SwitzerlandDepartment of Visceral Surgery and Medicine, Bern University Hospital, University of Bern, Bern, SwitzerlandMaurice Müller Laboratories, Department for Biomedical Research, University of Bern, Bern, SwitzerlandDepartment of Visceral Surgery and Medicine, Bern University Hospital, University of Bern, Bern, SwitzerlandMaurice Müller Laboratories, Department for Biomedical Research, University of Bern, Bern, SwitzerlandCrohn’s and Colitis Center, Gastroenterologie Beaulieu, Lausanne, SwitzerlandDepartment of Visceral Surgery and Medicine, Bern University Hospital, University of Bern, Bern, SwitzerlandMaurice Müller Laboratories, Department for Biomedical Research, University of Bern, Bern, SwitzerlandDepartment of Visceral Surgery and Medicine, Bern University Hospital, University of Bern, Bern, SwitzerlandMaurice Müller Laboratories, Department for Biomedical Research, University of Bern, Bern, SwitzerlandShotgun metagenomic sequencing is a powerful tool for studying bacterial communities in their natural habitats or sites of infection, without the need for cultivation. However, low microbial signals in metagenomic sequencing can be overwhelmed by host DNA contamination, resulting in decreased sensitivity for microbial read detection. Several commercial kits and other methods have been developed to enrich bacterial sequences; however, these assays have not been tested extensively for human intestinal tissues yet. Therefore, the objective of this study was to assess the effectiveness of various wet-lab and software-based approaches for depleting host DNA from microbiome samples. Four different microbiome DNA enrichment methods, namely the NEBNext Microbiome DNA Enrichment kit, Molzym Ultra-Deep Microbiome Prep, QIAamp DNA Microbiome kit, and Zymo HostZERO microbial DNA kit, were evaluated, along with a software-controlled adaptive sampling (AS) approach by Oxford Nanopore Technologies (ONT) providing microbial signal enrichment by aborting unwanted host DNA sequencing. The NEBNext and QIAamp kits proved to be effective in shotgun metagenomic sequencing studies, as they efficiently reduced host DNA contamination, resulting in 24% and 28% bacterial DNA sequences, respectively, compared to <1% in the AllPrep controls. Additional optimization steps using further detergents and bead-beating steps improved the efficacy of less efficient protocols but not of the QIAamp kit. In contrast, ONT AS increased the overall number of bacterial reads resulting in a better bacterial metagenomic assembly with more bacterial contigs with greater completeness compared to non-AS approaches. Additionally, AS also allowed for the recovery of antimicrobial resistance markers and the identification of plasmids, demonstrating the potential utility of AS for targeted sequencing of microbial signals in complex samples with high amounts of host DNA. However, ONT AS resulted in relevant shifts in the observed bacterial abundance, including 2 to 5 times more Escherichia coli reads. Furthermore, a modest enrichment of Bacteroides fragilis and Bacteroides thetaiotaomicron was also observed with AS. Overall, this study provides insight into the efficacy and limitations of various methods for reducing host DNA contamination in human intestinal samples to improve the utility of metagenomic sequencing.https://www.frontiersin.org/articles/10.3389/fgene.2023.1184473/fullgut micobiomehost DNA depletionmetagemonicphyloseqhuman small intestinemicrobial enrichment |
| spellingShingle | Dmitrij Marchukov Jiaqi Li Jiaqi Li Pascal Juillerat Pascal Juillerat Pascal Juillerat Benjamin Misselwitz Benjamin Misselwitz Bahtiyar Yilmaz Bahtiyar Yilmaz Benchmarking microbial DNA enrichment protocols from human intestinal biopsies Frontiers in Genetics gut micobiome host DNA depletion metagemonic phyloseq human small intestine microbial enrichment |
| title | Benchmarking microbial DNA enrichment protocols from human intestinal biopsies |
| title_full | Benchmarking microbial DNA enrichment protocols from human intestinal biopsies |
| title_fullStr | Benchmarking microbial DNA enrichment protocols from human intestinal biopsies |
| title_full_unstemmed | Benchmarking microbial DNA enrichment protocols from human intestinal biopsies |
| title_short | Benchmarking microbial DNA enrichment protocols from human intestinal biopsies |
| title_sort | benchmarking microbial dna enrichment protocols from human intestinal biopsies |
| topic | gut micobiome host DNA depletion metagemonic phyloseq human small intestine microbial enrichment |
| url | https://www.frontiersin.org/articles/10.3389/fgene.2023.1184473/full |
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