Metagenomic Sequencing for Microbial DNA in Human Samples: Emerging Technological Advances
Abstract
:1. Introduction
2. The Growing Need for Microbial DNA Enrichment Prior to Metagenomic Sequencing
3. Current Approaches to Host DNA Depletion
3.1. Removal of the Host Cells before DNA Extraction
3.2. Separating the Microbial DNA from the Host Background
3.3. Limitation and Controversy
4. Application in Microbiome Research and Clinical Metagenomics
5. New Strategies to Facilitate Metagenomic Sequencing in Samples with Overabundant Host DNA
5.1. The Removal of Unwanted High Abundance Species in Sequencing Libraries
5.2. Selective Sequencing
6. Conclusions and Future Perspectives
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Kit | Principle | Pros | Cons | Hands-On Time Per Sample | Cost Per Sample (USD) | Ref. |
---|---|---|---|---|---|---|
QIAamp DNA Microbiome (Qiagen, Hilden, Germany) | Lysis of host cell by saponin, degrade extracellular DNA with Benzonase nuclease | Ultra-clean columns to minimise contamination risk | Requires fresh sample | 160 min | 13 | [43] |
MolYsis™ Complete/Ultra-Deep Microbiome Prep (Molzym, Bremen, Germany) | Chaotropic lysis of host cell, degrade extracellular DNA with MolDNase | Applicable for body fluids, tissue and swab samples. Enrichment of bacterial and fungal DNA | Fresh sample is recommended | 120 min | 11 | [44] |
HostZERO Microbial DNA Kit (Zymo Research, Irvine, CA, USA) | Lysis of host cell, degrade extracellular DNA with microbial selection enzyme | Protocols for both tissue and liquid samples are provided | Requires intact (living) bacteria cells | 30 min | 10 | [45] |
NEBNext Microbiome DNA Enrichment (New England BioLabs, Ipswich, MA, USA) | Capture methylated host DNA | Can retain cell-free DNA from dead organisms to avoid DNA loss | Requires high molecular weight intact DNA. Bias to high CpG-methylated microbes | 30 min * | 39 * | [46] |
LOOXSTER Enrichment Kit (Analytik Jena GmbH, Jena, Germany) | Capture non-methylated CpG dinucleotides | Can retain cell-free DNA from dead organisms to avoid DNA loss | Requires high molecular weight intact DNA. Bias to high CpG-methylated microbes | 75 min * | 34 * | [47] |
Sample Type | Potential Clinical Indication | Sample Size | Depletion Method | Sequencing Platform | Reads Number | Ref. |
---|---|---|---|---|---|---|
Cerebrospinal fluid | Infectious aetiology identification | 13 | Selective lysis by a bead-beater tissue homogeniser followed by a Benzonase nuclease treatment | Ion Torrent PGM | N/A | [23] |
Prosthetic joint sonicate fluid | Pathogen identification | 408 | MolYsis basic kit | Illumina HiSeq | 2.8 million, mean | [38] |
Urine | Pathogen identification | 10 | Differential centrifugation and MolYsis kit | MinION | 0.026 million, median | [66] |
Urine | Antimicrobial resistance marker identification | 13 | NEBNext microbiome kit | Ion Torrent PGM | N/A | [67] |
Sputum | Pathogen detection | 6 | Microfluidic separation followed by DNase digestion | Illumina HiSeq | 36.3 million, mean | [12] |
Sputum, bronchoalveolar lavage and endotracheal aspirates | Diagnosis of known and unknown infections | 40 | Saponin-based differential lysis followed by HL-SAN DNase digestion | MinION | 0.041 million, mean | [20] |
Cerebrospinal fluid | Diagnosis of known and unknown infections | 95 | NEB Microbiome Enrichment Kit | Illumina HiSeq | 5~10 million | [68] |
Endotracheal aspirates | Pathogen identification | 22 | Saponin-based differential lysis followed by HL-SAN DNase digestion | MinION | 6628, median | [69] |
Synovial fluid | Pathogen detection | 168 | MolYsis basic kit | Illumina HiSeq | 30 million, mean | [70] |
Bone and joint infectious tissue | Pathogen detection and antibiotic susceptibility prediction | 24 | Ultra-Deep Microbiome Prep kit | Illumina HiSeq | 20 million, mean | [71] |
Valve tissue | Pathogen identification | 1 | Ultra-Deep Microbiome Prep kit | Illumina MiSeq | 1.4 million, mean | [72] |
Hepatic tissue | Diagnosis of unknown infections | 1 | Ultra-Deep Microbiome Prep kit | Illumina MiSeq | 1.1 million, mean | [73] |
Blood culture bottles inoculated with prosthetic joint tissue | Pathogen identification | 9 | MolYsis basic kit | Illumina MiSeq | 10.3 million, mean | [74] |
Blood | Pathogen detection | 8 | MolYsis complete kit and WGA | Illumina HiSeq | 27.5 million, mean | [75] |
Whole blood | Diagnosis of infection | 101 | MolYsis complete kit | Ion Torrent | N/A | [76] |
Sputum | M. tuberculosis detection and antibiotic susceptibility prediction | 40 | MolYsis basic kit | Illumina MiSeq and MinION | 3.6 million, mean | [77] |
Prosthetic joint sonication fluid | Diagnosis of prosthetic joint infections | 97 | A 5 μm pore size filter | Illumina MiSeq | N/A | [78] |
Urine | Pathogen detection and antimicrobial susceptibility prediction | 40 | NEB Microbiome Enrichment Kit | Ion Proton | N/A | [79] |
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Shi, Y.; Wang, G.; Lau, H.C.-H.; Yu, J. Metagenomic Sequencing for Microbial DNA in Human Samples: Emerging Technological Advances. Int. J. Mol. Sci. 2022, 23, 2181. https://doi.org/10.3390/ijms23042181
Shi Y, Wang G, Lau HC-H, Yu J. Metagenomic Sequencing for Microbial DNA in Human Samples: Emerging Technological Advances. International Journal of Molecular Sciences. 2022; 23(4):2181. https://doi.org/10.3390/ijms23042181
Chicago/Turabian StyleShi, Yu, Guoping Wang, Harry Cheuk-Hay Lau, and Jun Yu. 2022. "Metagenomic Sequencing for Microbial DNA in Human Samples: Emerging Technological Advances" International Journal of Molecular Sciences 23, no. 4: 2181. https://doi.org/10.3390/ijms23042181