Metagenomic Sequencing for Microbial DNA in Human Samples: Emerging Technological Advances

Abstract

Whole genome metagenomic sequencing is a powerful platform enabling the simultaneous identification of all genes from entirely different kingdoms of organisms in a complex sample. This technology has revolutionised multiple areas from microbiome research to clinical diagnoses. However, one of the major challenges of a metagenomic study is the overwhelming non-microbial DNA present in most of the host-derived specimens, which can inundate the microbial signals and reduce the sensitivity of microorganism detection. Various host DNA depletion methods to facilitate metagenomic sequencing have been developed and have received considerable attention in this context. In this review, we present an overview of current host DNA depletion approaches along with explanations of their underlying principles, advantages and disadvantages. We also discuss their applications in laboratory microbiome research and clinical diagnoses and, finally, we envisage the direction of the further perfection of metagenomic sequencing in samples with overabundant host DNA.

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“…More than 90% of the DNA fragments from samples such as blood, saliva and milk can come from the host genomes [ 38 , 39 ]. In shotgun metagenomics, since all the genetic material including host DNA is sequenced, a large amount of sequencing output is wasted on such contaminating host DNA [ 40 , 41 ]. This can lead to a high number of microbial species being unclassified, incorrectly classified and/or under-represented, thus causing serious inaccuracies in sample microbial community profiling [ 42 , 43 ].…”

“…Therefore, host DNA depletion is often advantageous when preparing gDNA for shotgun sequencing. Host depletion in amplicon-based approaches is not required as the PCR step is selective and amplifies only target microbial DNA regions [ 41 ].…”

“…The latter is increasingly employed due to its wide range of operating conditions compared to DNase I. Once host DNA is degraded, microbial gDNA extraction is performed [ 41 ]. Commercial kits such as MolYsis (Molzym, Bremen, Germany) use this pre-extraction method with a proprietary DNase called MolDNase B, while the QIAmp DNA Microbiome kit (Qiagen, Hilden, Germany) performs host depletion using Benzonase [ 45 ].…”

“…The drawbacks observed for pre-extraction methods are: (i) the likely destruction and consequent under-representation of sensitive microbes such as Mycoplasma spp. and parasites during selective lysis, and (ii) for PMA-based methods, biasing towards Gram-positive bacteria due their increased susceptibility to PMA treatment compared to Gram-negative bacteria [ 34 , 41 , 47 , 48 ]. The second approach to host DNA depletion takes place post-DNA extraction, and uses differences in methylation characteristics between microbial and host or eukaryotic genomes.…”

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“…It is specific to each bacterium, by which the bacterial species is identified, its precise function understood, and thus, how the microorganisms interact with the specific site (microbial ecology) [ 4 ]. This is useful for evaluating diseases in which specific bacterial strains predominate [ 5 , 6 ]. Targeted technologies with transcriptomics (microarrays) and proteomics (electrophoresis, chromatography, mass spectrometry) respectively study gene expression by transcribed RNAs and all proteins synthesized from mRNA in an organism [ 7 , 8 ].…”