Introduction Over the past decade, a dramatic reduction in DNA sequencing costs has propelled shotgun metagenomic sequencing into the forefront. Shotgun metagenomic sequencing is an indispensable tool for characterizing bacterial populations across diverse environments and host systems, bypassing the need for target-specific primers.
Shotgun metagenomic sequencing uses next-generation sequencing (NGS) technology to provide not only information on the taxonomic annotations of each organism, but also the functional profiling, gene prediction and microbial interaction of the whole community. However, in the analysis of host-derived samples, this broad-spectrum approach poses a challenge. Samples of metagenomic sequencing with higher host content typically include those obtained from host-associated environments or tissue samples where the microbial load is relatively low compared to the host DNA [1-3].
Current Host DNA Depletion Approaches
The development of a reliable method to systematically deplete host reads in shotgun sequencing is imperative for virtually all studies focusing on host-derived microbiomes. Here, we summarized the frequently used host DNA depletion methods [4,5].
Physical separation (microfiltration and centrifugation): Host cells are significantly larger than microbial cells, making it possible to separate human-derived cells using methods such as membrane filtration, low-speed centrifugation, or flow cytometry (Fig. 1B).
Microbial DNA enrichment: This methodology leverages the discrepancy in cytosine methylation frequency between eukaryotic and prokaryotic DNA. Utilizing MBD-Fc-bound magnetic beads, CpG-methylated host DNA sequences can be selectively captured and removed by a magnetic field while leaving the non-CpG-methylated microbial DNA in the supernatant available for downstream applications (Fig. 1C).
Enzymatic and chemical treatments (selective host cell lysis and DNase/PMA treatment): The prevailing pre-extraction method for eliminating host DNA involves initially subjecting human cells to a selective lysis buffer (i.e., saponin), followed by treatment with DNA nucleases (Fig. 1D) or propidium monoazide (PMA) as photoreactive DNA-binding dye (Fig. 1E).
Bioinformatic approaches:
Sequence alignment for removal: This method involves aligning metagenomic sequencing data with known host genome sequences and removing sequences that are highly similar to the host genome. This requires efficient alignment software such as Bowtie2 [7], BWA [8], etc.
K-mer based methods: Independent of reference genomes, this approach identifies and removes host sequences by comparing the frequency distribution of short sequence fragments (k-mers). This method is suitable for situations where the host genome is unknown or incomplete [6, 9].
Key advantages of Novogene’s shotgun metagenomic sequencing
1. Outstanding end-to end service with fast-turnaround time, top-notch quality and cost-effective pricing.
2. Expert bioinformatics analyses provide comprehensive data on annotated genes, metabolic pathways and antibiotic resistance genes profiles.
3. Our strategy synergizes both deep shotgun sequencing and efficient shallow shotgun sequencing on short-read platforms, serving diverse applications to unveil comprehensive analysis.
