Abstract
BackgroundEnzyme-based host depletion significantly improves the sensitivity of clinical metagenomics. Recent studies found that real-time adaptive sequencing of DNA molecules was achieved using a nanopore sequencing machine, which enabled effective enrichment of microbial sequences. However, few studies have compared the enzyme-based host depletion and nanopore adaptive sequencing for microbial enrichment efficiency.ResultsTo compare the host depletion and microbial enrichment efficiency of enzyme-based and adaptive sequencing methods, the present study collected clinical samples from eight children with respiratory tract infections. The same respiratory samples were subjected to standard methods, adaptive sequencing methods, enzyme-based host depletion methods, and the combination of adaptive sequencing and enzyme-based host depletion methods. We compared the host depletion efficiency, microbial enrichment efficiency, and pathogenic microorganisms detected between the four methods. We found that adaptive sequencing, enzyme-based host depletion and the combined methods significantly enriched the microbial sequences and significantly increased the diversity of microorganisms (p value < 0.001 for each method compared to standard). The highest microbial enrichment efficiency was achieved using the combined method. Compared to the standard method, the combined method increased the microbial reads by a median of 113.41-fold (interquartile range 23.32–327.72, maximum 1812), and the number of genera by a median of 70-fold (interquartile range 56.75–86.75, maximum 164). The combined method detected 6 pathogens in 4 samples with a median read of 547, compared to 5 pathogens in 4 samples with a median read of 4 using the standard method.ConclusionThe combined method is an effective, easy-to-run method for enriching microbial sequences in clinical metagenomics from sputum and bronchoalveolar lavage fluid samples and may improve the sensitivity of clinical metagenomics for other host-derived clinical samples.
Highlights
Enzyme-based host depletion significantly improves the sensitivity of clinical metagenomics
We showed a median of 113.41-fold microbe enrichment using the combination method of enzyme-based host depletion and adaptive sequencing
For the “unblock” and “no decision” reads, we found that except for the P4 and P6 samples, the “unblock” reads and bases were higher than the “no decision” reads and bases in the Host depletion with adaptive sequencing (HD_ADS) group (Fig. S3B)
Summary
Enzyme-based host depletion significantly improves the sensitivity of clinical metagenomics. Few studies have compared the enzyme-based host depletion and nanopore adaptive sequencing for microbial enrichment efficiency. A high background of host DNA in clinical samples impedes the detection of pathogens [9]. This shortage of clinical metagenomics may be overcome via microbe enrichment [3, 9,10,11]. The direct enrichment of viruses using spiked primers achieved a median of tenfold enrichment [15] This type of method only enriches limited number of microbes, which nullifies the major advantage of metagenomic sequencing. Experimental host DNA depletion methods enable relative microbe enrichment [3, 16, 17]. Charalampous et al used the saponin-based differential lysis method to deplete host DNA, which resulted in a maximum 104-fold depletion of host DNA and maximum 100-fold enrichment of microbe DNA [3]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
