Human methylated DNA removal method and comparison with Devin®

One of the constraints in sequencing-based methods of rapid pathogens detection is the presence of human DNA in the DNA samples. The size of human genome (3 billion bp) is very huge compared to the bacterial genome (average 4 million bp). So, the presence of a few human cells in the samples can result in overwhelming background noise in sequencing-based methods of microbial identification. So, there is a need to remove human DNA from the specimens. Without this the majority of the sequencing results would be generated from host instead of pathogen DNA. Nowadays there are several methods of human DNA removal, including differential cell size- based methods, chemical/enzymatic treatment methods etc. (Fig. 1) In this article we overview human methylated DNA removal methods and compare their efficacy with recently launched Devin® filter from Micronbrane Medical.

 

Figure 1. Contemporary Depletion Techniques

Abundance of methylated CpG domains in human DNA and extreme scarcity of CpG methylated sites in microbial DNA is utilized in many commercial methods to deplete host DNA via methylation. The commercial microbial DNA enrichment kits specifically target these sites to remove CpG human methylated DNA from clinical specimens. The method usually uses MBD2 (Methylated CpG specific binding protein) coupled with Fc region of human Immunoglobulin G. The Fc region of IgG binds with Protein A, that is coated on the magnetic beads. This is how human methylated DNA is selectively removed with the application of magnetic field leaving the non-methylated microbial DNA in the sample (Fig. 2).

Figure 2. A flow diagram showing removal of CpG methylated human DNA for microbial enrichment

New England Biolab’s NEBNext Microbiome DNA Enrichment kit utilizing method above claims to be able to remove human methylated DNA up to 94 % and increases the reads from 8 to 43 folds.

However, the efficacy of this method of microbial DNA enrichment has been tested in a research study which highlights certain limitations of this method (1). NEBNext® Microbiome DNA Enrichment kit was used for microbial enrichment from sino-nasal swabs. The study showed reproducible results (Confidence interval <100%), but the samples treated with NEBNext® Microbiome DNA Enrichment kit produced insufficient sequences for subsequent downstream analysis. Another drawback of this method reported is that the microbial community profile can be altered due to the magnetic field mediate removal of host DNA. The NEBNext Microbiome DNA Enrichment Kit can remove up to 95% of the human methylated DNA, but there are no significant increases in the amount of microbial DNA extracted as compared with control groups where microbial DNA was extracted using manual methods. Major limitation of this method is that it is expensive and requires high molecular weight genomic DNA input (> 15kb). This method is not suitable for specimens like saliva, serum, urine, plasma and samples that are likely to harbor no cellular portion (1).

The methylation dependent bacterial DNA enrichment requires pre-extraction of whole DNA from the clinical samples. The samples that do not have high cellular count such as saliva, urine and nasal swabs will result in small quantity of DNA. The minimum amount of DNA required for efficient removal of host DNA through CpG domains requires more than 3kb DNA, to which MBD-linked conjugated magnetic beads can bind. The low starting DNA materials result in inefficient binding of magnetic beads which in turn increase the background noise during sequencing and metagenomic analysis (5).

Other methods of microbial DNA enrichment include immunoprecipitation of host DNA that is mediated with methyl specific restriction enzymes. This method can remove human DNA up to 94% (2) but it requires long enzyme incubations hence no commercial kit working on this principle has been introduced into the market yet.

In comparison with human methylated DNA removal Devin® filter utilizes completely new technique of ZISC technology. Zwitterionic Interfaced Self-assemble Coating (ZISC) in Devin® membrane specifically binds with human leukocytes irrespective of the filter pore size and retain them without clogging the pores. The Devin® can deplete over 95% of human nucleated cells within just 5 minutes and possesses over 99% passing microbial efficiency (Fig. 3). Both bacterial cells and viral particles can easily pass through the filters (Fig. 4). Combined with PaRTI-Seq® it may increase microbial reads in the sample by 10-1000 folds compared with unfiltered samples. It is an efficient method for all types of samples, as it does not require pre-extraction of DNA so  there is no minimum limit of DNA required for microbial DNA enrichment (3).

Figure 3. Leukocyte reduction efficacy.

Figure 4. Microbial passing efficacy of Devin® filters

The NGS Nanopore sequencing and qPCR analysis of microbial DNA extracted via Devin® shows significant reduction in host contamination and improved microbial reads in NGS sequencing analysis as compared to methylation dependent human DNA removal and differential lysis method (4) (Fig. 5).

Figure 5. Comparison of efficacy of results of depletion method via NGS Nanopore sequencing and qPCR.

The comparison of microbial DNA extraction with Devin® and methylate dependent microbial DNA enrichment has been summarized in Table 1 below.

Table 1. comparison of Devin® and methylation dependent microbial enrichment method

Methyl dependent Microbial DNA enrichmentMicrobial DNA enrichment with Devin®
This method takes 120 minutes to complete the enrichment process and prior DNA extraction is needed for further treatment with MBD2-Fc protein.Can remove over 95% of human DNA contamination within just 5 minutes.
Microbial DNA profile can be altered due to application of magnetic field.No alterations in microbial DNA profile, because sample is not treated with magnetic beads.
Does not produce sufficient sequences for downstream analysis.Sufficient sequences are produced for any type of downstream analysis.
No suitable for samples i.e., saliva, serum urine and plasma.Suitable for all types of specimens.

 

CONCLUSION

Devin® microbial DNA enrichment method is an advanced method to remove background noise created due to human DNA in sequencing, metagenomic analysis and PCR based microbial detection in clinical samples. This method has high efficacy (99%) and takes significantly less time (5 minutes) as compared to methylation method which requires more than 120 minutes to process sample for microbial enrichment. Furthermore, there are chances of DNA damage in methylation dependent method because Host DNA is removed under the influence of magnetic field, which can greatly alter the bacterial profile. Devin® microbial DNA enrichment method promises damage free extraction of bacterial DNA.

 

 

REFERENCES

  1. Mackenzie, B. W., Waite, D. W., Biswas, K., Douglas, R. G., & Taylor, M. W. (2018). Assessment of microbial DNA enrichment techniques from sino-nasal swab samples for metagenomics. Rhinol. Online, 1, 160-193.
  2. Glassing, A., Dowd, S. E., Galandiuk, S., Davis, B., Jorden, J. R., & Chiodini, R. J. (2015). Changes in 16s RNA gene microbial community profiling by concentration of prokaryotic DNA. Journal of microbiological methods119, 239-242.
  3. Novel Human Cell Depletion Method Enables Rapid Pathogen Identi­cation by Next Generation Sequencing.
  4. Clarisse A. Marotz, Jon G. Sanders, Improving saliva shotgun metagenomics by chemical host DNA depletion; 2018 February 27
  5. Feehery, G. R., Yigit, E., Oyola, S. O., Langhorst, B. W., Schmidt, V. T., Stewart, F. J., … & Pradhan, S. (2013). A method for selectively enriching microbial DNA from contaminating vertebrate host DNA. PloS one, 8(10), e76096.

 

 

 

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