Comparative Study of Host DNA Depletion Methods for Microbial Metagenomics

One of the biggest obstacles in applying metagenomics in the clinical field is the increase of human genome reads in the resulting sequences. In this study, three depletion methods were compared that utilize the methylation of cytosine in human DNA as a mechanism of action. Two commercial kits NEBNext Microbiome DNA Enrichment Kit and MethylMiner™ Methylated DNA Enrichment Kit bind methylated cytosines to specific antibodies, along with MspJI restriction enzyme that digests methylated cytosines. The depletion methods were applied to a HeLa cell suspension (as a source for the human genome) spiked with well-characterized bacterial isolates of Staphylococcus aureus, ‎‎Klebsiella pneumoniae and Cryptococcus neoformans. The products of the depletion methods were run in parallel with undepleted samples as controls on Miseq. The outcome of the NEBNext Microbiome DNA Enrichment Kit showed the most significant decrease in human genome (p = 0.005) compared to undepleted samples and a significant increase in both K. pneumoniae (p = 0.005) and S. aureus reads (p = 0.014). While MethylMiner™ Methylated DNA Enrichment kit showed a significant increase for C. neoformans (p = 0.007) besides a significant increase in the percent coverage of the references for C. neoformans and K. pneumoniae (p = 0.046 and p = 0.004, respectively). Results showed that both kits could help in depleting human DNA and enriching spiked pathogens in the study sample. The findings of this study are recommended to be validated on a large number of clinical samples.