A Method for Selectively Enriching Microbial DNA from Contaminating Vertebrate Host DNA

DNA samples derived from vertebrate skin, bodily cavities and body fluids contain both host and microbial DNA; the latter often present as a minor component. Consequently, DNA sequencing of a microbiome sample frequently yields reads originating from the microbe(s) of interest, but with a vast excess of host genome-derived reads. In this study, we used a methyl-CpG binding domain (MBD) to separate methylated host DNA from microbial DNA based on differences in CpG methylation density. MBD fused to the Fc region of a human antibody (MBD-Fc) binds strongly to protein A paramagnetic beads, forming an effective one-step enrichment complex that was used to remove human or fish host DNA from bacterial and protistan DNA for subsequent sequencing and analysis. We report enrichment of DNA samples from human saliva, human blood, a mock malaria-infected blood sample and a black molly fish. When reads were mapped to reference genomes, sequence reads aligning to host genomes decreased 50-fold, while bacterial and Plasmodium DNA sequences reads increased 8–11.5-fold. The Shannon-Wiener diversity index was calculated for 149 bacterial species in saliva before and after enrichment. Unenriched saliva had an index of 4.72, while the enriched sample had an index of 4.80. The similarity of these indices demonstrates that bacterial species diversity and relative phylotype abundance remain conserved in enriched samples. Enrichment using the MBD-Fc method holds promise for targeted microbiome sequence analysis across a broad range of sample types.

源自脊椎动物皮肤、体腔及体液的DNA样本，同时包含宿主与微生物DNA；其中微生物DNA通常占比极低。因此，微生物组样本的DNA测序往往会同时获得目标微生物的测序读段，但宿主基因组来源的读段占绝大多数。在本研究中，我们基于CpG甲基化密度的差异，利用甲基-CpG结合域（methyl-CpG binding domain, MBD）分离甲基化的宿主DNA与微生物DNA。将融合了人抗体Fc区域的MBD（MBD-Fc）与蛋白A顺磁微珠强力结合，形成高效的一步法富集复合物，用于从细菌及原生生物DNA中去除人类或鱼类宿主DNA，以便后续进行测序与分析。我们对人类唾液、人类血液、模拟疟疾感染的血液样本以及黑摩利鱼的DNA样本开展了富集实验。当将测序读段比对至参考基因组时，比对至宿主基因组的序列读段数量减少了50倍，而细菌与疟原虫的DNA序列读段数量则提升了8至11.5倍。我们针对唾液中的149种细菌物种，计算了富集前后的香农-威纳多样性指数（Shannon-Wiener diversity index）。未富集的唾液样本该指数为4.72，富集后的样本则为4.80。这两个指数的相近性表明，富集后的样本中细菌物种多样性与相对系统型丰度得以保留。采用MBD-Fc方法的富集技术，有望在多种样本类型中实现靶向微生物组测序分析。