Molzym Blog

Comparison of Microbial DNA Enrichment Tools for WGS

27 April 2018

blog 36438728 s Banden 142x142In this study, Thoendel et al. [1] addressed the problem of generation of vast amounts of non-target reads by human sequences in metagenomic whole genome sequencing (MWGS) of low load microbial infections. For this purpose, two commercially available microbial DNA enrichment kits, NEBNext® Microbiome DNA Enrichment kit (New England Biolabs, USA) and the MolYsis™ Basic kit (Molzym, Germany) have been compared on MWGS of sonicated fluids from prosthetic joint infections (PJI). The NEBNext® Microbiome DNA Enrichment kit separates microbial DNA by binding high CpG methylated human DNA to modified Protein A-bound magnetic beads. The other kit, MolYsis™ Basic utilizes selective lysis of human cells and enzymatic degradation of human DNA prior to extraction of microbial DNA. For both tested kits, DNA isolation was performed with Mobio BiOstic Bacteremia DNA Isolation Kit (Qiagen, Germany). The isolated DNA was amplified by multiple displacement amplification with the Illustra GenomiPhi V2 whole genome amplification kit (GE Healthcare Bio-Sciences, USA). This led to sufficient amounts of DNA for library preparation. Sequencing was performed on the Illumina HiSeq 2500.

Compared to qPCR of total DNA extracts of S. aureus-spiked samples, enrichment factors of 6 (NEBNext®) and 76 (MolYsis™) were observed. MWGS analysis of PJI samples without enrichment showed that 0.0062 to 0.016% of the total reads were from known pathogens. Enrichment with the NEBNext® and MolYsis™ improved this yield to 0.18 to 0.53% and 7.0 to 59.4%, respectively. These results represent a 13- to 85-fold (NEBNext®) and 481- to 9580-fold (MolYsis™) enrichment of bacterial DNA. Thus, both methods were able to enrich bacterial DNA from PJI sonicate samples, although the MolYsis™ kit provided the greater enrichment factor and more bacterial sequencing reads in MWGS.

The authors concluded that microbial DNA enrichment methods such as those tested are powerful tools for the identification and characterization of pathogenic or commensal bacterial metagenomes.

[1] Thoendel M,Jeraldo PR, Greenwood-Quaintance KE, Yao JZ, Chia N, Hanssen AD, Abdel MP, Patel R (2016) Comparison of microbial DNA enrichment tools for metagenomic whole genome sequencing. J Microbiological Methods 127, 141-145.(link)
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