Standardised Method for Infection Diagnosis with NGS

An important and often discussed problem for microbiome analysis by Next Generation Sequencing (NGS) is the missing standardised workflow. The huge overload of host DNA in low microbial load specimens leads to diverse results and workflows for the same type of samples. The removal of host DNA can significantly increase the rate of microbial reads and provide deeper insights in the diversity of microbiomes. However, an increased sensitivity of microbial reads bears the risk of interference of DNA contaminants with the analysis. Thus, particularly pure reagents and consumables help to generate high quality data of NGS.

At European Congress of Clinical Microbiology and Infectious Diseases held in Madrid, Spain, in 2018, one topic of the poster sessions was 16S metagenomics. In that category, Edelmann and her team [1] presented a recent study comparing the impact of different DNA extraction kits regarding depletion of human DNA. All 4 DNA extraction kits were further analysed with two master mixes for bacterial DNA contaminations. The authors stated that DNA extraction and master mix reagents have a huge impact on the contamination signature in sequencing reads.

The following DNA extraction kits were compared: i) QIAamp® UCP Pathogen Mini Kit (Qiagen, Germany), ii) QIAamp® DNA Microbiome Kit (Qiagen, Germany), iii) QIAamp® UCP Pathogen Mini Kit in combination with Looxster® Enrichment Kit (Analytik Jena, Germany) and iv) automated MolYsis-SelectNA™plus kit (Molzym, Germany). Total DNA extraction of whole blood spiked with S.aureus showed only 0.1% of bacterial reads (i). The removal of non-target human DNA lead to a significant increase of bacterial reads of 51% (ii), 88% (iii) and 91% (iv), whereby, MolYsis-SelectNA™plus (iv) most efficiently depleted the human DNA.

Regarding the master mixes, superior contaminant purification was found for NGS Mastermix 16S V3/V4 (Molzym, Germany) in contrast to KAPA HiFi PCR Kit (Roche, USA). The number of bacterial contaminations (reads) with KAPA HiFi PCR Kit were multiple times over the one achieved with NGS Mastermix 16S V3/V4, but need to be viewed together with the DNA extraction kit.

The authors resumed that MolYsis-SelectNA™plus together with NGS Mastermix 16S V3/V4 aid an efficient workflow for NGS towards a standardised method in microbiome infection diagnosis.

For more detailed information please find a link to the poster in the reference.