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Shotgun metagenomic sequencing or metagenomic whole genome sequencing is a genome-wide sequencing approach to explore bacterial communities directly from their habitat or sites of infection. However, host DNA contamination in metagenomic sequencing overwhelm low biomass of microbial signals and decrease sensitivity for microbial detection. In this study, we evaluated the host DNA depletion efficiency of four different microbiome DNA enrichment methods (NEBNext Microbiome DNA Enrichment kit, Molzym Ultra-Deep Microbiome Prep, QIAamp DNA Microbiome kit and Zymo HostZERO microbial DNA kit) in diabetic foot infection (DFI) tissue samples using quantitative real-time PCR and their effect on bacterial community composition by 16S ribosomal RNA amplicon sequencing. The host DNA depletion ratio (18S/16S rRNA), the percentage of bacterial DNA component and the microbial community profile of DFI were compared before (control) and after each microbiome DNA enrichment method. There was a significant difference in the 18S/16S rRNA ratio among different microbiome DNA enrichment methods (p <.001). QIAamp and HostZERO method reduced 18S/16S rRNA ratio by 32 and 57 fold than control method respectively. The percentage of bacterial DNA component increased more than ten-fold in QiaAmp (71.0 ± 2.7%) and HostZERO (79.9 ± 3.1%) method respectively than those in control method without host DNA depletion (6.7 ± 0.1%). It demonstrated the host DNA contamination was efficiently depleted and bacterial DNA was effectively enriched in HostZERO and QIAamp methods, attesting to the efficacy of these two methods in shotgun metagenomic sequencing studies. Overall, bacterial community composition of DFI samples was similar between control and microbiome enriched DNA samples.
- 16S rRNA sequencing, microbiome
- Diabetic foot infection
- DNA extraction
- Host DNA depletion
- Metagenomic whole genome sequencing
- Microbiome DNA enrichment
- Shotgun metagenomic sequencing
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- 2 Finished
1/01/17 → 30/06/19