[A] DNA (10 ng) was purified from buccal mucosa swabs with the QIAamp DNA Microbiome Kit or kits from 3 alternative suppliers and the V4 region was amplified. After 25 cycles, amplification of samples isolated with the QIAamp DNA Microbiome protocol was most efficient, demonstrating the benefit of host DNA removal. [B] After sequencing on the Illumina MiSeq platform, the V4 amplicons of 2 samples purified with the QIAamp DNA Microbiome Kit were mapped to reference genomes derived from the Ribosomal Database Project. Bacterial composition was representative of the human oral microbiome.
Differential lysis and enzymatic treatment remove host DNA during the purification process, enabling enrichment of bacterial microbiome DNA.
The optimized protocol of the QIAamp DNA Microbiome Kit reduces bias due to differential susceptibility of microbes to lysis. A model microbiome was created by adding 6 cultured bacterial species to buccal swab samples. Community composition was quantified by plate counts. Whole metagenome shotgun sequencing was performed on the Illumina MiSeq platform and output reads were compared to input amounts. Relative composition was maintained most closely using the QIAamp DNA Microbiome Kit for sample preparation.
DNA isolated from buccal swabs using the QIAamp DNA Microbiome Kit or solutions from alternative suppliers was analyzed by whole metagenome shotgun sequencing on the Illumina MiSeq platform. [A] The percentage of human-derived reads was quantified and compared between the QIAamp DNA Microbiome Kit and alternative solutions with (Supplier B) and without (Supplier A) host DNA depletion protocols. Without host DNA removal, over 90% of reads mapped to the human genome. Compared to the kit from Supplier B, the effective removal of host DNA afforded by the QIAamp DNA Microbiome Kit resulted in over 90% reads for microbiome analysis. [B] To monitor recovery of bacterial species, buccal swab samples were spiked with an artificial sample of 6 bacterial species. Again, the effective host DNA removal achieved with the QIAamp DNA Microbiome Kit increases the coverage of bacterial species compared to a solution that affords no host DNA depletion (Supplier C).