DNA was prepared using the QIAamp UCP DNA Micro Kit and kits from other suppliers. The 16S gene was amplified using QuantiTect SYBR Green PCR Master Mix treated with propidium monoazide (PMA) to minimize DNA background from the PCR step. The QIAamp UCP DNA Micro Kit’s proprietary decontamination process ensures minimal background compared with kits from other suppliers. 30 columns from each supplier were tested and contamination was defined as >62.5 16S copies (relative to standard curve) in the eluate.
DNA was prepared using kits from various suppliers, as per manufacturer’s instructions and 100 copies of two different bacterial DNAs (Escherchia coli and Bacillus subtilis) were spiked into the eluates to allow quantification of the bacterial sequencing reads. The 16S gene was amplified using the QuantiTect Probe PCR Master Mix (QIAGEN). A library was constructed and sequenced in triplicates using next-generation sequencing on the Illumina MiSeq. The QIAamp UCP DNA Micro Kit had fewer microbial background reads compared with kits from other suppliers. A Bacterial reads as a percentage of total reads. Spiked bacterial reads are shown in dark blue, other (background) reads in light blue. B Further analysis of the microbial community at the family level, excluding spike. Results shown as a percentage of total reads.
Yields obtained from 10 µl whole blood using the QIAamp UCP DNA Micro Kit and standard (non-ultraclean) sample prep kits from various suppliers. The yields were comparable between all four methods.