QIAamp BiOstic Bacteremia DNA Kit (50)

Protocol tips

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	Use of additional blank negative controls with no sample to exclude DNA contamination during extraction and from reagent DNA traces

Protocol tips
Use of additional blank negative controls with no sample to exclude DNA contamination during extraction and from reagent DNA traces

Publication protocol

DNA was extracted by using four commercial DNA extraction kits. The first was the Maxwell® 16 FFS DNA Purification kit referenced as Maxwell (Promega, Charbonnière-les-Bains, France). This method required the Maxwell® 16 Instruments. This automat purifies samples using paramagnetic particles providing a mobile solid phase that optimizes DNA capture, washing and elution through purification reagents in prefilled cartridges. This purification system allows an automatic, standardized and simultaneous extraction of 16 samples at the same time. To our knowledge, no metabarcoding studies focused on CSS and FPP surface bacteria have been performed using this DNA Maxwell® 16 system extraction method. Two kits were purchased from Qiagen company (Qiagen, Courtaboeuf, France): Qiagen DNeasy PowerFood Microbial (formerly known as Mobio Powerfood Microbial) and Qiagen QIAamp BiOstic Bacteremia, referenced as QPFM and Q2B2, respectively. The fourth kit used, Zymo Quick-DNA Fecal/Soil Microbe Miniprep Kit referenced as ZFS, was purchased from Zymo (Ozyme, Saint-Cyr-l’Ecole, France). Qiagen and Zymo kits have already been used in previous surface and food microbial ecology studies (Bokulich et al., 2015; Stellato et al., 2016). The DNA purification technology is based on DNA selective binding to a spin column containing silica-based membrane, followed by several washing steps and a DNA elution. Both Qiagen kits use the MoBio PCR inhibitors removal technology. A first step of mechanical cell lysis using glass beads was achieved using a FastPrep (MPbiomedicals, Illkirch, France) for 30 s at a frequency of 6 m/s. When the Qiagen kits were used, the mechanical lysis was performed using glass beads provided by the kits. For the other DNA extraction methods, Maxwell and Zymo, 0.3 g of zirconium beads (100 μm diameter) were used (Scientific Industries, New-York, USA). DNA was extracted from three biological replicates from each batch. A Qubit® 2.0 fluorometer using the Qubit® dsDNA BR Assay Kit (Life technologies, Thermo Fisher Scientific, Villebon-sur-Yvette, France) was used to quantify DNA. Additional blank negative controls with no sample were used to exclude DNA contamination during extraction and from reagent DNA traces.

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Manufacturer protocol

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