A Rapid Bacteriophage DNA Extraction Method

Bacteriophages (phages) are intensely investigated as non-antibiotic alternatives to circumvent antibiotic resistance development as well as last resort therapeutic options against antibiotic resistant bacteria. As part of gaining a better understanding of phages and to determine if phages harbor putative virulence factors, whole genome sequencing is used, for which good quality phage DNA is needed. Traditional phage DNA extraction methods are tedious and time consuming, requiring specialized equipment e.g., an ultra-centrifuge. Here, we describe a quick and simple method (under four hours) to extract DNA from double stranded DNA (dsDNA) phages at titers above 1.0 × 1010 plaque-forming units (PFU)/mL. This DNA was suitable for library preparation using the Nextera XT kit and sequencing on the Illumina MiSeq platform.


Introduction
Bacteriophages (phages) are of great interest because of their ability to specifically attack bacteria. Phages are approved to be used in the food industry to control foodborne pathogens [1]. Moreover, animal and human clinical trials have demonstrated that phages can be efficiently used as alternative to antibiotics [2], including treatment of infections caused by antibiotic resistant bacteria. However, in order to ensure the safety of the phages, i.e., phages do not harbor putative virulence factors or antibiotic resistance genes that could be transferred to bacteria, whole genome analyses are performed [2], requiring extraction of phage DNA. Traditional extraction methods include precipitation of phages, ultra-centrifugation, the use of hazardous solutions, e.g., chloroform, phenol [3], and are time consuming, e.g., the protocol described in Sambrook takes two days [4]. While viral DNA extraction kits based on spin column technology are commercially available, we describe a simple and fast method using the spin column based DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany), which is used to extract DNA from blood, tissues, bacterial lysates, insects, and yeast. We chose this kit since it is our preferred in-house kit for extraction of bacterial DNA for whole genome sequencing. We applied a modified protocol to extract DNA from streptococcal phages. For propagation of our phages, we used a double agarose overlay plaque assay as previously described [5] with some modifications. It should be noted that this method may not apply to all phages or bacteria, hence users should use their preferred method of phage propagation.

Experimental Design
(a) To prepare the bottom layer, BHI broth containing a final concentration of 0.8% agarose is heated to melt the agarose, and then cooled to 56 • C before adding CaCl 2 to a final concentration of 5 mM. Twenty-five milliliters are dispensed to sterile petri dishes. Solidified plates are stored at 4 • C until use.
(b) To prepare the top layer, BHI broth containing a final concentration of 0.4% agarose is heated to melt the agarose, and then cooled to 56 • C before adding CaCl 2 to a final concentration of 5 mM.

Propagation of Streptococcal Phages by the Plate Method
For propagation of our phages, we used a double agarose overlay plaque assay as previously described [5] with some modifications. It should be noted that this method may not apply to all phages or bacteria, hence users should use their preferred method of phage propagation.
(a) To prepare the bottom layer, BHI broth containing a final concentration of 0.8% agarose is heated to melt the agarose, and then cooled to 56 °C before adding CaCl2 to a final concentration of 5 mM. Twenty-five milliliters are dispensed to sterile petri dishes. Solidified plates are stored at 4 °C until use.
(b) To prepare the top layer, BHI broth containing a final concentration of 0.4% agarose is heated to melt the agarose, and then cooled to 56 °C before adding CaCl2 to a final concentration of 5 mM.
(c) Prepare SM buffer containing gelatin as described previously [5]. Transfer the mixture to the DNeasy Mini spin column placed in a 2 mL collection tube (provided with the kit) and centrifuge for 1 min at 6000× g. If larger volumes are used, repeat this step several times. Do not load more than 750 µL at a time onto the column. Discard the flow-through and the collection tube.

4.
Place the DNeasy Mini spin column in a new 2 mL collection tube (provided), add 500 µL Buffer AW1, and centrifuge for 1 min at 6000× g. Discard the flow-through and the collection tube.

5.
Place the DNeasy Mini spin column in a new 2 mL collection tube (provided), add 500 µL Buffer AW2, and centrifuge for 3 min at 20,000× g to dry the DNeasy membrane. Discard the flow-through and the collection tube. Place the DNeasy Mini spin column in a new 2 mL collection tube (provided) and centrifuge for an additional 1 min at 20,000× g to make sure there is no carryover of ethanol. 6.
Place the DNeasy Mini spin column in a sterile 1.5 mL or 2 mL microcentrifuge tube (not provided in the kit), and pipette 30 µL AE Buffer directly onto the DNeasy membrane. 7.
Incubate at room temperature for 1 min, and then centrifuge for 1 min at 6000× g to elute the DNA. OPTIONAL STEP Repeat the elution step using the eluate from the first elution.
The concentration and quality of the DNA was assessed using the Nanodrop and Qubit according to the manufacturer's instructions. Five microliters of DNA was also run on a 1% agarose gel. Preparation of the sequencing libraries was done according to Kot et al. [7] and sequenced on the Illumina MiSeq platform [8] using MiSeq Reagent Kit v2 (500-cycles) and manufacturer's instructions.

Expected Results
We successfully extracted DNA from 16 phages active against Streptococcus uberis. The DNA quality and quantity are shown in Table 1 as measured with the Nanodrop, Qubit, and by gel electrophoresis (Figure 1). We have also included the results for the Salmonella (S112) and Escherichia coli (EC11 and EC80) phage DNA extracted using our method for comparison. The DNA concentration varied from 4.56-287.35 ng/µL. There was no linear correlation observed between the starting concentration of phages and the quantity of DNA extracted. Based on our results, we observed that the optimal concentration of our phages for successful extraction of DNA was above 1.0 × 10 10 PFU/mL, as no measurable DNA was obtained when titres lower than this were used (e.g., SU11, starting titer = 6.0 × 10 9 PFU/mL, nanodrop concentration = −8.59 ng/µL). The purity of DNA was determined by the A260/280 ratio. Using this measurement, 16/19 DNA samples were of high quality. The integrity of the extracted DNA was determined by gel electrophoresis (Figure 1). An intact band suggests that the DNA is not contaminated with RNA nor degraded.
our results, we observed that the optimal concentration of our phages for successful extraction of DNA was above 1.0 × 10 10 PFU/mL, as no measurable DNA was obtained when titres lower than this were used (e.g., SU11, starting titer = 6.0 × 10 9 PFU/mL, nanodrop concentration = −8.59 ng/µL). The purity of DNA was determined by the A260/280 ratio. Using this measurement, 16/19 DNA samples were of high quality. The integrity of the extracted DNA was determined by gel electrophoresis (Figure 1). An intact band suggests that the DNA is not contaminated with RNA nor degraded. Figure 1. Five µL of extracted DNA was loaded on 1% agarose gel. L-GeneRuler 1 kb DNA Ladder (250-10,000 bp, Thermo Scientific, Roskilde, Denmark); S13-S16 phage DNA.
Despite the low quality scores for a few samples, we were able to prepare libraries and sequence on the Illumina Miseq platform all phage DNA samples. De novo sequence assembly of the trimmed raw reads was performed in the CLC Genomics Workbench v.7 (Qiagen Hilden, Germany) and for each phage, ≥99.85% of reads were assembled into one contig representing the phage genome (Table 2).  Despite the low quality scores for a few samples, we were able to prepare libraries and sequence on the Illumina Miseq platform all phage DNA samples. De novo sequence assembly of the trimmed raw reads was performed in the CLC Genomics Workbench v.7 (Qiagen Hilden, Germany) and for each phage, ≥99.85% of reads were assembled into one contig representing the phage genome (Table 2).

Conclusions
The DNeasy Blood and Tissue kit from Qiagen is commonly used in research laboratories for extraction of bacterial or eukaryotic DNA. Here, we demonstrate that with some modifications it can also be used for purification of phage DNA that can be sequenced on the Illumina MiSeq platform.
Limitations to the technique. The quality and concentration obtained in this study was sufficient for sequencing on the Illumina Miseq platform. However, this DNA might not be of sufficient quality for use on other sequencing platforms such as non-amplification, long read technology e.g.,