Introduction

This protocol describes a method to extract genomic DNA (gDNA) from pond water as an example of an environmental water sample. We tested multiple extraction kits and compared performance using ZymoBIOMICS Microbial Community Standard, a commercial sample of known composition.

Our initial experiments were carried out using ZymoBIOMICS Microbial Community Standard diluted in sterile water and filtered through a vacuum before the gDNA was extracted directly from the filter. We tested the extraction kits:

• ZymoBIOMICS DNA Miniprep Kit
• Zymo Quick-DNA HMW MagBead Kit
• QIAGEN MagAttract
• QIAGEN Puregene
• QIAGEN PowerWater

The extraction kit that yielded the community representation closest to the expected control was ZymoBIOMICS DNA Miniprep Kit. All the other methods showed a deviation from the expected species distribution.

To validate the method further, we used a sample of water from a residential garden pond (containing a known fish species). The sample was extracted and prepared for sequencing with and without PCR amplification using the Rapid PCR Barcoding Kit and the Ligation Sequencing Kit, respectively. DNA was also amplified using QIAGEN REPLI-G Midi kit for whole genome amplification following our recommended protocol; Ligation sequencing gDNA - whole genome amplification. All samples were sequenced on MinION and the reads generated were analysed using Centrifuge run on raw reads. Results showed that the expected fish species were detected in all samples. In addition, it was also possible to detect other eukaryotic species that are present in the garden such as rodents, birds, and plants, alongside diverse species belonging to the rich microbial community that can be found in an environmental sample.

Materials

• ZymoBIOMICS Microbial Community Standard
• ZymoBIOMICS DNA Miniprep Kit
• Vacuum filter unit (e.g. Thermo Scientific™ Nalgene™ Reusable Filter Units)
• Vacuum pump
• 0.2 µm cellulose nitrate filter (e.g. Cytiva Whatman™ Cellulose Nitrate Membrane Filters)
• ZR BashingBead Lysis Tubes
• Tweezers
• 1.5 ml Eppendorf tubes
• Vortex mixer
• Microcentrifuge

Method

1. Prepare the vacuum filter unit with a 0.2 µm cellulose nitrate filter.
2. Pour 500 ml of pond water through a vacuum filter unit.
3. Once all the solution has passed through vacuum, disassemble the vacuum filter unit, and use tweezers to remove the filter.
4. Carefully roll the filter with tweezers and transfer it directly to a ZR BashingBead™ Lysis Tube.
5. Add 750 µl of ZymoBIOMICS™ Lysis solution, and ensure the tube is properly closed.
6. Place the tube in a tube holder, vortex adapter or simply tape the tubes to the vortex. Agitate at maximum speed for 40 minutes.
7. Follow the recommended protocol from steps 4-13 (pages 6-7 from the ZymoBIOMICS DNA Miniprep kit handbook.

Results

DNA yield: 1.5 - 2 µg OD A260/280: 2.73 OD A260/230: 0.49

Sequencing performance

• Read length profile:

Species identification

Figure 1. ZymoBIOMICS Microbial Community Standard species abundance (percentage of base pairs) according to the theoretical composition and the different kits that were tested.

Figure 2. A Krona graphical representation of the species detected with Centrifuge on the pond sample. The number of unclassified reads is within expectation for a real sample as there will be species present for which a reference genome does not yet exist. Sequencing the native DNA shows the microbial and eukaryotic species diversity present in this pond.

Figure 3. A representation of the species (minimum abundance cut-off of 0.1%) detected with EPI2ME FASTQ WIMP (What’s In My Pot) workflow. WIMP provides the identification of bacteria, fungi, viruses, and archaea, being ideal for users focused on microorganisms.