Animal Housing Conditions
The animals used in this exploratory study will be Peromyscus maniculatus (deer mice), Mus musculus (C57/B6 mice), and Rattis norvegicus (Fischer rats). deer mice, C57/B6 mice, and Fischer rats were held in BSL2 cages, equipped with ventilation systems, at the University of Northern Colorado Animal Facilities. BSL2 cages The deer mice were held in cages of two with each cage containing female mice from the same litter. The C57/B6 mice were held in cages of four with each cage containing female mice from the same litter. And the Fischer rats were held in cages of two with each cage containing female rats from the same litter. The rodents were maintained in a room temperature room with a light to dark ratio of 12:12. Each cage had Envigo Teklad laboratory grade sani-chips as bedding. All rodents were fed ad libitum with the food and water being replenished every morning. The H2O container volume per cage was about 200 mL. All rodents were fed Envigo Teklad 16% protein rodent diet and supplied tap water. All rodents were taken out of their cages once a week and transferred to a clean cage with fresh bedding. The prairie rattlesnakes, Syrian golden hamsters, and Jamaican fruit bats were also kept at the University of Northern Colorado Animal Facilities. All animals apart of this research come from established mice, rat, bat, and snake colonies from the University of Northern Colorado.
Fecal Sampling
The fecal sampling procedure remained the same for the deer mice, C57/B6 mice, and Fischer rats. The rodents were removed from their cages one by one and placed in a different sterile cage when the fecal sample was needed. Once there was fecal matter to collect in the cage, the rodent was placed back in her original cage. The fecal matter was collected using forceps and placed in a 2mL tube. This procedure was performed for all four samples of each species of rodent.
The fecal matter was collected using the procedure above for the Syrian golden hamsters. For the snakes, droppings from each individual prairie rattlesnake’s cages were removed from the snake cages. Forceps were used to cut the feces of the snakes into more sizeable pieces and to find fecal matter with less mouse fur. The fecal sample was then placed into a 2mL tube. In order to sample the bats, an anal swab was taken using a scientific swab and was then directly submerged in the Powerbead Tube from the DNA extraction kit. The swab was twisted around inside of the Powerbead Tube to allow material and DNA from the swab to transfer into the tube for the first step of the DNA extraction process.
The fecal matter was collected using the procedure above for the Syrian golden hamsters. For the snakes, droppings from each individual prairie rattlesnake’s cages were removed from the snake cages. Forceps were used to cut the feces of the snakes into more sizeable pieces and to find fecal matter with less mouse fur. The fecal sample was then placed into a 2mL tube. In order to sample the bats, an anal swab was taken using a scientific swab and was then directly submerged in the Powerbead Tube from the DNA extraction kit. The swab was twisted around inside of the Powerbead Tube to allow material and DNA from the swab to transfer into the tube for the first step of the DNA extraction process.
DNA Extraction
The Qiagen DNEasy PowerSoil Kit was used to extract the DNA from all fecal samples. The manufacturer's instructions were followed. For more information about the Qiagen product, click this link: https://www.qiagen.com/us/products/kits-for-instruments/qiacube-connect/power-kits/dneasy-powersoil-kit/#orderinginformation
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DNA Purity Test
A ThermoFischer Scientific NanoDrop 2000/2000c was used to conduct all of the DNA purity measurements. For more information about the ThermoFischer Scientific product, click this link: https://www.thermofisher.com/order/catalog/product/ND-2000 To make sure the DNA was pure enough to be sent to Novogene in China, we needed the following measurement values:
- Atleast 5 ng/μL of nucleic acid: This is the amount Novogene needs to sequence the sample of DNA.
- 260/280 ratio measurement of about 1.8: This denotes that the DNA sample is pure. If the measurement is significantly lower or higher, it could indicate contamination in the sample such as protein, phenol, and others.
- 260/230 ratio measurement of about 2.0 to 2.2: This value shows that the nucleic acid in the DNA sample is pure. If the value is significantly lower or higher, there are probably mistakes in the structure of the nucleic acid itself. For instance, if the ratio is lower, it might mean that the contaminant in the sample absorbs at the lower frequency of 230nm as opposed to the 260nm that the nucleic acids absorb at.
Novogene Process
Novogene is a company in China and they were able to sequence our data from the DNA we sent them. They sequenced the 16S gene at the V4 variable region. They implement their own pre-sequencing process which can be seen in their flow chart and they they specifically used ion torrent sequencing on our DNA samples.