For mice and rats, we recommend placing the animal in an empty autoclaved cage (containing no bedding) and waiting until the animal defecates naturally. Using sterile toothpicks or syringe needles, spear the fecal pellet and place the sample directly in a sterile 1.5 mL Eppendorf tube, pre-labeled with any identifying information (e.g., animal ID, treatment group, etc.). Once samples have been collected, close tube and transfer immediately to a -80°C freezer. The main reason for prompt cooling of samples is to prohibit any proliferation of rapidly dividing bacteria and subsequent skewing of data. See also Sample Collection SOP.
Samples should be evacuated no more than a few minutes prior to sample collection. When collecting samples from a cage (even seemingly “fresh” samples), there is the chance that certain bacterial species with rapid doubling times will proliferate, resulting in falsely elevated levels. Additionally, there is nuclease activity in fecal material which can lead to degeneration of microbial DNA. Prompt collection and cooling of samples will minimize the nuclease activity and optimize the quality of DNA extracted from samples.
Regarding the quantity of fecal material, we request a minimum of two normal fecal pellets per mouse sample, i.e., two fecal pellets per Eppendorf tube. For rats, we request one normal fecal pellet per sample. For other species, please contact us to discuss an appropriate amount for sample collection.
Due to the inherent variability in species richness and composition of fecal samples, the answer to this question depends on the animals in question and the goals of the project. We recommend that you contact us to discuss your study. You may also want to view the Example Data for an idea of the inherent variability in the sequencing data generated from a set of fecal samples from sex-, age-, strain-, and vendor-matched mice.
We have not performed experiments to answer this question definitively. To minimize any potential for a confounding effect, we recommend that samples that have come in contact with urine or other biological materials be discarded and fresh samples collected.
No, we have not seen any appreciable difference in the microbial profile of sequential fecal pellets from the same mouse.
We have seen diurnal fluctuations in the composition of the fecal microbiota and thus recommend collecting all samples at a uniform time of day to minimize any such differences.
Samples should be shipped overnight in a container with sufficient dry ice to ensure that samples remain completely frozen during transport. Samples should be shipped to:
Attn: Aaron Ericsson
MU Metagenomics Center
University of Missouri
4011 Discovery Drive
Columbia, MO 65201
Turn-around times will depend on our current sample load. Our goal is to return data to users within 4 to 6 weeks of sample submission.
Along with the raw data, we provide several standard bioinformatics analyses. Sequence data will be annotated to a database of known microbial 16S rRNA gene sequences. Thus, the abundance of microbes at each taxonomic level (i.e., phylum, class, order, etc.) will be depicted in bar chart form with the relative abundance of each taxa listed below in a tabular format. Multiple principal component analyses (PCA) will be performed for the first three principal components. Two-dimensional PCAs depicting PC1 v. PC2, PC1 v. PC3, and PC2 v. PC3 will be generated using weighted and unweighted algorithms. Additionally, a three-dimensional representation of PC1, PC2, and PC3 will be provided. Rarefaction curves will be generated to provide an estimate of the adequacy of sequencing depth, as well as overall species richness. Rarefaction curves will be generated using multiple algorithms (chao1, observed species, PD whole tree, and Shannon). The Example Data will give you an idea of the data provided.
We do not routinely provide any biostatistical analyses beyond the informatics described above. Post hoc biostatistical analyses (e.g., high definition heat maps or correlation to phenotypic data) are very time-intensive and typically require consultation with an experienced biostatistician. We are happy to provide consultation regarding what types of analyses would be of use to your projects.
Our PCR primers and sequencing platform allow us to annotate many sequences to the species level, however, many are resolved to the genus, family, or higher level. Thus, we refer to the lowest level of annotation as an “operational taxonomic unit” or OTU. The Example Data shows the OTU-level annotation from a typical murine fecal sample.
Raw sequence data will be maintained in perpetuity.
We can archive fecal samples at -80°C for an additional fee. Please contact us prior to sample submission to discuss the number of samples and cost of sample storage.
While not part of the standard output, FASTQ files can be provided.
This will obviously depend on the number of samples submitted and type of sequencing performed. We are able to sequence up to 96 samples per lane, resulting in the generation of approximately 1GB of data, including the informatics analyses (bar charts, PCAs, rarefaction curves), from 16S rRNA amplicon sequencing. One lane of whole genome shotgun sequencing typically generates several GB of data.
You will require current versions of Java and Google Chrome to view the informatics analyses (bar charts, PCAs, rarefaction curves). A current version of Excel is required for manipulation of the raw data and taxa abundances.
Yes, we can re-annotate sequence data at an additional cost. Please contact us to discuss if you have questions regarding the cost and/or benefit of such analyses.
We have worked with fecal samples from other species but would request that you contact us to discuss your project goals before submitting samples from species other than mouse or rat.
We generally acquire between 80,000 and 120,000 reads per sample, although this may vary depending on the amount and quality of DNA extracted from the original sample. If you require deeper sequencing, we recommend that you contact us to discuss your project goals.
The primers we use target the hypervariable V4 region of the 16S rRNA gene and result in an approximately 300 bp amplicon.
For 16S rRNA amplicon sequencing, we use the Illumina MiSeq platform in conjunction with a set of 96 bar-coded primers.
Contiguous DNA sequences are assembled using FLASH software and representative OTUs are selected using Qiime v1.8. Selected OTUs are annotated to the Greengenes database of 16S rRNA gene sequences using BLAST.