The following FAQ topics can be expanded to provide guidance as you navigate through submission to data retrieval:
1. If some of my samples fail to amplify can the lab try other conditions to improve amplification?
Yes, we will ask whether you would like us to troubleshoot your samples. Additional charges apply.
2. Are there protocols I can follow to prepare my samples for submission/shipping?
Yes, please refer to the relevant service’s page for links to detailed instructions.
3. Is there a form to fill out prior to submitting samples for sequencing?
Yes, please login to your MiCores account and fill out a service request.
4. How do I get a MiCores account?
Please consult this User Help Guide to sign up for a MiCores account.
5. How long will you keep my DNA (submitted or extracted)?
We will keep DNA for 60 days after submission; samples will be disposed after this period. If you are an external client and have sent in samples for DNA extraction, we will ship your DNA back at cost to you. For internal clients, please pick up DNA when receiving your USB containing the data. We are not responsible for DNA that was not claimed and disposed after the grace period.
It is best practice to submit an aliquot of your DNA for submission and keep a set for yourself.
6. Can I use your services if I am not affiliated with the University of Michigan?
Yes, we do take external clients. There is a surcharge for external clients.
7. What is the Illumina Nano Kit?
The MiSeq Nano kit is a smaller version of the full 500-cycle Illumina kit we use for 16S rRNA gene sequencing. It yields fewer reads (~1.5 Million reads vs the full 500v2 kit’s estimated 20 Million reads), but this output is enough for data analysis. This kit is a great option for clients submitting a single plate.
8. Do you have a template for the plate map I need to submit electronically?
Yes, the plate map can be found here.
9. Why am I asked if I do metabolic mouse work on the submission form?
We are part of the National Mouse Metabolic Phenotyping Centers (MMPC). The MMPC is a NIH-sponsored resource that provides experimental testing services to scientists studying diabetes, obesity, diabetic complications, and other metabolic diseases in mice. By initiating your order through the MMPC, your submission ensures continued financial support for the Core services our lab provides.
Nucleic Acid Extraction
1. Do I have to add my samples to the Bead Plate or do you do it?
We do not add samples to the Bead Plate. We feel it is best that the client adds the samples as this reduces the risk of error.
2. How do I get a Bead Plate?
Instructions can be found in our MiCores service request forms (specifically in ‘Community Analysis Submission’ or ‘Stand-alone DNA Isolation Service’).
3. How much sample should I add to each well of the Bead Plate?
250 uL or 0.25 g can be added to each well. Please seal well with the provided silicone mat provided before returning to the Core.
4. Where should I add my samples to the Bead Plate?
A biohazard hood using appropriate personal protective equipment.
1. What are your average reads per sample?
For samples that have amplified well, the average is 20K reads per sample on the Nano 500v2 flowcell and 15-20K per sample on the standard 500v2 flowcell. However, this cannot be guaranteed as many factors can influence average reads/sample.
2. Do I have to pay for an entire plate if I am submitting less?
Yes, if you do not have an entire plate you are still responsible for the cost of the entire plate. On the plus side, you will receive more coverage of those samples than if you submitted a full plate.
3. Should I quantify my DNA?
It is not necessary, but recommended. If quantifying the sample, please use a fluorescence-based assay such as Picogreen and not the Nanodrop.
4. What is touchdown PCR and how does it differ from standard PCR?
The touchdown program is modified from the standard program as the initial annealing temperature is higher than the optimal Tm of the primers and is gradually reduced over subsequent cycles until the Tm temperature or “touchdown temperature” is reached. This increases specificity and sensitivity in PCR amplification as described here. Additionally, after 20 rounds of touchdown PCR, another 20 rounds of standard PCR are performed.
Touchdown is a great tool for low biomass samples, but should used with caution as any trace amounts of 16S will amplify including that in the negative controls.
1. What is the Mock control and why do I need it?
The mock community used is commercially produced ZymoBIOMICS Microbial Community DNA Standard (cat# D6306), which is a mixture of genomic DNA extracted from pure cultures of eight bacterial and two fungal strains. This mock community can be used for error analysis (see: Mothur Wiki). The FASTA file for this mock can be found here.
2. What type of data will I receive?
3. Are the indices removed from the FASTQ files?
4. How do I receive the data?
All sequencing project data will be transferred on Illumina’s BaseSpace website. For an additional cost, you may request the data also be transferred to a USB.
5. Why am I having trouble accessing my data on Basespace?
You need to open an account using your email and include the email address in your service request form.
6. What if I lose my data?
We keep backups of data for six months so if you lose your USB or delete your BaseSpace run within this time frame, we will likely have a backup copy. A fee of $75 will be charged to retrieve your data.
7. What is in the workbook?
The standard workbook contains a methods sheet, your plate map, the PCR conditions for each of your samples, any troubleshooting conditions, post-PCR QC results, a summary of run metrics, and how well your samples indexed.
8. How do most of your clients analyze their 16S rDNA data?
9. Do you provide a service to analyze the data?
We do not currently provide analytical support for external clients at this time. For clients within the University of Michigan Medical School, we can provide a list of resource for analytical support.
Citing the Microbiome Core
We ask that all publications and presentations of research results supported by the Microbiome Core contain the following (or equivalent) acknowledgement: "This research was supported by work performed by The University of Michigan Microbiome Core"
Please let us know as soon as you have an article accepted for publication, poster, or meeting presentation which has received Microbiome Core support. Early notification helps us to track the activities being supported by the Microbiome Core. Submit your notifications by email to firstname.lastname@example.org. Be sure to include a copy of the presentation abstract or complete accepted article.