Frequently Asked Questions (FAQs)
This page is dedicated to providing answers to frequently asked questions (FAQs) for the Underground Explorers Program.
See Molecular and Sequencing FAQs
General FAQs¶
When should I start my expense report?
The expense report is not due until the end of the grant period. However, you should keep all receipts that you received for purchases that you made for your project. You can make purchases even before you receive your funding and before the kick-off, so long as the purchases are directly for your project and you keep the receipt to report out on in the expense report.
Can I use my funding to purchase a computer?
Please check with the UEP team if you are considering using funding to subsidize the purchase of a computer, and we can discuss on a case-by-case basis.
What happens if I am paying a field guide and do not have a receipt from them?
We expect that some transactions in the field will be “informal”, meaning that they are cash payments and there is no receipt. In this case, we have a receipt template that you can fill out with the amount you paid, to whom you paid (“To the order of”), and the reason for the transaction.
I am delayed in my project. What should I do?
If you are delayed with your project, that is okay. We just ask that you contact us (rachel@spun.earth) to notify us if you are delayed and that you do not expect to complete a grant requirement in time. Please let us know when you think you will be able to be back on track.
What kind of camera should I purchase to record photo and video for this project?
We recommend you take footage with your smartphone and with a higher quality camera such as a GoPro Hero 9. If you are considering buying a different kind of recording device such as a drone or a more expensive camera that is not included in your budget, just check with the SPUN team first. You can email rachel@spun.earth and magda@spun.earth.
Molecular and Sequencing FAQs¶
Can I carry out my sequencing through SPUN?
SPUN does not have a laboratory and carries out all sequencing through two different sequencing facilities. We offer our UEP grantees in the Americas the opportunity to use the SPUN sequencing pipeline at Scripps Research in the US; and UEP grantees in Africa, Asia, and Europe to use the SPUN sequencing pipeline at QIB in the UK for samples taken for their specific UEP project. Both facilities carry out the PCRs and sequencing for both the ITS2 and SSU regions (two amplicons) for identification of both EcM and AMF. See the SPUN Molecular and Sequencing Protocol for more in-depth information on SPUN sequencing. Please note that only samples that are part of the UEP project can be sequenced via a SPUN facility, and due to low staff capacity at Scripps and QIB, a maximum of 80 samples can be sent. Other samples which are not accounted for in the originally proposed UEP sample number that was approved by SPUN, need explicit approval from SPUN in order to be sequenced via a SPUN partner facility. Please keep this in mind as a very high volume of samples from one project can significantly delay the data return for multiple other projects.
How much does sequencing through SPUN cost, and what is included?
Sequencing through SPUN costs $50 per sample. This includes PCR amplification of both the ITS2 and SSU regions using specific fungal primers, library preparation, and sequencing of both amplicons. The shipping cost of samples to the SPUN partner facility is not included and shipping will need to be covered and organized by the grantee.
How do I send samples to the SPUN sequencing partner? Do I send soil samples, or DNA?
Our sequencing partners will only receive extracted DNA samples, and soil samples cannot be shipped to them. DNA extracted from soil samples must be shipped at room temperature, stabilized in TE Buffer. If you have not previously discussed sequencing via SPUN, please email the team and we can send you the form to fill out via Good Grants. This form will include all information you will need to send your samples for sequencing via SPUN.
Do I have to consider permits when sending DNA samples to SPUN's sequencing partner?
No import permits are required for the DNA samples to arrive in the USA. Most countries also do not have export restrictions for shipping of DNA samples out of the country. However, some countries require permits or permissions for DNA to be exported. It is your responsibility to check whether your country of sending has restrictions on exporting DNA, and, if so, that you comply with arranging these permissions. SPUN cannot help with DNA export permits or permissions. If documents are required by the receiver, you may contact SPUN to arrange these for you.
When can I expect to receive my data back from SPUN's sequencing partners-- what is the sequencing timeframe?
Due to the volume of demand for this mycorrhizal-specific and low-cost sequencing, we cannot guarantee specific timeframes for return of sequencing data and we do not take responsibility for delays in data return as this is determined by the capacity at the specific sequencing facility. It may be 2-5 months before sequencing data is returned to UEP awardees. We do not have a process for checking the status of sample sequencing, but we will let UEP awardees know as soon as their data has been generated. We will of course be flexible in regards to delayed project reports if you experience delays with your samples.
How will SPUN return my data to me if I sequence via SPUN?
When your sequencing data has been generated. you will be notified, and you can use SPUN’s sequencing data app to download your data by following the instructions under “Download your Data”.
Can I carry out 16S sequencing to identify bacteria through SPUN partner facilities?
SPUN is a mycorrhizal only organization, and we do not have any protocols or methods for 16S sequencing. We will not be able to facilitate any sequencing for 16S.
If I am carrying out my sequencing through my own sequencing facility or collaborators, are there restrictions on molecular protocols? There are no restrictions on the molecular methods you use to amplify fungal DNA or sequence. However, SPUN asks that you consider the best available methods and primers for identifying fungi from soil samples. SPUN has protocol guidance to help you decide on the best primer sets for your research, in addition to guidance on choosing a sequencing platform and reagents, and these can be found in the SPUN Molecular and Sequencing Protocol.
When sequencing independently, how do I send sequencing data to SPUN? When you receive your sequencing data, you can use SPUN’s sequencing data app to upload your data to SPUN by following the instructions under “Upload your Data”.
I have low DNA concentrations after DNA extraction, how do I improve them? To improve concentrations during DNA extraction, you can try the following:
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Try eluting with a smaller volume of elution buffer than recommended in the protocol (many recommend 50-100uL). Try eluting with 30uL. This tends to work well to concentrate the DNA a little more in very low input samples.
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Use a homogeniser/beadbeater (like a FastPrep grinder and lysis system) rather than a vortex for the initial homogenisation step. This tends to lyse fungal spores and hyphae better than a vortex, as recommended in many soil extraction kits.
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Carry out multiple extractions per soil sample, and pool the replicate samples.
DNA concentrations can be particularly low if DNA is being extracted from dry or arid ecosystems.
What do I do if some of my DNA extractions still have very low concentrations? If some of your DNA has low concentrations, you may still send it for sequencing, as we may still identify interesting mycorrhizal fungi from these samples. Samples with as little as 200ng total DNA may still be sequenced, though if you have less DNA than this you may try re-extracting DNA from the same soil sample. Consult the SPUN DNA Shipping Instructions for more information. These instructions may be useful even if you are not sequencing through a SPUN partner facility.
Do I need to send my negative DNA extraction samples for sequencing? Yes, even if your negative DNA extraction samples have very low or undetectable levels of DNA, you should send these for sequencing. Please send the maximum volume of DNA you can. This is so that we can detect any contaminating fungal DNA that may be present in your samples and remove it from analyses. Consult the SPUN Molecular and Sequencing Protocol for more information about negative DNA extraction controls.
Can SPUN help me with soil chemistry analyses? Unfortunately, SPUN does not currently work with any facilities for soil chemistry analyses and cannot help with this. However, we encourage UEPs to carry out soil chemistry analyses on soil samples where possible.
Should I keep backups of DNA samples? We recommend that for every sample, even those with low DNA concentrations, you keep aliquots or small amounts of DNA for each sample frozen in case of issues with shipping or sequencing. While these issues are unlikely, this will allow you to re-send or re-sequence if necessary. We also recommend keeping backups of soil samples frozen until you are sure you have sufficient yields of DNA extracted from each sample.