Send 4 test samples for metabolomics to UW metabolomics

AHuffmyer commented 1 month ago

Details available on metabolomics sheet here: https://docs.google.com/spreadsheets/d/1iFsVfp1vix9IfNcqSajQfLgXXDg0CCPT/edit?gid=1811659955#gid=1811659955

Send 4 test samples to UW metabolomics using the following procedure.

Potential test fragments include the following. These are samples that are the same species as our sequencing samples but are not going to be used for sequencing/metabolomics. Pick 4 total fragments (n=1 per species and then select one more that is a small fragment to test low tissue input).

P. tuahiniensis (POC-68, POC-48, POC-47, POC-44, or POC-43) P. evermanni (POR-81, POR-82, or POR-70) A. pulchra (ACR-140, ACR-165, or ACR-175)

Use the following protocol to prepare the tissue:

Keep everything as COLD as possible; keep tubes on ice at all times when not in centrifuge

Thaw fragment on ice and weigh mass of fragment
Airbrush with ice cold 10X PBS
Homogenize on ice (plastic pestle in tube to avoid lipid oxidation - do not use immersion homogenizer)
Separate fractions by centrifuging at 3,000g for 5 min at 4°C
Move supernatant to new tube and try to keep/scrape lipid films to keep with the supernatant
Vortex briefly and centrifuge again at 3,000g for 5 min at 4°C
Move supernatant to new tube, this is the host tissue; keep any lipid films as much as possible
Return to -80°C
Weigh mass of airbrushed fragment and record difference in weight
Ship to UW on dry ice directly to facility

They prefer to have ~5mg of tissue input. Because we have to airbrush, I am not sure of the best way to obtain a mass of tissue. We want to avoid lyophilizing as this is not necessary and is another potential step for changes to the metabolites in the tissue. I propose that we weigh the fragment before airbrushing and after, and that difference in weight should give us an estimate of total tissue removed. Our tissue input will be less than this because we are only keeping host tissue, but we could at least estimate total tissue as a starting point and use our calculated host/symbiont AFDW ratios to estimate proportion of total tissue that is likely host.

Estimated Symbiont:Host biomass ratios from E5 work are: POC = 0.25 POR = 0.3 ACR = 0.35

I'll add shipment address below when received. There is no specific date that they request to have samples sent. I think the sooner the better would be great.

AHuffmyer commented 1 month ago

The address to send samples will be:

Danijel Djukovic The Northwest Metabolomics Research Center University of Washington Medicine SLU 850 Republican St., Room S140 Seattle, WA 98109 Office Phone: (206) 685-4753

They asked that we verify the shipping date before sending so they can make sure someone is there to receive them.

hputnam commented 1 month ago

@AHuffmyer is there anyone there who has time for the extractions? I have already asked Jill and Zoe to prioritize all the nucleic acid work for E5 and I anticipate it will take them some time on top of their regular work. Would we be able to just ship the test fragments to you or Sam?

AHuffmyer commented 1 month ago

I will be traveling for the remainder of August, so I will not be able to do the tissue preparation until early September. I can check with Sam, but I am not sure if we are set up to do airbrushing in the lab here. I can look into this further. I would be happy to process samples for the full sample set following successful tests.

If possible, I think having Jill and Zoe prepare the tissue for the 4 test samples would be best. We do not need to do any metabolite extraction, we only need to airbrush, separate host and symbiont, and ship. This should take <2 hours total to do or less. What do you think @JillAshey?

I think because the priority is to get the test samples run as soon as possible it would be best to do at URI and ship directly to UW Metabolomics.

sr320 commented 1 month ago

Sam is out for next two weeks - so not really set up to do here to do test samples. - Also it would be ideal to get those test samples done this week given the timeline..

JillAshey commented 1 month ago

Yes I think I'll be able to prep tissue for 4 samples this week, shouldn't take too long. If the tests are successful and we want to move forward with more samples, we can revisit the conversation about preparing them.

hputnam commented 2 weeks ago

@AHuffmyer can you confirm what you think will be the benefit of UW data types and a new method, versus Rutgers data types and the established protocol we have used in the past?

AHuffmyer commented 2 weeks ago

UW has a more extensive in house library. Metabolomics data can capture 300+ annotated compounds and lipidomics data can capture 1000 + lipids. This is more compounds than I recieved previously (~190 identified compounds). I have attached example data from UW.

The other major advantage of UW is that they would extract and prepare metabolites and lipids for analysis. This would greatly cut down our times and they will do a better job optimizing extractions for the small tissue input that we will likely have. Extractions for ~100 samples would take ~10-15 days if we did it ourselves. We also do not have a lipidomics extraction protocol tested and confirmed, so we would have to develop that if we did it ourselves. They can return data within 2 months since they are a high throughput facility. Rutgers turn around times have been longer.

Billing would also be easy at UW and does not require a PO and quote in advance.

They will also do 4 samples for free for lipids and metabolies. 2024-05-20_TargetedData-Tissue.xlsx 2024-06-28_DataSample_Tissue.xlsx

hputnam commented 2 weeks ago

Thank you @AHuffmyer ! Do you think we should test host, symbiont, and holobiont fractions?

AHuffmyer commented 2 weeks ago

I do not think we have enough material - the samples we have to work with are small, so we should prioritize host tissues. If we think there is a nice pellet we could also test symbiont fraction. We have 4 free samples to use - 3 should be host tissue with one from each genera and we could use the 4th as a symbiont sample?

If we had more material I would be in favor of testing symbiont more thoroughly, but I don't think we do.

It would also double the cost to add symbiont samples.

hputnam commented 2 weeks ago

I agree with logistics and cost, but I am also thinking about what we will be able to say with the data in terms of disentangling host and symbiont. In particular since we are airbrushing the samples. Do you think there is something strategic we can do at this earliest stage to deal with the holobiont nature? What do you think we will be able to interpret from the test samples?

AHuffmyer commented 2 weeks ago

Agreed that we could disentangle host and symbiont if we added symbiont samples. From host samples, we will be able to look at host metabolism and the presence of photosynthates (e.g., glucose) would give us an indication of translocation and host control of nutrients. I remember we were interested in looking at cofactors for methylation, etc. and host tissue would give us information on host responses.

The problem is that I don't know how much of a pellet would be needed to detect symbionts, but we could add it into our test round.

Test samples will tell us if we can detect compounds we are interested in reliably and will tell us if we even have enough tissue for their analysis of metabolites and lipids. We can't proceed further until we know if we even have enough material to give them. If we don't we may need to reach out to CRL or others who have done some analysis with small tissue input.

hputnam commented 2 weeks ago

Thanks @AHuffmyer These questions came up in the mechanisms meeting today. Lets keep moving forward with UW with your original plan above.

@JillAshey please proceed with the extractions. Thanks!

JillAshey commented 2 weeks ago

Test samples have been airbrushed and separated into host and symbiont fractions (see post). Samples will be sent out on Tuesday, 9/3.

AHuffmyer commented 1 week ago

What is the expected arrival day and time to UW? I'll confirm someone can receive them.

JillAshey commented 1 week ago

I will send them overnight on Tuesday (9/3) so they should get to UW on Wednesday morning (9/4).

JillAshey commented 1 week ago

Tracking numer 778325107120 for test metabolomics samples sent to address above. They should arrive on Wednesday morning

AHuffmyer commented 1 week ago

Estimated 5-25 mg host tissue in each sample. UW is ready to receive.

JillAshey commented 1 week ago

Samples were delivered successfully!

AHuffmyer commented 1 week ago

Samples arrived without dry ice and at room temperature. We will need to submit another aliquot of samples and send in different packaging.

AHuffmyer commented 1 week ago

Jill will send a new round of samples on Monday (9/9). Print and include the attached sample manifest with the shipment. NWMRC SampleSubmissionForm.xlsx

hputnam commented 1 week ago

@JillAshey can we double the amount of dry ice, or send in a dry shipper if needed?

JillAshey commented 1 week ago

Yes I will double the amount of dry ice and secure the container better

JillAshey commented 2 days ago

New round of samples was sent today (tracking number 778446649371). Samples were sent in a styrofoam container with double the amount of dry ice as the previous shipment. Sample manifest was printed and included with the shipment. Packaging was labeled with appropriate dry ice labels. Package should arrive to UW metabolomics center tomorrow morning (9/10)

AHuffmyer commented 14 hours ago

Samples were received at UW today in good condition. Will close this issue and will follow up with results when received.

urol-e5 / timeseries

Send 4 test samples for metabolomics to UW metabolomics #74