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pool 6 new crab libraries and submit to NWGC #798

Closed grace-ac closed 4 years ago

grace-ac commented 5 years ago

do we have a quote from NWGC @kubu4 ?

kubu4 commented 5 years ago

NOPE! Emailed them again today to see what the status is on getting a quote... Initiated the request nine days ago.They responded, but never provided me with a quote.

Questioning our sanity in going back to them.

grace-ac commented 5 years ago

ha oh nooooooo :( i'll find you and steven tomorrow and we can talk about it

grace-ac commented 5 years ago

This is the facility Kerry Naish's lab uses: https://gc3f.uoregon.edu/about

One of her current grad students said that typical turnaround time to receive data is ~2 weeks.

kubu4 commented 5 years ago

Two quotes for 50M reads per sample.

Standard (~12 weeks): $1700

Expedited (~6 weeks): $11,700

sr320 commented 5 years ago

10k for 6 weeks? On Nov 22, 2019, 11:23 AM -0800, kubu4 notifications@github.com, wrote:

Two quotes for 50M reads per sample. Standard (~12 weeks): $1700 Expedited (~6 weeks): $11,700 — You are receiving this because you are subscribed to this thread. Reply to this email directly, view it on GitHub, or unsubscribe.

kubu4 commented 5 years ago

Yep. The expedited service requires them to use an entire 48 samples library prep kit. So, we're paying for the entire kit, even though we'd only be using 6 samples.

kubu4 commented 5 years ago

Although, the cost might not be terribly far off from going to an outside facility (e.g. Genewiz) with a similar turnaround time...

grace-ac commented 5 years ago

Pooled samples and there's 1000+ ng of RNA in each pooled sample:

trtmnt_tank day infection_status total-yield_ng elution_vol vol_for_pool total_vol_ul qubit_vol_ul qubit_sample_ng total_ng_RNA_pool final_sample_vol
NA 9 0 287.3 13 4.977375566 52.65550411 2 21 1063.765586 50.65550411
NA 9 1 305.5 13 4.680851064 57.13842355 2 21.5 1185.476106 55.13842355
cold 12 0 412.1 13 3.470031546 51.61757335 2 23.6 1170.974731 49.61757335
cold 12 1 280.8 13 5.092592593 39.22471819 2 27.2 1012.512335 37.22471819
warm 12 0 380.9 13 3.754266212 40.86453844 2 26.9 1045.456084 38.86453844
warm 12 1 230.1 13 6.214689266 41.00446376 2 26.3 1025.817397 39.00446376
grace-ac commented 5 years ago

Are we going with NWGC? or Genewiz? Other?

@sr320 @kubu4

sr320 commented 5 years ago

These go to NWGC but in Dec we need to start working on getting quotes from GW.

kubu4 commented 5 years ago

Expedited or not?

sr320 commented 5 years ago

not

kubu4 commented 5 years ago

Quote has been signed and returned. Sample prep instructions will be sent along once they enter project into their system.

sr320 commented 5 years ago

Submitted yet?

kubu4 commented 5 years ago

Nope. Already have a follow-up email out to them to see when our project is going to be entered in the system...

kubu4 commented 5 years ago

Still haven't heard anything back. I've sent an email to the lab manager (the person who helped straighten things out the last time). Hopefully she can get the ball rolling.

kubu4 commented 4 years ago

I've forwarded sample prep and submission form info to @grace-ac .

grace-ac commented 4 years ago

Pooled samples need to be concentrated. NWGC requires 50ng/ul --> currently all six are around 25ng/ul.

Called zymo to learn how best to concentrate. Recommended RNA Clean & Concentrator Kit R1013.

It has same spin columns as extraction kit (which we have plenty of), but the clean and concentrator kit has RNA cleaning, concentrating, and RNA binding buffers.

They have a free sample kit that either has 5 or 10 preps (guy on phone wasn't sure how many exactly). I only have 6 samples that need to be concentrated.

I'll order the free samples, yes?

grace-ac commented 4 years ago

Ordered the free sample kit (3 business days)

In the meantime, per Sam's suggestions, I will:

grace-ac commented 4 years ago

Pooled some extra RNA samples together, and then attempted to concentrate using the Zymo Microprep Kit starting from Step 2, and continuing on. Samples were eluted in 35ul of TE (NWGC requires 50ng/ul of RNA eluted in at least 30ul of TE).

Results:

Pool 1:

ambient, day 12 tube numbers: 325 (1); 303 (1); 321 (0); 301 (1) Expected pooled RNA (ng): 2251.25

After attempted concentration to 35ul (TE + RNA) Qubit of 2ul: 25.7 ng/ul Total yield (25.7 * (35-2)) = 236.94 ng of RNA in remaining pooled sample

Pool 2:

ambient, day 12 tube numbers: 343 (0); 312 (0); 357 (1); 329 (0) Expected pooled RNA (ng): 1673.59

After attempted concentration to 35ul (TE + RNA) Qubit of 2ul: 7.18 ng/ul Total yield (7.18 * (35-2)) = 848.1 ng of RNA in remaining pooled sample

Summary:

I don't think it will work for my 6 pools, because they can't afford to lose a bunch of RNA in the concentrating process. They are all currently at at least 1,0000 ng of RNA in the total pooled samples.

For pool 1, I would have expected around 2000 ng of RNA, but ended up with 236.94 ng of RNA in remaining pooled sample. For pool 2, I would have expected at least 1000 ng of RNA, but ended up with 848.1 ng of RNA in remaining pooled sample. Pool 2 was closer to working that pool 1. Still, loss of RNA.

kubu4 commented 4 years ago

Did you measure the pools of RNA before processing?

grace-ac commented 4 years ago

Nope! Did not even occur to me. I should have done that. :(

I have lots more extra samples. I can re-do and make sure I run pools on Qubit before going through the process.

kubu4 commented 4 years ago

It's a pretty quick procedure. I would do that, just so you're confident on how much RNA is actually going in.

grace-ac commented 4 years ago

will do that right now. thanks!

grace-ac commented 4 years ago
  1. Pooled
  2. Qubit 2ul
  3. Concentrate and elute in 35ul TE
  4. Qubit 2ul

Results:

Pool 1:

tubes: 310, 315, 341, 337 pooled all together initial vol: 41 ul qubit 2 ul —> 25.2 ng/ul total RNA remaining (ng) => (25.2 * (41-2)) = 982.8

after concentrate into 35ul TE qubit 2ul —> 23.7 ng/ul total RNA remaining (ng) => (23.7 * (35-2)) = 782.1

Pool 2:

tubes: 201, 203, 220, 237 pooled all together initial vol: 40ul qubit 2 ul —> 16.4 ng/ul total RNA remaining (ng) => (16.4 * (40-2)) = 632.2

after concentrate into 35ul TE qubit 2ul —> 14.1 ng/ul total RNA remaining (ng) => (14.1 * (35-2)) = 465.3

sr320 commented 4 years ago

@grace-ac status?

grace-ac commented 4 years ago

Details in this post: here

Short answer:

Didn't put in the correct volume of RNA Binding Buffer when I was trying to concentrate, so I lost some RNA in concentration process.

Currently all 6 pools need some extra RNA to bring pools up to NWGC standards of at least 1500ng (1.5micrograms) RNA, concentrated to 50ng/ul of RNA in at least 30ul of TE.

kubu4 commented 4 years ago

Email them, explain the situation, and see what they'll accept. I'm fairly certain I sent them <1.5ug of RNA for a couple of the samples last time. Don't have time, atm, to see how many/which samples.

grace-ac commented 4 years ago

Will do!

On Fri, Jan 24, 2020 at 10:39 AM kubu4 notifications@github.com wrote:

Email them, explain the situation, and see what they'll accept. I'm fairly certain I sent them <1.5ug of RNA for a couple of the samples last time. Don't have time, atm, to see how many/which samples.

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHub https://github.com/RobertsLab/resources/issues/798?email_source=notifications&email_token=ADR6CKXFZK2KEAODNSU3J5LQ7MYT7A5CNFSM4JQKYKZ2YY3PNVWWK3TUL52HS4DFVREXG43VMVBW63LNMVXHJKTDN5WW2ZLOORPWSZGOEJ3WILY#issuecomment-578249775, or unsubscribe https://github.com/notifications/unsubscribe-auth/ADR6CKQYBCXQJ25A634HJVLQ7MYT7ANCNFSM4JQKYKZQ .

-- Grace Crandall [Pronouns: she, her, hers (more info https://www.mypronouns.org/what-and-why)] OneHealth Certificate Student (info https://deohs.washington.edu/cohr/graduate-certificate-one-health) Graduate Student, Roberts Lab http://faculty.washington.edu/sr320/ School of Aquatic and Fishery Sciences University of Washington, Seattle (206) 221-0978 | graceac9@uw.edu | grace-ac.github.io

grace-ac commented 4 years ago

Update: Sent NWGC manifest of our samples just now.

Once they receive that, they'll make a sample plate and send it over. Then we plate the samples, send them over to NWGC. They will run QC on samples, then be in touch about sequencing options.

kubu4 commented 4 years ago

Where are your samples stored?

grace-ac commented 4 years ago

I think they're in Rack 7, Col 3, Row 4...

grace-ac commented 4 years ago

Plate is ready for pickup. I can pick it up this Friday morning if @kubu4 is unable to do it.

kubu4 commented 4 years ago

That'd be great!

grace-ac commented 4 years ago

submitted 6 pooled samples to NWGC for QC