Laura’s Notebook: QuantSeq Library Generation Batch 2

Generated libraries on my second batch of ctenidia RNA samples.

Samples processed, volumes used for RNA and DEPC-treated water, and total RNA used.

Sample order in plate Sample No. [RNA] (ng/ul) Vol RNA used Vol H2O to add ng RNA used
1 343 114.0 3.07 1.93 350
2 345 190.0 1.84 3.16 350
3 303 95.2 3.68 1.32 350
4 346 43.6 5.00 218
5 302 62.4 5.00 312
6 336 94.8 3.69 1.31 350
7 292 29.6 5.00 148
8 NTC1 -B2 5.00 0
9 317 158.0 2.22 2.78 350
10 322 44.6 5.00 223
11 332 65.8 5.00 329
12 334 64.8 5.00 324
13 349 82.0 4.27 0.73 350
14 337 194.0 1.80 3.20 350
15 341 89.6 3.91 1.09 350
16 313 72.4 4.83 0.17 350
17 309 170.0 2.06 2.94 350
18 327 85.2 4.11 0.89 350
19 319 77.6 4.51 0.49 350
20 326 130.0 2.69 2.31 350
21 306 136.0 2.57 2.43 350
22 323 102.0 3.43 1.57 350
23 314 42.2 5.00 211
24 316 146.0 2.40 2.60 350
25 339 77.2 4.53 0.47 350
26 293 39.6 5.00 198
27 329 162.0 2.16 2.84 350
28 296 180.0 1.94 3.06 350

Volumes of solutions needed – I aliquoted volumes into 7 pcr tubes, so I could then add to samples using a multichannel pipette.

Step, Chem. Vol per rxn (uL) Total + 10% or 15% Vol per aliquot (n=7) Vol per sample
Step 3 MM: FS2 9.5 305.9 46 10
Step 3 MM: E1 0.5 16.1
Step 6: RS 5 154 22 5
Step 7: SS1 10 308 44 10
Step 9 MM: SS2 4 128.8 23 5
Step 9 MM: E2 1 32.2

PCR Plate setup

1 2 3 4 5 6 7 8
A 343 345 303 346 302 336 292
B
C NTC1 -B2 317 322 332 334 349 337
D
E 341 313 309 327 319 326 306
F
G 323 314 316 339 293 329 296
H

Notes

  • I worked in 4 rows of 7 (27 samples + 1 NTC).
  • I accidentally used 4.51 uL of sample #326, which is ~585 ug of RNA (exceeds the 500 ug max). I proceeded anyway, and will see if it influences the library quality.
  • Protocol was slightly improved by shortening the amount of time samples sit at 42C at step 2/3 (from 15 mins to ~7 minutes), and I cut the PCR plate to only have 8 of the 12 columns, which freed up space for pre-warming master mix #1 (steps 2/3).
  • I aliquoted 20uL of each sample to new tubes and placed in -80 freezer.

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Laura’s Notebook: QuantSeq Library Generation Batch 1

Began my full QuantSeq library prep today. I am processing ctenidia samples first, and since there are 53 samples I’m doing ~half at a time. Today I generated double stranded cDNA for 26 samples + 2 NTC (28 total). I loaded samples onto a PCR plate in 4 rows of 7.

Samples processed, volumes used for RNA and DEPC-treated water, and total RNA used.

cDNA synthesis 12/5/19 # samples + 2 NTC 28 Work in 4 rows of 7
Sample order in plate Sample No. [RNA] (ng/ul) Vol RNA used Vol H2O to add ng RNA used
1 328 156.0 2.24 2.76 350
2 299 50.4 5.00 252
3 301 75.8 4.62 0.38 350
4 342 162.0 2.16 2.84 350
5 331 42.2 5.00 211
6 307 89.4 3.91 1.09 350
7 295 34.8 5.00 174
8 304 200.0 1.75 3.25 350
9 305 75.2 4.65 0.35 350
10 311 158.0 2.22 2.78 350
11 NTC2 – B1 5.00 0
12 298 182.0 1.92 3.08 350
13 348 54.4 5.00 272
14 315 148.0 2.36 2.64 350
15 344 25.0 5.00 125
16 325 180.0 1.94 3.06 350
17 338 81.6 4.29 0.71 350
18 347 69.0 5.00 345
19 312 90.6 3.86 1.14 350
20 321 148.0 2.36 2.64 350
21 333 78.6 4.45 0.55 350
22 291 158.0 2.22 2.78 350
23 308 73.6 4.76 0.24 350
24 NTC1 – B1 5.00 0
25 335 180.0 1.94 3.06 350
26 318 174.0 2.01 2.99 350
27 294 110.0 3.18 1.82 350
28 324 172.0 2.03 2.97 350

Volumes of solutions needed – I aliquoted volumes into 7 pcr tubes, so I could then add to samples using a multichannel pipette.

Step, Chem. Vol per rxn (uL) Total + 15% Vol per aliquot (n=7) Vol per sample
Step 3 MM: FS2 9.5 305.9 46 10
Step 3 MM: E1 0.5 16.1
Step 6: RS 5 161 23 5
Step 7: SS1 10 322 46 10
Step 9 MM: SS2 4 128.8 23 5
Step 9 MM: E2 1 32.2

PCR Plate setup

1 2 3 4 5 6 7 8 9 10 11 12
A 328 299 301 342 331 307 295
B
C 304 305 311 NTC2 – B1 298 348 315
D
E 344 325 338 347 312 321 333
F
G 291 308 NTC1 – B1 335 318 294 324
H

Notes

  • Samples were thawed on wet ice, and vortexed once before use.
  • I loaded the first 3 samples, 328, 299 and 301, onto the PCR plate but then had to wait a few minutes (~3-5) for the rest to thaw.
  • Step #2: held samples at 42C for 14 minutes while preparing master mix. Probably a bit too long.
  • I used a whole PCR Plate, which took up the entire thermocycler space. Next time, I should use partial PCR plates to leave room for a PCR strip to pre-warm the master mix needed for step 3 & 4.
  • The centrifuge gradually cooled as I was using it, which was weird since I didn’t change the temp. I think I need to set temperature to 22C every time I use it, b/c it may default to 4C.
  • I aliquoted 20uL of each sample to new tubes and placed in -80 freezer.

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Laura’s Notebook: Oly methylation analysis, Nov 20, 2019

Revisiting Oly methylation data. We now have two lists of loci:

  • 1) DMLs between two Olympia oyster populations, Hood Canal and South Sound, which were identified using MethylKit.
  • 2) Loci where methylation status is associated with oyster shell length, filtered by a) loci have 10x coverage in all samples, and b) loci have 10x coverage in any sample.

Today I re-plotted heatmaps using MACAU loci, based on feedback from Steven & Katherine:

  • Only use loci with 10x coverage
  • Add heatmaps where samples are NOT ordered by cluster analysis, but instead by 1) tree from MethylKit, and 2) shell length. See my notebook, 06-analyzing-MACAU-results-rev1.html, and here’s one of the new heatmaps, with samples (columns) ordered by shell length, and a barplot of shell length below (red = Hood Canal oysters, green = South Sound oysters).

69304438-6e9ddb00-0bd5-11ea-950a-64b24e80fbf4.png69304447-78bfd980-0bd5-11ea-995a-149f7b6a6b83.png

Then I used bedtools to see where DMLs and MACAU loci are located, see my notebook here:
07-DML-MACAU-annotation.ipynb.

Finally, I began annotating loci locations for DMLs and MACAU loci; see my notebook: 08-Annotations.html. Here’s a barplot showing which features overlap with the DMLs: image

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Laura’s Notebook: November 2019 goals

Tasks that must be completed in November

  • GRIP application (Due Dec. 4)
  • NSF INTERN program (No due date, sooner the better)
  • Address Ecological Applications formatting changes & submit
  • Jackie class – lots of writing
  • Finish larval measurements for Oly temp/food paper
  • Finish final revisions on Polydora MS

Longer-term tasks

  • Revise Oly temp/food paper to incorporate larval size differences
  • Can I automate oocyte measurements for Oly temp/food paper to get time series of oocyte size?
  • Get cracking on the QuantSeq library prep!
  • Revisit Oly methylation data and begin next steps in analysis. Need to determine steps, probably visualize results from MethylKit + MACAU + SNPs together, describe locations including function if possible.
  • Oly methylation data –> what’s the angle in my Aquaculture America talk?
  • Revisit/revise QuantSeq pipeline using Salmon and the Oly genome
  • Make sure Christian knows which samples are which
  • Identify possible Aquaculture 2020 funding

Other responsibilities

  • Start Polydora research position (goes through January)
  • Help with GSS
  • NSA quartlerly newsletter
  • Any Baltimore tasks?

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Laura’s Notebook: Temperature data from Clam Bay, Mud Bay, Fidalgo Bay

Over the past 2 years I have accumulated temperature data from a few locations in Puget Sound, WA. Using HOBO data loggers, I collectet temperature (& some light intensity data) from Clam Bay, which is where the Manchester research station is located, from Mud Bay, which is near Bremerton and has a very productive Olympia oyster bed, and from Fidalgo Bay, which is near Anacortes and the location of an assemblage of Olys that are uniquely large.

The following screenshots from the HOBOware plots are saved in this GitHub repo, and HOBO/.csv files.

Clam Bay Data, various dates Aug. 2017 – Sept. 2019

Data Files: Clam-Bay-Temperatures

Loggers held alongside Olympia oysters hanging off dock ~1-3 meters below surface (“dock”), and inside a tumble bag attached to the racks installed on the beach (accessible below -1’). Clam bay is located at the NOAA Manchester Research Station

Clam-bay-beach-jun2018-jun2019.png

Clam-bay-dock-jun2018-sept2019.png

Clam-bay-dock_aug2017-jun2018.png

Fidalgo Bay Temperature, Winter 2017-2018

Data Files: Fidalgo-Bay-Temperatures

Deployed attached to a sunken raft at (48°28’41.7”N 122°34’26.6”W) aka (48.478238, -122.574057)

Fidalgo-bay-beach-Nov2017-Jun2018.png

Mud Bay, Winter 2017-2018

Data Files: Mud-Bay-Temperatures

Deployed at 3 locations in Mud Bay, Dyes Inlet, approximately here: (47°35’22.9”N 122°40’22.2”W) aka (47.589681, -122.672831). Exact deployment coordinates for the 3 probes are here: 2017-11-06_Mud Bay-Temp-Logger-Locations.kml

Mud-Bay-intertidal-winter2017-2018_depl1

Mud-Bay-intertidal-winter2017-2018_depl2

Mud-Bay-low-intertidal-winter2017-2018

Mud-Bay-subtidal_winter2017-2018

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Laura’s Notebook: Analyzing MACAU results, take 3

New and improved with the following:

  • Included a False Discovery Rate correction as per this paper doi:10.3390/genes10050356
  • 2 heat maps created with % methylation:
    1) excluding loci for individual samples where coverage <5x (retained for other samples), and
    2) excluding loci for all samples if any had <5x coverage
  • Barplot of lengths in same order as 2nd heat map

See new RMarkdown notebook: 006-analyzing-MACAU-results-rev1

Preview of new plots

image

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Laura’s Notebook: Analyzing MACAU results, take 2

I revisited the MACAU result again to:

  • re-do heat maps with only loci that has 10x or greater coverage
  • Generate heat maps of % methylation (and coverage >= 10x)
  • Generate PC plot using count data (>= 20x coverage)

Check out my RMarkdown notebook, 06-Analyzing MACAU results

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