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).

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:

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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

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)

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

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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

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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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