Laura’s Notebook: QuantSeq Library Generation Batches 3 and 4

Batch 3 Sample Information

Sample order in plate	Sample No.	[RNA] (ng/ul)	Vol RNA used	Vol H2O to add	ng RNA used
1	35	94.4	3.71	1.29	350
2	524	63.0	5.00	–	315
3	453	196.0	1.79	3.21	350
4	554	188.0	1.86	3.14	350
5	442b	69.8	5.00	–	349
6	489	68.0	5.00	–	340
7	462b	106.0	3.30	1.70	350
8	533	6.5	5.00	–	32.6
9	522	32.2	5.00	–	161
10	474	77.2	4.53	0.47	350
11	452b	97.2	3.60	1.40	350
12	443	60.2	5.00	–	301
13	477	164.0	2.13	2.87	350
14	526	138.0	2.54	2.46	350
15	562	126.0	2.78	2.22	350
16	432	74.0	4.73	0.27	350
17	37	74.6	4.69	0.31	350
18	413	130.0	2.69	2.31	350
19	45	25.1	5.00	–	125.5
20	561	28.0	5.00	–	140
21	542	32.8	5.00	–	164
22	527	124.0	2.82	2.18	350
23	492	82.6	4.24	0.76	350
24	475	27.4	5.00	–	137
25	541	44.4	5.00	–	222
26	565	31.2	5.00	–	156
27	B3 – NTC1	NA	–	5.00	#VALUE!
28	B3 – NTC2	NA	–	5.00	#VALUE!

Batch 3 Plate Configuration

1	2	3	4	5	6	7	8
A	35	524	453	554	442b	489	462b
B
C	533	522	474	452b	443	477	526
D
E	562	432	37	413	45	561	542
F
G	527	492	475	541	565	B3 – NTC1	B3 – NTC2
H

Batch 4 Sample Information

Sample order in plate	Sample No.	[RNA] (ng/ul)	Vol RNA used	Vol H2O to add	ng RNA used
1	571	LOW	5.00	–	NA
2	525	140.0	2.50	2.50	350
3	563	47.0	5.00	–	235
4	404	112.0	3.13	1.88	350
5	484	58.4	5.00	–	292
6	531	95.4	3.67	1.33	350
7	34	104.0	3.37	1.63	350
8	490	186.0	1.88	3.12	350
9	523	71.4	4.90	0.10	350
10	473	124.0	2.82	2.18	350
11	485	19.1	5.00	–	95.5
12	402	114.0	3.07	1.93	350
13	487	118.0	2.97	2.03	350
14	476	39.2	5.00	–	196
15	421	57.6	5.00	–	288
16	553	186.0	1.88	3.12	350
17	41	108.0	3.24	1.76	350
18	46	98.6	3.55	1.45	350
19	551	96.4	3.63	1.37	350
20	486b	148.0	2.36	2.64	350
21	445	160.0	2.19	2.81	350
22	43	156.0	2.24	2.76	350
23	506	29.2	5.00	–	146
24	482	22.2	5.00	–	111
25	412	31.2	5.00	–	156
26	488	60.0	5.00	–	300
27	47	128.0	2.73	2.27	350
28	B4 – NTC2	NA	–	5.00	0

Batch 4 Plate Configuration

1	2	3	4	5	6	7	8
A	571	525	563	404	484	531	34
B
C	490	523	473	485	402	487	476
D
E	421	553	41	46	551	486b	445
F
G	43	506	482	412	488	47	B4 – NTC2
H

Notes

• Aliquoted 20 uL RNA into new tubes and froze in -80. Except, for the following samples I aliquoted 7 uL: 34, 35, 41, 46, 46
• Sample #35 was not fully sealed during either the RNA removal step or the second strand synthesis step, and I believe I lost some volume to evaporation. I proceeded normally, but will see how that library looks.
• Not sure if I mentioned this previously, but I’m using 350 ng RNA, except for low concentration samples (I use 5 uL to get the maximum amount of RNA possible).
• To “quickly spin down” samples at room temperature I put the sample plate in the centrifuge, push start, then push stop immediately when the speed does not read 0. I also set the temperature to room temperature so it doesn’t actively cool.

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Laura’s Notebook: QuantSeq Purification and qPCR Assay Batches 1 & 2

ds cDNA purification, pre-PCR

I purified the ds cDNA from batches 1 and 2. Notes on how to improve that process:

• Bubbles! Bubbles make it difficult to use a mutichannel pipette. Need to improve handling to minimize bubble formation.
• Should reduce amount of time I keep samples on magnet and to dry. The beads seem to crack a bit, and it’s difficult to resuspend them in the final elution step.
• Max # of PCR plate columns for purification step = 8

qPCR assay for optimal endpoint PCR cycles

I performed the qPCR assay on all the ctenidia samples (batches 1 and 2). Here are the amplification curves:

Batch 1 end-point PCF cycle calculations

Sample No.	50% max	No. Cycles @ 50%	No. Cycles @ 50% minus 3 cycles	Cycles, round down
328	1,662	16.51	13.51	13
299	1,603	18.54	15.54	15
301	1,572	18.66	15.66	15
342	1,673	18.85	15.85	15
331	1,572	18.94	15.94	16
307	1,001	28.77	25.77	25
295	1,498	21.86	18.86	18
qPCR NTC	1,352	29.1	26.1	26
304	1,392	18.05	15.05	15
305	1,334	19.41	16.41	16
311	1,449	19.09	16.09	16
NTC2 – B1	1,521	25.92	22.92	23
298	1,437	18.27	15.27	15
348	1,394	19.33	16.33	16
315	1,496	18.74	15.74	15
qPCR NTC	1,483	28.23	25.23	25
344	1,545	18.19	15.19	15
325	2,029	17.64	14.64	14
338	1,572	19.31	16.31	16
347	1,547	17.9	14.9	15
312	1,510	18.77	15.77	15
321	1,561	18.03	15.03	15
333	1,651	18.07	15.07	15
291	1,265	18.06	15.06	15
308	1,297	19.1	16.1	16
NTC1 – B1	1,538	25.23	22.23	22
335	1,449	18.86	15.86	15
318	1,586	19.01	16.01	16
294	1,444	19.3	16.3	16
324	1,648	19.34	16.34	16

Batch 2 end-point PCR cycle calculations

Sample No.	RFU @ endpoint	50% max	No. Cycles @ 50%	No. Cycles @ 50% minus 3 cycles	Cycles, round down
343	3583	1791.5	16.62	13.62	13
345	4129	2,065	17.31	14.31	14
303	3543	1,772	17.28	14.28	14
346	2823	1,412	17.75	14.75	14
302	3153	1,577	16.81	13.81	13
336	3300	1,650	22.82	19.82	19
292	3253	1,627	18.6	15.6	15
NTC1 -B2	3079	1,540	20.67	17.67	17
317	3576	1,788	18.8	15.8	15
322	2540	1,270	19.31	16.31	16
332	2689	1,345	20.02	17.02	17
334	2851	1,426	20.78	17.78	17
349	2825	1,413	18.57	15.57	15
337	3325	1,663	18.65	15.65	15
341	3152	1,576	17.61	14.61	14
313	3184	1,592	18.47	15.47	15
309	2962	1,481	18.05	15.05	14
327	2973	1,487	18.89	15.89	15
319	3037	1,519	20.88	17.88	16
326	2810	1,405	18.12	15.12	15
306	3698	1,849	21.19	18.19	18
323	3537	1,769	18.47	15.47	15
314	2996	1,498	20.58	17.58	17
316	2929	1,465	19.02	16.02	16
339	2761	1,381	19.33	16.33	16
293	2213	1,107	19.67	16.67	16
329	2164	1,082	18.72	15.72	15
296	2182	1,091	18.1	15.1	15

Notes

• The highest quality RNA samples require 12 cycles. According to my QuantSeq rep, the very lowest quality and concentration RNA samples require 25 cycles.
• I should use the optimal cycle number for end-point PCR, BUT some people that are in a hurry run all samples using the average of three consecutive cycles. For instance, if some samples need 14, some need 15, and some need 16, one can run all samples for 15 cycles. However, that is not the optimal protocol. I will proceed, but may need to re-generate a few libraries, which I will determine using Qubut and high sensitivity DNA chip on the Bioanalyzer.

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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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Laura’s Notebook: Analysis of QuantSeq Larval Sizes

Check out my RMarkdown notebook where I analyze larval size upon release by parental pH and temperature treatment: laura-quantseq/notebooks/Larval-size-on-release.html

Here’s a sneak peak at one of the plots generated in this notebook:

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