Re-submitted the Oly Population Structure Paper to Marine Ecology.
Did more Oly seed morphology measurements.
Link to image folder: here
Link to spreadsheet: here
Silo 9, Day 11
Silo 3 and 9 Day 13
After my oysters are done conditioning, I will strip spawn them and rear larvae. Understanding which males and females to cross, and how many to use, are important to ensure genetic diversity in the F1 generation.
From each tank, I will pool eggs, but keep sperm separate. I will cross the pooled eggs from one tank with sperm from all males from all tanks. The next day, I will measure hatch success, then pool D-hinge larvae by treatment.
Table 1. Number of males available for crosses, assuming there is a 50/50 male-to-female ratio in each tank. Tanks 1-3 were exposed to low pH conditions, and tanks 4-6 had ambient water conditions. Number of oysters from each tank can be found here.
|Tank||Number of Males|
With 56 males to cross and 7 different “tanks”, there are 392 possible crosses. If I were to limit the number of males used, I would have less crosses.
Table 2. Number of crosses for varying numbers of males used.
|Number of Males||Number of Crosses|
After measuring hatch rate, I can pool my individual crosses into groups based on treatments.
Table 3. Treatment pool combinations. In total, there are nine possible pools.
|Parent 1||Parent 2|
|Low Female||Ambient Male|
|Low Male||Ambient Female|
|Low Female||Heat Shock Male|
|Low Male||Heat Shock Female|
|Ambient Female||Heat Shock Male|
|Ambient Male||Heat Shock Female|
|Low Female||Low Male|
|Ambient Female||Ambient Male|
|Heat Shock Female||Heat Shock Male|
Each pool will be transferred to 28 micron silos, so I will need replicates. Assuming I can fit four totes on each level of the table at Manchester, and each tote can fit 8 silos, I can have a maximum of 64 silos. The number of replicates I need will depend on how many larvae hatch and my stocking density. Molly recommends having 15-25 larvae per mL of silo volume.
Table 4. Number of silos needed based on replicates used for each pool.
|Replicates||Total Silos Needed|
Here’s what I need to do next:
|6/30/17||15 ºC||490 mL|
|7/1/17||16 ºC||700 mL|
|7/2/17||17 ºC||700 mL|
|7/3/17||18 ºC||750 mL|
|7/4/17||19 ºC||750 mL|
|7/5/17||20 ºC||800 mL|
This week, I’ve been at Manchester trying to balance temperature, flow and pH in the conditioning set-up. On June 25, Laura told me that the pH in my tanks was reading very low (around 7.3). With all of the excess feeding, the oysters are respiring so much that they’re modifying the chemistry in the tanks.
I went ot Manchester to adjust the flow rate and raise the pH. First, I vacuumed the tanks and checked mortality. One oyster died recently from Tank 6A. I increased the flow to 1.5 L/min from 1.2 L/min, which raised the pH to 7.7. I then fed the oysters 490 mL of Reed’s Shellfish Diet in 200 L of freshwater, dosing at 55%. When I left, the pH was between 7.65 and 7.7.
After monitoring pH for a day, I found it was still dropping below 7.6 in my conditioning tanks. Additionally, the 800 W heater was unable to maintain my desired temperature with the increased flow. I drained and cleaned the oyster tanks, finding one dead oyster in Heat Shock A. I increased the flow to 2.4 L/min which stabilized the pH once more, but caused temperature to drop.
First, I vacuumed the oyster tanks and bleached the algal line. No mortalities found!
Table 1. Current oyster count in conditioning set-up.
While feeding the oysters, I stabilized the pH at 7.7-7.8 by increasing flow to 2.7 L/min.
Figure 1. pH and temperature reading after flow stabilization.
I took another 800 W heater from the lab and added it to my header tank. I set the original heater at 16 ºC. The second heating unit’s control box was in Farenheit, so I set it to 68 ºF to see what would happen.
Figure 2. In order to plug in the second heater, Olivia and I had to move the AVTECH unit to a different outlet.
Figures 3-4. Temperature settings on controllers. I wanted to the heated water temperature to be 16 ºC, so I set the old heater setpoint to 16 ºC and the new one to 68 ºF.
Figure 5. AVTECH computed pH values over the past 30 days. The pH dropped below 7.6 on June 17. It is now holding around 7.73.
Figure 6. AVTECH temperature readings for the past 10 days. The temperature held around 18ºC until June 25. Temperature reached a peak of 20ºC on June 26, dropping after an increase in flow. Temperature is now at 15.43ºC.
Now that I’ve stabilized pH, my next step is to revise my modified conditioning plan.
Took pictures of slides 17-21 today. Took a really long time to figure out which oysters were which because 19 was another split slides, and almost all the oysters were rearranged. The ink and Laura’s pictures online continue to be helpful though!
Oly 17 slide: no issues
Oly 18 slide: no issues
Oly 19 slide: Split slide
Oly 20 slide: SN-6_31 and NF-6_31 have no images as I could not find any gonad tissue
Oly 21 slide: no issues
Here is the link to the google drive where I have been uploading all the images!
So I set up Metassembler to run on the BGI and Platanus assemblies to try and merge them in to a single assembly. It ran for a while, and then returned this error:
---------- Run bash command ---------- nucmer BGI_Plat_MetAssemb/BGI/BGI.fa BGI_Plat_MetAssemb/Platanus/Platanus.fa: nucmer --maxmatch -l 50 -c 300 -p BGI_Plat_MetAssemb/Metassembly/QPlatanus.BGI/Q Platanus.BGI BGI_Plat_MetAssemb/BGI/BGI.fa BGI_Plat_MetAssemb/Platanus/Platanus. fa ... 1: PREPARING DATA 2,3: RUNNING mummer AND CREATING CLUSTERS # reading input file "BGI_Plat_MetAssemb/Metassembly/QPlatanus.BGI/QPlatanus.BGI .ntref" of length 777071945 # construct suffix tree for sequence of length 777071945 # (maximum reference length is 536870908) # (maximum query length is 4294967295) # process 7770719 characters per dot /gscratch/srlab/programs/MUMmer3.23/mummer: suffix tree construction failed: tex tlen=777071945 larger than maximal textlen=536870908 ERROR: mummer and/or mgaps returned non-zero ----------------------------------------
After some googling it turns out that MUMmer naturally compiles as a 32 bit program, and since we have so much data (it ***is*** possible to have too much data) we have to recompile it in 64 bit mode to have larger intergers available to us.
Oh well, going to recompile and start over!
Took pictures of slides 12-16 today. Ran into a few problems as one of the cassettes (14) isn’t photographed or just wasn’t included in the file I’ve been looking at on Laura’s GitHub. This ALSO happens to be a slide that was split into two slides and contained oysters from two different treatments. So there really is no way to tell which oysters are which on slide 14.
Summary of issues/notes on the slides I worked on today:
Oly 12 slide: There are 6 oyster locations listed on the histology key, but only 5 oysters on the slide/cassette. All the of the supposed 6 oysters were HL-6, so I am not too concerned.
Oly 13 slide: HL-10_18, not sure if I captured gonad tissues
Oly 14 slide: Split slides. 3 smaller oysters on the first slide and two larger oysters on the second. From Laura’s key it looks as if the largest oyster was supposed to be NF-10_26 (third numerically on slide 14), but it was on the second slide. I called the inked oyster and middle oyster on slide one HL-10_20 and HL-10_21, the super large oyster on the second slide NF-10_26, the third oyster on the first slide I called NF-10_25, and the smaller oyster on slide two I called NF-10_27. Unless the original picture can be found though this is just my best guess.
Oly 15 slide: no problems
Oly 16 slide: no problems
I will go back through once I’m done and accumulate a list of the oysters I was unsure about or couldn’t find gonad tissue for. I should be done by the end of this week.
****note: 7/7/17 Found picture of slide 14, fixed image titles on google drive and all tissues have been identified
Today I scrounged around for missing data from Heare et. al paper (Evidence of Ostrea lurida Carpenter, 1864 population structure in Puget Sound, WA).
Am now going through all the revisions and making sure that they were addressed. Sending the final version to Brent and Steven today, and will resubmit by Monday!
Sean’s Notebook: BWA-Meth Output for EPI-135.
Found a different methylation aligner, BWA-meth, that’s based on a Burrows Wheeler aligner that’s supposed to deal better with gap alignment than Bowtie2. Fired it up with the EPI-135 and 10k Geoduck genome. It gave an answer, but I *really* don’t believe it. The
bamtools stats output I believe claimed 80% mapping rate. Compared to the 6% from Bismark.
sean@emu:~/Documents/Geoduck_Rerun/bwatest$ bamtools stats -in bwa-meth.bam ********************************************** Stats for BAM file(s): ********************************************** Total reads: 55253237 Mapped reads: 48098666 (87.0513%) Forward strand: 30884874 (55.8969%) Reverse strand: 24368363 (44.1031%) Failed QC: 16970759 (30.7145%) Duplicates: 0 (0%) Paired-end reads: 55253237 (100%) 'Proper-pairs': 30054258 (54.3937%) Both pairs mapped: 47463686 (85.9021%) Read 1: 27626448 Read 2: 27626789 Singletons: 634980 (1.14922%)
.bam file is available: here
Sean’s Notebook: Starting GARM meta-assembly of PacBio and BGI assemblies for Olys.
The Pilon polishing for the CANU assembly finished last night, and it seems pretty small, but hopefully between that, the BGI assembly, and the Platanus assembly, we can assemble ourselves up one decent genome. Fingers crossed at least.
Assembly stats on the Polished assembly:
D-69-91-159-59:BGI_Oly_Genome Sean$ assembly-stats oly_polished_.fasta stats for oly_polished_.fasta sum = 46364927, n = 3388, ave = 13685.04, largest = 61211 N50 = 14126, n = 1230 N60 = 12962, n = 1573 N70 = 11906, n = 1947 N80 = 10932, n = 2352 N90 = 9590, n = 2803 N100 = 2074, n = 3388 N_count = 1 Gaps = 1
1 gap is interesting, but with the assembly size being at least 1, of not two orders of magnitude smaller than the expected genome size, I think we’re short on coverage to allow for conservative error correction levels. Will have to reassemble with looser standards and see if we can bump it up.
Polished CANU assembly found: here
Pilon output file: here
Next step is GARM, to see what that gives us. I think I’ll also re-assemble the PacBio stuff with much less stringent error correction to see if that gives any measurable difference in outputs.
Edit: Also, I finished the –non-directional runs for Bismark, no change in mapping rates and less than 1% complementary mapping, so it looks like the regular arguments are correct. Output .bam files are found here with the NonDir tag.