Geoduck sample selection for next round of MS/MS
This is what I have to work with:
Steven, Yaamini & I decided to focus on the DNR Trial #1 for the next round of sample extraction/protein analysis.
To narrow down my sample selection further, I see that the geoduck did not fare well at the Skokomish site’s bare treatment (likely due to predation), as only 2 specimens survived to be sampled, AND Micah said that the eelgrass bed @ that site was in a freshwater seep, and therefore the salinity was significantly different there. I am therefore not moving forward with the SK site
I will select 6 samples from each site/treatment for this next round, ~2 per enclosure. To do so, I used a random number generator to select 2 to 3 replicates from each enclosure.
The file with my list of selected samples is located in my DNR Repo
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My computer really didn’t like dealing with this much data, but I started organizing it all into pivot tables and it seemed to work?
1. First I created a pivot table that summed all the rows labeled with the same protein name giving me one total number for each protein per site. I copied this data into a new sheet.
2. Then I summed each row and got protein totals across all the sites, which I then sorted by value from largest to smallest.
I put the file with my results in my folder on OWL:
Still working on figuring out how to upload the other documents.
- Aeration @ larval table not working; turned airstones up and that worked, so somehow the airline on the larval table lost some pressure overnight.
- Valves 6, 7 & 8 on the bottom shelf’s manifold the larval table consistently bubbling from air in the FSW line, other buckets don’t have the same bubbling.
- Banjos moderately dirty, not clogged though
- K-10 amb larval dripper clocked, bucket only 1/2 full
- Larvae present in catchment buckets:
- K-6 amb: a bit
- SN-6 low B: a lot
- SN-6 low A: a lot
- SN-10 amb A: a lot
- SN-10 amb B: a lot
- NF-10 low B: a lot
- Measured volume actually in 5 gal bucket with banjos: 15 L = 4 gallons
- Stocking density (as per PSRF / FAO manual): 1 larva/mL for 1 tank turnover/day
- (24 hrs / (Tank Vol (L) / (flow rate)))1 larvae/mLTank Vol (L) * 1000mL/L = # larvae/mL to stock in bucket
- e.g. (24hrs/(15L/8 L/hr))*1 larva/mL * 15 L * 1000 mL/L = 192,000 larvae. I’m going to round up to 200,000 larvae
- Imaged larvae collected 5/19 & 5/20
- Counted larvae collected 5/20
- Plumbed in new algae input for broodstock.
- I have noticed that the water color on the top shelf on the broodstock table is light. I’m thinking that, because the algae injection point is very close to where the pipe splits, the algae could be “channeling” to the bottom shelf.
- Cut the 1/2 pipe ~3 feet back from the current injection point; glued in new 1/2” threaded coupler. Let dry for ~1 hour. Flushed for ~45 minutes post gluing.
- The same issue may be happening on the larval table; there isn’t pipe available to plum in new injection point. Researched mixing valve/connection for larval line, but I didn’t find one. Need to chat with Stuart. Could just use Y’s to only use 4 middle valves and split them
- Cleaned all banjos
- Fed with live algae:
- Used 1/2 Tiso/Ciso mix + CGW
- Completed top shelf of larval table: g
- Got new banjos finished and installed on the top shelf of the larval table
- Replaced drippers on all lines
- Replaced air stones as needed
- Screened new larvae, sampled for counting & imaging
- Screened new larvae through 200um screen onto sorting table filled w/ FSW
- Placed 2x100um screens under sorting table- top one clean and vortexed
- Drained sorting table onto 100um screen
- Soaked larvae in freshwater @ 18degC for ~1 minute.
- Collected larvae in tripour filled to 800mL for dense amounts, and to 250mL for small amounts. Labeled with tape, left on bench while I collected the other groups
- Vortexed sorting table & screens between groups
- Sampled triplicates from each group; counted 1 well only for stocking purposes. Fixed all wells with Lugols for counting tomorrow.
- Stocked larval buckets:
- Some groups that were already fully stocked on the bottom shelf spawned today, so I decided to use the top shelf of larval rearing table. This Tuesday when I screen/size/count I can either 1) separate out 2 size classes (e.g. <160um, >160um) and add new larvae to the small size class bucket until full, or 2) combine larvae from both buckets, save 200k max for one bucket, then use the empty bucket for new round of larvae. This will depend on how the larvae grow and the mortality.
- Sketched out a method to stock buckets gradually.
- As per PSRF, only clean/screen larvae 2x per week to minimize handling, e.g. on Tuesdays/Fridays: screen on Tuesday to size & count; then use the following calculation:
- (200,000 – # larvae stocked on Tuesday) / 3 days until Friday = # larvae to stock on Tuesday afternoon, Wed, & Thurs.
- E.g. stock ~50k per day on Fri, Sat, Sun & Mon for a total of 200k, then on Tuesday screen, count, and take stock of what’s left.
- To measure out volume needed from each group, I used plunger to mix while simultaneously pouring into separate tripour to desired vol.
To Do tomorrow:
- Measure dripper rate @ larval tank.
- Make connection for freshwater
- Collect new larvae, sample, count, stock
- Determine how I should collect larval samples, and how frequently
- Determine if I should fix larval samples in formalin moving forward as opposed to Lugols. Lugols dyes the larvae dark, so it’s not possible to decipher between live & dead. It’s helpful for counting & (I would imagine) imaging purposes, though. Ask Rhonda/Grace what was used on gigas larvae.
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Arrived @ 9:30am
- lab meeting
- can steal 2 HOBOs from gigas, leave 1
- Brent suggested that it will take ~24 hrs of dual-bucket system to successfully collect only live larvae. This is a good idea, however I don’t expect there to be sufficient room – but maybe, if larval collections are staggered… ?
- NEED TO: purchase more 5-gal buckets!
- Imaged larvae collected on 5/17, and also imaged larvae collected and saved over past week.
- Images include triplicate samples from each collection/group (see larval collection data sheet): 1x, 2x, 3x of each on Nikon SMZ645
- Will save to GoogleDrive, then to GitHub
- Checked catchment buckets
- SN-10 amb B: little bit
- NF-10 amb B: teensy amount
- SN-10 low B: a bit
- SN-6 amb B: little bit
- K-10 low: some!
- HL-10 low: maybe, probably just spermy though
- All except HL-10 have spawned within the past 4 days, and since there is no significant amount of larvae here I will not keep them- I have a suspicion that these are from the same fertilization event, and they are trickling out of the female. Will collect for real tomorrow…
- Stole 2 HOBOs from gigas (left 1 there); downloaded the data from the HOBOs and emailed to Yaamini, then reprogrammed and launched to record T every 15 minutes, installed in broodstock buckets.
- Counted larvae collected on 5/17 & imaged larvae for size analysis later
- PSRF has agreed to collect/sample my larvae tomorrow (Friday, 5/19) while I’m on campus. I pre-labeled larval rearing buckets, got water flowing, and left instructions with Jade & Alice.
- Met with Alice to get a summary of her larval husbandry practices. PSRF hasn’t updated theirs in a couple years, and there are many lessons learned. See write-up in my GitHub (I will update as needed).
- Screened and counted larvae, imaged. Larvae went down drain today.
- Temperature is holding around 17.5
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It’s been 14 full days since we moved broodstock to their separate “chambers.” From the literature, oysters release larvae on average 10-12 days after fertilization. Today I will clean all broodstock, larval catchment buckets, after which I will plan to collect larvae to save/rear.
- Arrived 9:30am
- Installed 2nd immersion heater (500 watt) that I borrowed from Jon and set to 19degC. Changed set point for the other immersion heater to 19degC. Water temp seems to cool down a bit from the header tank b/c room temperature gets below 18degC at times (at night, when PSRF is trying to spawn abalone/sea cucumbers)
- Dumped and cleaned larval buckets on east/larval table: I had left water flowing through this system overnight to test it and rinse it.
- Cut new outflow tube for broodstock buckets- the old ones were dirty
- Tested method of collecting only live larvae. Set up a small larval catchment bucket with banjo, removed banjo from 5-gallon larvae bucket, and increased flow using the 26 L/Hr dripper. Tested this on the SN-10 low pH B group since there were lots of larvae, and I kept an eye on things over the day. After 6 hours, I could see larvae that remained on the bottom of the bucket, which I collected and there were still lots and lots of live ones (see video). I was surprised!
- Deployed 8 new HOBO loggers, one for each broodstock tank
- Cleaned gigas – NOTE: no more stringy stuff!
- Cleaned Oly broodstock:
- Stopped flow to bottom shelf
- Collected larvae in catchment buckets
- Sampled larvae and fixed in Lugols
- Collected larvae that remained in 5-gal bucket
- Rinsed broodstock with freshwater
- Cleaned all buckets with Vortex solution
- Moved broodstock to other side of table, placed them randomly along manifold.
- Screened and sampled larvae that I had been keeping alive so I could take images of them, and perhaps also to count. Dumped them down the drain!
- I will upload and tag all images, and save in GitHub/Google Drive. Link to image location TBD.
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Sean’s Notebook: Olympia Oyster genome assembly in Platanus.
I’ve been trying to incorporate the new PacBio data we recently got and we’ve settled on a combination Platanus/Redundans approach for assembling a new genome from scratch.
I’ve finished the first portion surprisingly quickly. Hooray for Hyak I guess?
Some stats on the final gap closed Platanus assembly:
stats for /Users/Sean/Documents/RobertsLab/Oly__gapClosed.fa
sum = 4121175, n = 28721, ave = 143.49, largest = 1971
N50 = 133, n = 10380
N60 = 122, n = 13618
N70 = 114, n = 17127
N80 = 109, n = 20829
N90 = 105, n = 24686
N100 = 100, n = 28721
N_count = 32133
Gaps = 975
Still a lot of gaps. Hopefully the Redundans run with the PacBio stuff will help with that.
Gap filled Assembly:
Now on to Redundans.
Sean’s Notebook: Bismark mapping efficiency with Hard trimmed C. virginica sample.
Yesterday Mackenzie Gavery came by and offered some suggestions to increase mapping rates for our Virginica BS-Seq data using Bismark. Her two suggestions were using the –non_directional flag to account for the PBATness of the data, which had a huge effect, and hard trim the first 16 bases in our samples, because they look weird.
I tried everything on a single sample for speed and finished it this morning.
Hard Trimming of the first 16 bases:
That cleans stuff up for sure. Unfortunately it didn’t have much of an effect on mapping rate, bringing us up from 28% to 28.3%. Was worth a shot though!
Final Alignment report
Sequences analysed in total: 12197930
Number of alignments with a unique best hit from the different alignments: 3456338
Mapping efficiency: 28.3%
Sequences with no alignments under any condition: 5760842
Sequences did not map uniquely: 2980750
Sequences which were discarded because genomic sequence could not be extracted:0
Number of sequences with unique best (first) alignment came from the bowtie output:
CT/CT: 181719 ((converted) top strand)
CT/GA: 166362 ((converted) bottom strand)
GA/CT: 1588675 (complementary to (converted) top strand)
GA/GA: 1519582 (complementary to (converted) bottom strand)
Final Cytosine Methylation Report
Total number of C's analysed: 61813350
Total methylated C's in CpG context: 12572131
Total methylated C's in CHG context: 4005979
Total methylated C's in CHH context: 12350257
Total methylated C's in Unknown context: 0
Total unmethylated C's in CpG context: 2271987
Total unmethylated C's in CHG context: 12442077
Total unmethylated C's in CHH context: 18170919
Total unmethylated C's in Unknown context: 5
C methylated in CpG context: 84.7%
C methylated in CHG context: 24.4%
C methylated in CHH context: 40.5%
C methylated in Unknown context (CN or CHN): 0.0%
Bismark output files located: here
now time to run the rest of them!