Daily Archives: November 5
Shelly’s Notebook: Fri. Nov 2, 2018
Pt. Whitney Geoduck broodstock experiment
Setup
- Troubleshoot offline Apex
- reset router by little reset button, nothing happened
- SOULUTION: hard reset router by unplugging power supply
- apex light went from orange to blue to orange; and reconnected to fusion
-
- need to set Apex to notify when Fusion connection is lost
- create conditional: if connection lost:
- switch off outlet 2_8 (outlet 8 on the EB832 Tank1_low) for 10 seconds
- switch outlet 2_8 back on
- another potential solution
- Set up electricity
- Apex now plugged in to UPS battery backup
- UPS backup plugged into upper right-side GFI behind wall apex is on)
- Connected 2 pumps in tanks 3 and 4
- surge protectors are mounted on wall above tank 1 and tank 3
- pumps in tank 1 and 2 are plugged into surge protector above tank 1
- pumps in tanks 3 and 4 are plugged into surge protector above tank 3
- surge protectors are plugged into extention cord that runs above tanks and silos and is plugged into outlet 6 in Apex 1 power block (outlet “EB_5”, 2_6)
- Recalibrated pH probe 3 with pH standards and placed it back in tank 3.
- Now it’s reading too low. Maybe replace this probe?
- Reduced gas flow in tanks 1 and 2 by slightly dialing back black knobs
- Feeding is happening in all tanks twice/day.
- food has a high pH
- Matt could fix this
- Variability in inflow water temp can be improved. The head tank is on a recirculating chiller right now and incoming water may be fluctuating more because of this. An HVAC guy is coming to fix this.
Remaining setup
- Apex data download; still need to figure this out for remote access
- Rig probe holders (to ensure probes go back in correct tanks and not under the crates)
- Install hooks to hang pumps on when tanks are being cleaned?
- decide on treatment pH values. Should the ambient treatment have pH control (set at 8) to reduce variation?
- method for turning off CO2 during tank cleaning
- number clams (didn’t get to this but have nail polish)
- Get square milk crates?
- connectors between venturi injector and gas lines are too large for tubing; exchange for smaller ones?
Water sampling
- following Sam’s protocol
- video of titrator initial setup
- ran titrator pH calibration 2x to get correct, 2nd time I used fresh pH 7.0 buffer
- ran CRMs with ‘CRM’ program, should have been ‘007’ program (not mentioned in titrator protocol)
- reran CRMs with ‘007’ program
- junk = 59.774g
- CRM1 = 59.9790g
- CRM2= 59.6699g
- CRM data
- calibrating Orion (Thermo) pH probe: 1) click mode to get to pH mode 2) click cal 3) follow instructions (need DI water for rinsing)
- reading tris buffer: T27
- T = 20.04
- pH = 8.23
- salinity = 33.68, 33.59 PSU/51.58mS/cm/25.28ppt (DI water = 0.073psu, 20.19ppm)
- did not do calibration with tris samples at different temps. need to ask Sam about this protocol
- Sampling from tanks
- only sampled from tanks 1 and 2, because feedings were happening in 3 and 4 at the time
- filled up measuring cup
- got pH, temp, and salinity
- pH meter seems off after calibrating:
- pH didn’t match logger probes
- pH of standard pH 4.00 was reading at 3.0
- need Sam’s help with Tris calibration/methodology
- pH meter seems off after calibrating:
- pour into sample bottles
- wash/wipe probes between tanks
- filter from glass bottles into labeled titrator cups on scale (59-61g); didn’t do this, syringe filters (0.2um) are very hard to push water through
- ran samples on calibrated titrator
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Shelly’s Notebook: Thur. Nov 1, 2018
Methylation analysis:
Geoduck alignments too slow, should be directional, and many chromosomal sequence extraction errors
- increase processors/cores to use from default(4) to 28 (i.e. -p 28)
- change to directional mapping because non-directional mapping should be 1:1:1:1 But instead the non-directional mapping shows ~ 10:1:1:10 as follows:
CT/GA/CT: 2758 ((converted) top strand)
GA/CT/CT: 350 (complementary to (converted) top strand)
GA/CT/GA: 316 (complementary to (converted) bottom strand)
CT/GA/GA: 2725 ((converted) bottom strand) - padded genome
- strigg/analyses/20181025/slurm-401715.out shows > 1M errors: ‘Chromosomal sequence could not be extracted’
- creating padded genome to see if this improves mapping
- Steven can run scripts from coenv : sbatch -p coenv -A coenv 20181101_bmrkPgenrGenmPadded.sh *needed to make my directory writable (chmod 777)
- tested effects of directional mapping and genome padding on mapping efficiency with one sample (103)
- jupyter notebook
- mapping reports to compare:
- original alignment (non-directional mapping, aligned to non-padded genome)
- /Volumes/web/metacarcinus/Pgenerosa/20181025/EPI-103_S27_L005_R1_001_val_1_bismark_bt2_PE_report.txt
Bismark was run with Bowtie 2 against the bisulfite genome of /gscratch/srlab/strigg/data/Pgenr/ with the specified options: -q --score-min L,0,-1.2 --ignore-quals --no-mixed --no-discordant --dovetail --minins 60 --maxins 500 Option '--non_directional' specified: alignments to all strands were being performed (OT, OB, CTOT, CTOB) Final Alignment report
- /Volumes/web/metacarcinus/Pgenerosa/20181025/EPI-103_S27_L005_R1_001_val_1_bismark_bt2_PE_report.txt
- original alignment (non-directional mapping, aligned to non-padded genome)
Shelly’s Notebook: Wed. Oct 31, 2018
-Geoduck hemolymph extraction
-Met with Jose – he has experience with developing Vitellogenin ELISA assays in fish – Options: – ELISA: quantitative – Western Blot: not quantitative – qPCR: quantitative – Calcium assay; calcium correlated with vitellogenin (vitellogenin binds Ca2+) – assay for Upstream vitellogenesis inducer? (i.e. estradiol, although Brent says Geoducks likely don’t have this) – To develop ELISA: – need 1) isolated vitellogenin and 2) antibody. Need isolated protein to generate standard curve and generate antibody – can isolate vitellogenin by: – fractionation? It’s large – inducing expression – cloning and doing bacterial expression – can generate antibody by sending protein (or protein fragment) to commercial antibody lab (they inject rabbits) – To develop Western: – only need antibody – Calcium assay
- To develop qPCR: - need primers - need sequence - VTG superfamily domain? -BLASTX to get DNA sequence? -revtrans on serial cloer to get DNA sequence? - BLAST to Geoduck genome - build BLAST index out of Geoduck genome - align VTG superfamily domain to genome - align protein to genome - samples: - no template - non-DNase treated sample or gDNA prep? - RNApreps - hemocytes from different stages - immature - early - middle - late - post spawn - gonad tissue from different stages - immature - early - middle - late - post spawn - hepatopancreas - immature - early - middle - late - post spawn
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Shelly’s Notebook: Tues. Oct 30, 2018
Broodstock experiment
need to:
- order KCl 3M 25mL pH probe storage solution, Mettler Toledo, # 51343180
- order CRMs (only 1 unopened bottle left)
- bring extension cord and power strip for adding pumps to tanks 3 and 4
- bring materials to label ducks (nail polish? superglue + labels?)
- something to fix probes in tanks?
setup status
- CO2 gas gauge
- tank 2 pH probe 3 placement may have been too close to the inflow
- Moved it to the front corner
- Moved it to the front corner
- tank 1 probe placement
- tank 4 probe placement
- tank 1 initial CO2 flow rate
- tank 2 initial CO2 flow rate
Monitoring water chemistry in OA treatments
- brought waste container and DI water back to Pt. Whitney
- using the titrator follow this protocol
- will take video next time I run it.
- did about 5 flushes with flicking
- ran pH calibration with pH standards
- ran CRMs
- ran samples
- used measuring cups to sample the water, poured it immediately into glass bottles
- TA calculation script
- discrete water sample pH, salinity, and temp measurements
-figure out how to download data from Apex Fusion Sam G’s code
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[code]#!/bin/bash ## Job Name #SBATCH...
#!/bin/bash ## Job Name #SBATCH --job-name=ks-oly-blast ## Allocation Definition #SBATCH --account=coenv #SBATCH --partition=coenv ## Resources ## Nodes (We only get 1, so this is fixed) #SBATCH --nodes=1 ## Walltime (days-hours:minutes:seconds format) #SBATCH --time=3-00:00:00 ## Memory per node #SBATCH --mem=100 ##turn on e-mail notification #SBATCH --mail-type=ALL #SBATCH --mail-user=sr320@uw.edu ## Specify the working directory for this job #SBATCH --workdir=/gscratch/srlab/sr320/analyses/1105 /gscratch/srlab/programs/ncbi-blast-2.6.0+/bin/blastn \ -task blastn \ -query /gscratch/srlab/sr320/data/oly/Trinity.fasta \ -db /gscratch/srlab/sr320/data/oly/Olurida_v081 \ -out /gscratch/srlab/sr320/analyses/1105/ks-trinity-v081.tab \ -evalue 1e-20 \ -outfmt 6 \ -num_threads 28