Nov. 7-9, Geoduck DMR feature analysis

Generate appropriate background

  • I previously used within-sample DMRs filtered for coverage in 3/4 individuals/group as the background.
    • HOWEVER, these did not include all sites that had the potential to be methylated
    • To create a more inclusive background, I need to look at all CG sites considered prior to determining within-sample DMRs
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Wed. Nov. 6, Geoduck DMR Summary and Feature Analysis

Summary of DMRs

  • Within-sample DMRs were called (mox script here: 20191024_DMRfindAllEPI.sh; DMR output files here: https://gannet.fish.washington.edu/metacarcinus/Pgenerosa/analyses/20191024/, see files ending in _DMR250bp_MCmax30_cov5x_rms_results_collapsed.tsv) and the total number of DMRs called for each of the following comparisons were:
    • All ambient samples over time: 249
    • All day 10 samples: 182
    • All day 135 samples: 109
    • All day 145 samples: 213
  • Filtered DMRs: Within-sample DMRs were filtered for those that show coverage in at least 3/4 individuals per experimental group (R script here: mcmax30_DMR_cov_in_0.75_SamplesPerCategory.R, Rproj here: DMR_cov_in_0.75_SamplesPerCategory.Rproj). After these steps the total number of DMRs that went into the ANOVA test for experimental group differences for each of the following comparisons were:
    • All ambient samples over time: 82
    • All day 10 samples: 99
    • All day 135 samples: 42
    • All day 145 samples: 26
  • Significant DMRs After running group statistics (post here: https://shellytrigg.github.io/209th-post/), the fraction of DMRs with an experimental group effect significant at a 1way ANOVA p.value of < 0.1 for each of the following comparisons were:
    • 38/82
    • 29/99
    • 13/42
    • 5/26
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Nov. 3-5, Salmon DMR analysis

Find DMRs

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Wed. Oct. 30, Geoduck filtered DMR validation

Visualizing filtered DMRs in IGV

DMRs have been filtered for 5x coverage in at least 3/4 samples per experimental group, then further filtered for showing a significant difference in % methylation across experimental groups at an uncorrected ANOVA p.value < 0.1

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Tues. Oct. 29, Geoduck DMR filtering

Performing group stats on DMRs

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Tues. Oct. 1, 2019, Geoduck Juv. OA DMR analysis

QC of DMRs

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Fri. Sept. 27, Pt. Whitney Juvenile Geoduck var.low pH experiment

Water chem

  • Water level was low in the low pH header tank
    • Appears to be the same thing that happened to the ambient tank last week. I turned flow up so it can maintain the water level
  • Took discrete measurements and TA between 10:45-11.
  • for TA, I took directly from the discrete measurment water after filtering it through 20uM. The discrete measurement water samples were directly from the headers and inside the silos (using clean TA cups)
    • everything looked as it should
  • 12-12:30pm calibrated all Apex probes
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Thur. Sept. 19, 2019 Pt. Whitney Juvenile Var.low pH experiment

Water chemistry

  • Took discrete measurements and TA around 2:30pm
    • for TA, I took directly from the discrete measurment water after filtering it through 20uM. The discrete measurement water samples were directly from the headers and inside the silos (using clean TA cups)
    • everything looked as it should
  • @5:40pm calibrated B5 which was off by 0.08. All other probes were within 0.01.
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Fri. Sept. 13, 2019 Pt. Whitney Juvenile Var.low pH experiment

Water chemistry

  • Took discrete measurments and TA
    • @11:40AM I moved Apex probes monitoring the trash cans directly into the silos
    • for TA samles I took directly from the discrete measurement water (filtered it). The discrete measurement water samples were directly from the headers and inside the silos (using clean TA cups)
  • will finish full water chem analysis ASAP, but generally things seem on point
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Aug 15 - Sept 9, 2019 Salmon-sea lice BS seq analysis

Sea lice bismark alignments:

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Fri. Sept. 6, 2019 Pt. Whitney Juvenile Var.low pH experiment

Respirometry

  • Ran 1 respirometry trial with 1 animal/vial; each animal from different silo
  • filling vials (tried something different):
    • filled 2.5qt HD buckets halfway with 20um filtered treatment water (this would probably be better with a vaccuum filtration unit, syringe filtering is pretty terrible)
    • placed 12 vials in 24-well plate (as holder to maintain silo ID) and submmerged in treatment water
  • added 1 animal to each vial
  • brought back to lab by carrying buckets in light protected box
  • @4pm capped vials submerged with corresponding numbered caps
  • checked for bubbles - dried with microfiber towel the kim wipe - placed randomly in 24 well reading plate
  • started trial @ 4:08pm and stopped @ 5:12pm
  • got total animal wet weight
    • data here:
  • got shell length
    • data here:
  • froze in liquid nitrogen
    • location: Roberts lab -80C rack 6 column 2 row 4
      • labeled “Geoduck Juveniles from Var.low vs. Amb pH parents 09-09-2019 Shelly Trigg”
  • paper on biomass vs size: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5270594/

Final check and clean up

  • calibrated all 4 probes
    • probe calibration looked good for totes 2 and 3
    • recalibrated tote 1 and tote 5 probes
  • checked on flow
    • flow looked good
  • removed macomas and total animals/silo
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Wed. Jul. 10, 2019 Salmon-Sea lice Zymo Pico Methyl prep + RRBS digest repeat

Zymo Pico Methyl kit prep

  • set up programs on PCR machine in 209 (called ZYM1,ZYM2, ZYM4, and ZYM5 under STRIGG folder)
  • Into a 48-well plate, I aliquoted out 20-50ng DNA and up to 20uL of nanopure H2O according to this sheet
  • I added 50ng of sea lice DNA:
    • Sea lice Female 1 = 67.2ng/uL (qubit HS, 7/10)
      • added 0.75uL + 19.25uL nanopure H2O
    • Sea lice Female 2 = 17ng/ul (qubit HS, 7/10)
      • added 3uL + 17uL nanopure H2O
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May 8, 2019 Geoduck broodstock hemolymph DMR calling

Running methylkit:

I had planned to run MethylKit on Emu’s RStudio web interface to find DMRs in the WGBS hemolymph. In reviewing a previous MethylKit R script, I realized I need sorted bam files, which my initial bismark mox script didn’t include. So I submitted a new mox job to sort my bams. Once the job finishes (after Mox maintenance), I will copy the sorted bams over to gannet and then on to Emu so I can run the Rstudio web interface (a little painful, but getting there!).

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Thurs. Mar 14, 2019, Oyster Seed Proteomics

Mapping to SR lab GO slim terms

  • I was unable to get simantic similarity for these terms (which is needed in order to relate proteins to one another through their terms) because they don’t map to terms in the goslim_generic.obo file or to the go data that ontologyX parses. So I’m not going to use these.

Cleaning up analysis for poster figs

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Wed. Feb 20, 2019, Geoduck genome versions, histology scoring, pt. whitney supplies

Geoduck gonad histology scoring:

  • worked with Kaitlyn to figure out scoring for geoduck gonad histology
    • we looked at Robert Marshall’s dissertation(he was in the Pearce lab at UBC), Grace’s geoduck gonad project, and Molly’s manuscript (from Brent) for some criteria on scoring .
    • so far, Kaitlyn has recorded the data here
    • for pictures and to find a scope with a ruler for measuring oocyte diameter, I contacted Lindsay in Jackie’s lab to see if we can use theirs.

Collected supplies to bring to Pt.Whitney tomorrow

  • heath stacks
  • liquid nitrogen in the dry shipper
  • pipette tips
  • DI water
  • Mercuric chloride waste container
  • microfuge tube racks (3)
  • 1 box large gloves
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Fri. Jan 11, 2019, Oyster Seed Proteomics

In trying to run NMDS analysis on technical replicate ADJNSAF data, I found discrepencies between the ADJNSAF values in Steven’s ABACUS_output021417NSAF.tsv and Sean’s Abacus_output.tsv. I compared Steven’s ABACUS_output021417.tsv file (from which he made ABACUS_output021417NSAF.tsv, see his jupyter notebook https://github.com/sr320/nb-2017/blob/master/C_gigas/04-Exploring-Abacus-out.ipynb) with Sean’s Abacus_output.tsv and found no difference:

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Wed. Nov 14, 2018

Geoduck Broodstock Experiment

FFAR meeting notes

  • Can’t untangle effects from high/low pH or static/fluctuating, so include additional fluctuating low pH treatment with new geoduck
    • Hollie has a code for this
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Thur. Nov 1, 2018

Methylation analysis:

Geoduck alignments too slow, should be directional, and many chromosomal sequence extraction errors

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Mon. Oct 29, 2018

from mox rsync –archive –progress –verbose 20181004/ strigg@ostrich.fish.washington.edu:/Volumes/web/metacarcinus/Cvirginica

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Sun. Oct 28, 2018

Methylation analysis: P. generosa juvenile OA response

bismark currently running on full data set

mapping and coverage analysis with subsetted samples (10K reads each)

bismark summary
- all samples have > 50% mapping efficiency with Bismark settings –score_min L,0,-1.2 -I 60 –non_directional
- deduplication removed very few reads, per usual
- % methylation is on average 20%

methylkit summary - coverage plots don’t appear to obviously show any particular sample or condition should be excluded due to low coverage.this may become more obvious in the full analysis or downstream analysis
- the genome region > 60000 seems to have low coverage in all samples
plot of genome regions with no coverage organized by length of treatment

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Tues. Oct 9, 2018

ASCA analysis with oyster proteomics temperature x time series dataset

  • Goal: To determine if proteins drive difference between temperature groups over time
    • we need to understand the model that ASCA creates from the data so we want to extract the loadings from the temperature factor ASCA and try to understand the behavior of the proteins with high loadings values
      • PC1 explains 100% of the variation; this seems strange so need to understand what is up with that
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Mon. Oct 1, 2018

My goals today

  1. Get Husky card
  2. Get bus pass
  3. Geoduck meeting @ 1:30p
    • notes from meeting:
      • How frequently to sample
      • Why are animals limited to 80?
        • because feeding of live algae is limiting
          • benefits of live algae vs. paste
            • paste better because doesn’t mess with pH and we can have more animals that way
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Friday. Sept 28, 2018

##My goals for today

  • work on Bismark alignment comparison
    • how does Bismark compare to Methylpy?
      • reference papers:
      • jupyter notebook
      • install methylpy
      • try bowtie vs. minimap
      • make genomes
    • Insert size alignment setting
      • in bowtie, this is the -I parameter
        • default is 0; in methylpy it’s 20.
  • Roberto’s last day lunch @12:30
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Thurs. Sept 27, 2018

My goals for today

  • FISH 546 9-10
  • Epigenetics reading group @ NOAA NWFSC 12:30-1:30
    • transgenerational of envronmental info
    • thoughts: stress opens heterochromatin => transposon/pseudo gene?/protogene? expression; does the cell just go crazy under stress and hope something works? Or is there a non-random mechanism that selects for advantageous changes?
    • siRNAs silence TEs: piRNAs specificly target transposons and this is heritable
    • should we be doing ChIPseq of H3K9me (heterochromatin mark) to complement methylation data if H3K9me marks change under stress like they do in the paper?
  • TGIT 4-6
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Wed. Sept 26, 2018

My goals for today

  • Work on Bismark alignments
    • Trying different alignment score settings:
      • Looked into bowtie2 alignment manual
      • alignement score is dependent on matches and mismatches
        • alignment score of 0 = 100% perfect match
        • 1 mismatch = -6 score
        • 1 ambinguous character = 1
        • if there’s a gap in the reference seq. or the read seq: 5 + 3*gap length
          • a 2bp gap in the read seq. = 2 insertions (relative to the read seq.)
          • a 2bp gap in the ref. seq. = 2 deletions (relative to the read seq.)
      • default in normal alignment mode is L, 0, -0.2 (where L = linear, the first number = the intercept, and the last number = the slope, and x = read length). The default specifies for reads that are 100bp the alignment score must be >= -20.
        • in –local alignment mode, G is used and G = natural log (i.e. f(x) = 1.0 + 5.4ln(x), where x is the length of the read). The min score default in local mode is 26 for 100 bp read.
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Tues. Sept 25, 2018

My goals for today

  • Get personal items from Mukilteo field station
    • done
  • Farewell lunch at noon
    • done
  • Get everyone’s thoughts on water chemistry data table for crab metabolomics paper
    • done
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Mon. Sept 24, 2018

My goals for today

  1. Lab meeting @ 10
  2. Meet with Steven to decide projects to focus on
  3. SAFS kickoff @ 2-5

Notes from lab meeting

  • Sam’s working on oyster transcriptome and genome assembly
  • Kaitlyn’s working on oyster proteomics temperature x time series analysis
    • pharmacokinetics toolkit applicable? (Brent asked)
    • RNA-seq time series analysis tool applicable?
  • Yaamini’s working on reviewer comments for JSR submission on oyster broodstock DMR comparison under OA conditions
    • what is the best way to vaildate methylation levels?
      • MSP vs. MBD or both? Need data relevant to physiology
  • Brent’s working on lots of grants and coordinating Pt. Whitney setup
    • Pt. Whitney: need to set a date, set up CO2 tanks
      • monitoring Geoduck over time by
        • measuring respiration weekly
        • hemolymph sampling (how contaminated with sea water will this be?)
          • where’s the best place to poke?
            • heart (Brent tried, not best survival rate)
            • aductor muscle (maybe too small, size of an eraser)
            • siphon
          • ELISA for vitellogenin to tell dev. stage
            • can we order an commercial ELISA kit to test on Geoduck?
            • can we test kit with old hemolymph sample where vitellogenin was expressed?
          • [SRM]https://pubs.acs.org/doi/full/10.1021/pr400444m)
        • non-lethal gonad biopsy (Emma’s paper)
        • histology? at least on some for validation of dev. stage
  • Laura’s working on summer histology analysis on OWL (protocol on Git). Austrailia work was temp. association with spawning and using histology to quantify eggs to tell stage: brooder vs. non-brooder. Also doing OA Oly. RNAseq data analysis pipeline
  • Emma’s working on hatchery microbiome with OA conditioned Geoduck. Comparing traditional assembly to MOCAT to 6-Gill and digging into unaligned reads. Larvae at pH 8.2 had ciliates in seawater, ciliates didn’t grow at lower pH treatments.
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Wed. Sept 19, 2018

Progress on first Bismark analysis:

-Found a way to do my analysis/write files to the server but keep my Jupyter notebook in my github desktop folder see Jupyter notebook
-still working on code for generating Bismark summary files

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