Laura’s Notebook: June 2019 goals

Accomplished last month:

O. lurida 2017-2018 project (OA/Temp carryover)

  • Revised MS based on co-author feedback and for general improvements
  • Sent for 2nd round of comments.

O. lurida 2018 project (Temp/Food carryover)

  • Finished 1st draft discussion
  • Correlated CV for larval shell length/width with # larvae released that day (is CV higher with more larvae?) Answer: no.

Bypass & admin:

  • Compiled draft application, including draft dissertation
  • Scheduled committee meeting

NSF GRFP

  • Met with Krista N. re: NSF GRIP, interesting Dungeness crab project looking at larval epigenomes in various pCO2
  • Submitted NSF GRFP funding request for next year (final year)

Oly methylation data:

  • Pulled matrices for Katherine

Oly QuantSeq data & future steps:

  • Expanded plan of attack. Would like to include some juvenile Port Gamble whole-body samples to compare those with high pCO2 parental histories vs. no history, in the location where parental history correlated with survival

Goals / To Do List

Admin

  • Prepare committee meeting presentation, meeting agenda, list of items needed / questions
  • Revise bypass application based on committee input
  • Submit bypass
  • Schedule qualifying exam
  • NSA quarterly newsletter

O. lurida 2017-2018 project (OA/Temp carryover)

  • Write cover letter for journal submission
  • Revise MS based on 2nd round of co-author feedback
  • Submit!

O. lurida 2018 project (Temp/Food carryover)

  • Calculate degree-days until larval release onset for all spawning tanks (if possible).
  • Figure out how to do broodstock survival analysis, then do it
  • Write Intro
  • Finalize plots
  • Send to co-authors

Oly QuantSeq:

  • Start testing QuantSeq analysis pipeline with Oly genome, Salmon (removing the multiple read/transcript auto-correct), Trinity’s isogroup designation file.
  • Schedule benchwork, order supplies

Oly epigenetics project

  • Wrap my head around action plan, steps for analysis

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Laura’s Notebook: May 2019 Goals

Accomplished last couple months:

  • O. lurida 2017-2018 project (OA/Temp carryover)
    • Revised O. lurida 2017 manuscript
    • Sent O. lurida 2017 manuscript to co-authors for review
  • O. lurida 2018 project (Temp/Food carryover)
    • Analyzed data
    • Drafted methods and results section
    • Began drafting Discussion
  • Polydora paper
    • Revised based on Brady’s feedback, sent back to Brady
  • Helped/facilitated Alanna with RNA extractions
  • Finished FSH 324 TA-ship/grading
  • Began FSH 310 TA-ship
  • Revised Multivariate Stats class paper for “publication” in EJAMS
  • Odds and ends

Goals / To-Do list:

  • O. lurida 2017-2018 project (OA/Temp carryover)
    • Revise O. lurida 2017-2018 MS co-author feedback
  • O. lurida 2018 project (Temp/Food carryover)
    • Finish Discussion
    • Write Intro
    • Finalize plots
    • Send to co-authors
  • Bypass:
    • Write and submit bypass!
  • NSF GRFP
    • Meet with Krista N. re: NSF GRFP
    • Confirm that I’ll take my final NSF GRFP funding next year, and submit to NSF
  • Oly methylation data:
    • Pull matrices for Katherine
    • Wrap my head around action plan
  • Oly QuantSeq data:
    • Revisit – plan of attack. Do I want to do some juvenile Port Gamble whole-body samples to compare those with high pCO2 parental histories vs. no history, in the location where parental history correlated with survival?
    • Start testing QuantSeq analysis pipeline with Oly genome, Salmon (removing the multiple read/transcript auto-correct), Trinity’s isogroup designation file.

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Laura’s Notebook: March goals

Laying down my monthly goals midway through March and towards the end of the quarter means that I need to be realistic/conservative.

Accomplished February/early March:

  • Re-worked Oly parental OA exposure paper methods/results/discussion
  • Sent Polydora paper to co-authors for final comments
  • TA duties maintained
  • Secured TA-ship for next quarter (shellfish biology with Jackie)
  • Prepared presentations on Oly OA parental effects
  • Presented twice at Aquaculture 2019 in New Orleans, the triennial NSA meeting. Presented Oly parental exposure & QuantSeq data in the Mollusc restoration session, and also in the Student Spotlight competition.
  • Fulfilled my Student Recruit Co-chair duties at the NSA meeting!
  • Identified how to use remaining grant money – will broaden my larval QuantSeq data, and perhaps do some samples from the 2018 winter temp/food experiment to look at gene expression in high/low performing larval families.
  • Also decided to keep an eye open for opportunities to sequence adult somatic tissues after Oly OA exposure.

To do, rest of March:

  • Outline of bypass proposal – start drafting during spring break downtime
  • Refine discussion section of Oly 2017 paper, outline introduction
  • NSA quarterly newsletter for Leroy
  • Start testing QuantSeq analysis pipeline with Oly genome, Salmon (removing the multiple read/transcript auto-correct), Trinity’s isogroup designation file.
  • Plan QuantSeq sample selection, RNA isolation, library prep, sequencing, etc.
  • Help/facilitate Alanna with RNA extractions
  • SO MUCH GRADING!!!!
  • Clean up teaching lab
  • Revise/finalize cover letter for Polydora paper

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Laura’s Notebook: First day of tissue prep, Alanna captstone / acute OA shock trial

Today Alanna and I got started processing juvenile Olympia oyster whole body tissues for RNA extraction. We are using the South Sound offspring (from Oyster Bay Cohort 1), from the 6C parents that were exposed to 7.3 pH and those that were unexposed, and which were held in control pH tanks (8.0), and acute low pH (7.0) for about 6 hrs.

We are processing 8 samples at a time, 2 per each treatment. First step is tissue disruption and homogenization in liquid nitrogen using a mortar and pestle, which we did today on the following samples:

Sample # Population Parental Treatment Offspring Treatment
100 SN 6-Amb control
106 SN 6-Amb control
112 SN 6-Amb acute low pH
115 SN 6-Amb acute low pH
124 SN 6-Low control
126 SN 6-Low control
136 SN 6-Low acute low pH
136 SN 6-Low acute low pH

Note: after grinding in liquid nitrogen, no more than 5 mg were allocated into tubes, then 350 uL of Buffer RTE Plus was added, the mixture was vortexted vigorously, then all was transferred to the QIAshredder columns, and spun at maximum speed (21,300 rcf) for 2 minutes. The homogenate, which the protocol says can be held in -70 for “months”, was then put into the -80 for RNA extraction tomorrow. samples are held in collection tubes in the -80, rack #1, column 2, row 3. remaining dry ice is in the middle -80 freezer, top shelf, in a styrofoam cooler.

The oysters are small, about 5mg total tissue. I was able to save about 1/2 the ground tissue for only some of the samples (106, 112, 115, 126). If we get enought RNA with less than 5 mg of tissue, I will see if Alanna can do this for the rest of the samples.

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Laura’s Notebook: February 2019 Goals

Accomplishments last month:

  • Submitted geoduck proteomics data to PRIDE
  • Published skyline project files on panoramapublic
  • Submitted geoduck eelgrass revision
  • Received feedback on polydora paper from Teri King
  • Oly 2017 paper:
    • Began draft introduction
    • Finished draft discussion, methods and results …
      • Received feedack from Steven, re-assessed data to include, changed paper’s angle, plots
  • Submitted travel grants for NSA conference
  • Agreed to WA Sea Grant symposium, but it was postponed due to gov’t shutdown. Will likely participate next fall.

Things to do, this short month

  • Oly 2017 paper:
    • Final edit of methods, results rewrite
    • Finish disucssion rewrite
    • Finalize plots
    • Finish Introduction
    • Draft abstract
  • NSA presentation, on Oly 2017 OA / QuantSeq project
    • Need full 12 minute presentation
    • Need 3.5 minute, abbreviated proposal, maximum of 5 slides for student spotlight session
  • Bipass proposal … getting antsy
    • Incorporate final geoduck paper
    • Draft list of proposal options with details
    • schedule deadline

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Laura’s Notebook: January 2019 Goals

  • Submit geoduck proteomics data to PRIDE
  • Publish skyline project files on panoramapublic
  • Submit geoduck eelgrass revision
  • Format polydora paper for JSR
  • Submit polydora paper to JSR
  • 2017 Oly paper, “Transgenerational carryover effects of parental low pH exposure in the Olympia oyster (Ostrea lurida)”
    • Identify journal
    • Finish Discussion
    • Finish Introduction
    • Write abstract
  • Bipass proposal
    • incorporate final geoduck paper, olympia transgenerational paper
    • draft list of proposal options with details
    • schedule deadline
  • Email Deborah about WA Sea Grant event
  • Poster for Sea Grant – update geoduck poster, OR do Oly QuantSeq project

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Laura’s Notebook: Assessing Oly gonad transcriptome assembly

The Trinity assembly is complete. Today I inspected it using transrate, in addition to running blast over the weekend to annotate genes using the Uniprot/Swissprot database. Transcriptome assembly quality, as per transrate using the trimmed/normalized reads, seem sub-par, as percent good mapping is only 10% … but I’ll investigate further. Check out my Jupyter notebook for more details: transcriptome-assess-annotate.ipynb

transcrate screen shot

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