Shelly’s Notebook: Mon. Oct 22, 2018

  • updated author list and re-approved Sci Reports manuscript
  • Corrected Pgenr Mox script to run Bismark on subset of Holly’s BS data, and submitted job (in queue because nodes are being used)
  • Submitted Bismark job on full Cvirg dataset on Mox with analysis time extended to 10 days because the previous time (1d15h) was not enough to finish the job, hence why it timed out.
    • copied over incomplete analysis to Cvirg metacarcinus folder on Gannet to try methylkit analysis while waiting for job to complete: pwd /Volumes/web/metacarcinus/Cvirginica

      rsync –archive –progress –verbose .

  • lab meeting @1pm
  • ELISA detection of vitellogenin in Geoduck:
    • emailed on 10-12-2018 about C. corteziensis Vtg/Vn polyclonal antibody from Fabiola’s paper
      • Fabiola’s paper: compared translated mRNA sequences of Vtg/Vn from Panopea globosa and Crassostrea corteziensis to see if C. corteziensis VTG/Vn antibody would be compatible. I wanted to see the percent mapping between these protein sequences to get a sense of how much alignment and what domain homology is needed, but I couldn’t find these sequences in the Genbank as the paper says.
    • Contacted TECO about their multispecies ELISA kit for vitellogenin detection in fish:
      • Elizabeth Mihal @ TECO said (Oct 11, 2018): “Unfortunately, we do not have any indication that the Multispecies VTG kit would cross react with shellfish. I have attached a cross reactivity chart for your reference where you can see what species of fish have been shown to cross react with each kit. It is my understanding that the VTG in shellfish is too divergent from the VTG of Japanese rice fish, Australian rainbowfish, cod and flounder so it would make for an inappropriate sample type.” Some info testing the kit compatibility with various fish species has been published
    • tried general protein alignment of Geoduck VTG with BLASTP

      Conclusions: Pgenr VTG aligns best with VTG from other bivalves; BLASTp returned no species listed in TECO kits

    • tried protein alignment of Geoduck VTG to VTG from species used in TECO kit:

    Conclusions: TECO species VTGs align much better to each other than Pgenr VTG aligns with TECO species VTG. Pgenr VTG is more similar to C. gigas than to any TECO fish species and Pgenr VTG is most likely too divergent from TECO fish species.

    mRNA expression may be the better way to go

Grace’s Notebook: Crab Project – try RNeasy Kit on some samples; Adding Qubit data to sample file

Today, Steven, Sam, and I discussed where we’re at with the crab samples and RNA extraction. We decided to extract some samples using the RNeasy Mini Plus Kit from Qiagen that Sam has already used and had decent results. In the meantime, I’ll be working on creating a little metadata analysis comparing the different methods used so far, the yields, whether the samples were lyophilized, etc. To do this, I had to figure out how to add the new Qubit data to the crab sampling file. I think I’ve figured out a general work flow in R, and Sam helped me out with some codes to fix some errors I was having.

Crab RNA Extraction history thus far

The Tri-reagent method hasn’t been super great becuase the yields have been so low. The RNAzol method from months ago wasn’t good either because the Qubit results at the time of extraction were really good, but after pooling and speed vac-ing, the Qubit yields were too low for the sequencing faciliy. I even tried the RNAzol protocol, but with 6x the reagents, but that didn’t change things.

Sam has used the RNeasy Mini Plus Kit from Qiagen on some samples that I have given him, and had okay yields that were clean (good bioanalyzer and nanodrop readings).

Since those looked so good, I gave Sam some more samples to do the RNeasy kit on. Of those 40, only 15 had quantifiable RNA. Those 15 pooled together are what I sent to be sequenced as our first library. I speed va-ed them, and then walked them over to NWGC (August 23, 2018).

We recieved the sequence data on October 15, 2018 (Owl/nightingales/C_bairdi/).

Crab Extraction Plan for this week

Extract RNA from a few more Day 26 samples using the Qiagen RNeasy Kit that Sam used and see what happens with yields and purity.

Make a visualization of the Crab RNA extraction metadata

For this, I need to streamline the workflow of adding new Qubit data to the hemosample_qubit_table.csv. With Sam’s help, I think I have a pretty good idea of how to do this. I just need to clean up the script a little and to figure out how to add the “total_yield_ng” column, though. I know how to add columns and put in information as long as every row has the same information. With this, though, I need the column contents to be the “Original_sample_conc_ng.ul” multiplied by the “total_sample_vol_ul”.

R script

from Grace’s Lab Notebook