Sam’s Notebook: DNase Treatment – Ronit’s C.gigas Ploiyd/Dessication Ctenidia RNA


After quantifying Ronit’s RNA earlier today, I DNased them using the Turbo DNA-free Kit (Ambion), according to the manufacturer’s standard protocol.

Used 1000ng of RNA in a 50uL reaction in a 0.5mL thin-walled snap cap tube. Samples were mixed by finger flicking and then incubated 30mins @ 37oC in a PTC-200 thermal cylcer (MJ Research), without a heated lid.

DNase inactivation was performed (0.1 volumes of inactivation reagent; 5uL), pelleted, and supe transferred to new 1.7mL snap cap tube.

Samples were stored on ice in preparation for qPCR to test for residual gDNA.

DNase calculations are here:

Samples will be permanently stored here (Google Sheet):

from Sam’s Notebook

Sam’s Notebook: Data Received – Chionoecetes bairdi RNAseq & FastQC Analysis


We received Grace’s 100bp PE NovaSeq (Illumian) RNAseq data from the Northwest Genomics Center today.

Data was downloaded via their Aspera browser plugin and rsynced to:

MD5 checksums were generated (md5sum on Ubuntu):

  321ec408ba7e0f0be1929ca44871f963 304428_S1_L001_R1_001.fastq.gz b95c69f755c9c42d9203429119d4234d 304428_S1_L001_R2_001.fastq.gz a0fd8db312057dedd480231d4d125fd3 304428_S1_L002_R1_001.fastq.gz c6e70ef7f3c8a866851a1b9453aef36a 304428_S1_L002_R2_001.fastq.gz  

FastQC analysis was run, followed by MultiQC.

Output folder (gannet/Atumefaciens):

MultiQC Report (HTML):

Nightingales spreadsheet was updated with file info and FastQC info:

from Sam’s Notebook

Sam’s Notebook: RNA Isolation – Ronit’s C.gigas diploid/triploid dessication/heat shock ctenidia tissues


Isolated RNA from a subset of Ronit’s Crassostrea gigas ctenidia samples (see Ronit’s notebook for experiment deets):

  • D01 C
  • D02 C
  • D19 C
  • D20 C
  • T01 C
  • T02 C
  • T19 C
  • T20 C

RNA was isolated using RNAzol RT (Molecular Research Center) in the following way:

  • Unweighed, frozen tissue transferred to 500uL of RNAzol RT and immediately homogenized with disposable pestle.
  • Added additional 500uL of RNAzol RT and vortexed to mix.
  • Added 400uL of 0.1% DEPC-treated H2O, vortexed and incubated 15mins at RT.
  • Centrifuged 12,000g for 15mins at RT.
  • Transferred 750uL of supernatant to clean tube (discarded remainder), added 1 volume (750uL) of isopropanol, vortexed, and incubated at RT for 10mins.
  • Centrifuged 12,000g for 10mins at RT.
  • Discarded supernatant.
  • Washed pellet with 75% ethanol (made with 0.1% DEPC-treated H2O).
  • Centrifuged 4,000g for 2mins at RT.
  • Discarded supernatant and repeated wash steps.

Pellet was resuspended in 50uL of 0.1% DEPC-treated H2O and stored @ -80oC in Ronit’s temporary box.

from Sam’s Notebook

Sam’s Notebook: SRA Submission – Olymia oyster Whole Genome BS-seq Data


Submitted our whole genome bisulfite sequencing data to NCBI Sequence Read Archive (SRA).

Relevant SRA info is below.

Have updated nightingales Google Sheet with SRA info.

SAMPLE SRA (Study) BioProject BioSample
1NF11 SRP163248 PRJNA494552 SAMN10172233
1NF15 SRP163248 PRJNA494552 SAMN10172234
1NF16 SRP163248 PRJNA494552 SAMN10172235
1NF17 SRP163248 PRJNA494552 SAMN10172236
2NF5 SRP163248 PRJNA494552 SAMN10172237
2NF6 SRP163248 PRJNA494552 SAMN10172238
2NF7 SRP163248 PRJNA494552 SAMN10172239
2NF8 SRP163248 PRJNA494552 SAMN10172240

from Sam’s Notebook

Sam’s Notebook: Installation – Microsoft Machine Learning Server (Microsoft R Open) on Emu/Roadrunner R Studio Server


Steven recently saw an announcement that Microsoft R Open now handles multi-threaded processing (default R does not), so we were interested in trying it out. I installed MLR/MRO on Emu/Roadrunner (Apple Xserve; Ubuntu 16.04). Followed the Microsoft installation directions for Ubuntu. In retrospect, I think I could’ve just installed MRO, but this gets the job done as well and won’t hurt anything.

I’ve set both Emu & Roadrunner R Studio Server to use this installation of R by changing the /etc/restudio/rserver.conf file to the following:

  # Server Configuration File # Use Microsoft R Open instead of default R version. # Comment out and restart R Studio Server (sudo rstudio-server restart) # to restore default R version. rsession-which-r=/opt/microsoft/ropen/3.4.3/lib64/R/bin/R  

I have confirmed that R Studio Server on both machines starts up and is using MRO instead of the default version of R.

from Sam’s Notebook

Sam’s Notebook: Ubuntu – Fix “No Video Signal” Issue on Emu


An issue with Emu cropped up a few weeks ago that was seemingly caused by upgrading from Ubuntu 16.04 to 18.04.

However, the problems only seemed related to using Emu via the GUI; users could still use Emu as a headless computer via SSH.

Today, I was upgrading some packages and noticed two things:

  1. When initially logging in to Emu.
      sam@swoose:~$ ssh emu Welcome to Ubuntu 16.04.5 LTS (GNU/Linux 4.4.0-57-generic x86_64) * Documentation: * Management: * Support: 0 packages can be updated. 0 updates are security updates. New release '18.04.1 LTS' available. Run 'do-release-upgrade' to upgrade to it. You have mail. Last login: Tue Oct 2 07:30:32 2018 from  

    This is showing that Emu is still running Ubuntu 16.04, not 18.04 as presumed!

  2. An error in the GRUB config generation process when upgrading packages.
  run-parts: executing /etc/kernel/postrm.d/zz-update-grub 4.4.0-134-generic /boot/vmlinuz-4.4.0-134-generic Generating grub configuration file ... Found linux image: /boot/vmlinuz-4.4.0-137-generic Found initrd image: /boot/initrd.img-4.4.0-137-generic Found linux image: /boot/vmlinuz-4.4.0-135-generic Found initrd image: /boot/initrd.img-4.4.0-135-generic Found linux image: /boot/vmlinuz-4.4.0-57-generic Found initrd image: /boot/initrd.img-4.4.0-57-generic Found linux image: /boot/vmlinuz-4.4.0-53-generic Found initrd image: /boot/initrd.img-4.4.0-53-generic error: syntax error. error: Incorrect command. error: syntax error. Syntax error at line 98 Syntax errors are detected in generated GRUB config file. Ensure that there are no errors in /etc/default/grub and /etc/grub.d/* files or please file a bug report with /boot/grub/ file attached. done Processing triggers for libc-bin (2.23-0ubuntu10) ...  

These two bits of information led me to believe the problem wasn’t that the system upgrade to 18.04 was incompatible with these old Apple Xserve hardware (since the upgrade didn’t actually get implemented) and instead was that the upgrade might have been initiated, but aborted, which modified the GRUB configuration file(s), breaking the GUI; much like the problem I previously addressed earlier this summer.

When I fixed the display/GUI issues with Emu and Roadrunner earlier this summer, I noted that the /etc/default/grub files on each of the computers were slightly different, despite the fact that these two computers should be identical. So, I replaced the /etc/default/grub file on Emu with the file from Roadrunner and rebooted Emu.

Contents of /etc/default/grub file on Emu/Roadrunner, for future reference:

  # If you change this file, run 'update-grub' afterwards to update # /boot/grub/grub.cfg. # For full documentation of the options in this file, see: # info -f grub -n 'Simple configuration' GRUB_DEFAULT=0 #GRUB_HIDDEN_TIMEOUT=0 GRUB_HIDDEN_TIMEOUT_QUIET=true GRUB_TIMEOUT=10 GRUB_DISTRIBUTOR=`lsb_release -i -s 2> /dev/null || echo Debian` GRUB_CMDLINE_LINUX_DEFAULT="quiet splash" GRUB_CMDLINE_LINUX="" # Uncomment to enable BadRAM filtering, modify to suit your needs # This works with Linux (no patch required) and with any kernel that obtains # the memory map information from GRUB (GNU Mach, kernel of FreeBSD ...) #GRUB_BADRAM="0x01234567,0xfefefefe,0x89abcdef,0xefefefef" # Uncomment to disable graphical terminal (grub-pc only) #GRUB_TERMINAL=console # The resolution used on graphical terminal # note that you can use only modes which your graphic card supports via VBE # you can see them in real GRUB with the command `vbeinfo' #GRUB_GFXMODE=640x480 # Uncomment if you don't want GRUB to pass "root=UUID=xxx" parameter to Linux #GRUB_DISABLE_LINUX_UUID=true # Uncomment to disable generation of recovery mode menu entries #GRUB_DISABLE_RECOVERY="true" # Uncomment to get a beep at grub start #GRUB_INIT_TUNE="480 440 1"  

Voila! Emu now has a functional display/GUI again!

from Sam’s Notebook

Laura’s Notebook: October goals

Before October 1st:

  • Submit geoduck oa paper to CBPD, special aquaculture issue
  • Also get DIA data onto Panorama (SRM already in)
  • Also submit paper as pre-print to Biorxiv


  • ASAP: Get my hands on QuantSeq RNA data from Katherine, and assembly/annotation pipeline
  • Write results of Oly 2017:
    • Confirm my stats approaches are sound and/or see if I can use 2017 Oly data in multivariate class …
    • Prepare figures
  • Identify options for 2018 Oly deployment sample processing: simple genetic analysis for genetic diversity (microsats)? Microbes? Expression?
  • Meet with Micah, determine sample processing
  • Trip to Manchester for 2018 Oly temp experiment:
    • Count survival in silos
    • Count survival in bags off dock
    • Measure oysters hanging off dock
    • ALSO: survey which oysters I have left from the 2017 experiment
  • O. angasi:
    • For female samples, measure egg size
    • For all questionable sex/stage samples: revisit
    • Develop method/standard for sexing/staging
    • ID what to do with frozen tissues
  • Committee meeting?
    • Can I get a stats specialist added to my committee?
  • Start preparing bypass proposal
  • Submit Polydora paper …

from The Shell Game