Sam’s Notebook: GOALS – July 2015

Below is a from Sam’s Lab Notebook and provides some insight into some of our genomic focused ocean acidification studies.

Before we check out this month’s goals, let’s have a quick review of last month’s goals and which, if any, I was able to accomplish:


 From June 2015:

Geoduck Reproductive Development Transcriptomics

Done. Data from this was received 20150629.

BS-Seq Illumina Data Assembly/Mapping – C.gigas larvae OA

Little progress. No response from Univ. of Oregon. However, it seems that Mac has been having a similar issue with libraries constructed using Epigentek kits. She has contacted Illumina and Epigentek for help.

Geoduck & Olympia Oyster Genome Sequencing

No progress. Purchasing personnel (both departmental and university) appear to have had difficulty contacting BGI. I have put both purchasing personnel in contact with the BGI rep (Frank Hu), so things are starting to progress. Still not certain why they were unable to accomplish this.

Miscellany

Goal(s):

  • Migrate Wikispaces notebook to this notebook
  • Add to GitHub code pages
  • Flesh out/create README files on server(s)
  • Lab cleanup tasks
  • Assist on Jonathan’s Capstone project
  • Establish go-to location for lab personnel questions
  • Teach Unix shell at Software Carpentry

Status:

  • Migrate Wikispaces notebook to this notebook – Minimal progress
  • Add to GitHub code pages – Updated “Commercial Protocols”, improved wget command for offline notebook backups
  • Flesh out/create README files on server(s) – Some progress, but mostly on the notebook backups and newly received transcriptomic data. No progress on older directories.
  • Lab cleanup tasks – Partial progress. Autoclaved multiple bags of old autoclave trash.
  • Assist on Jonathan’s Capstone project – Improved my time management working with Jonathan, but his sequencing results are a bit odd and he needs guidance on how to manipulate, and comprehend, what the data means.
  • Successfully taught both the shell and GitHub at Software Carpentry.

 

O.lurida RNA Isolation and Reverse Transcription

Need to isolate RNA from a set of Jake’s mechanically-stressed oyster tissue. Need to clarify with Steven if we want the 1hr post-mechanically stressed to “match” with the 1hr heat shock samples that were previously processed, or if we want the 24hr post-mechanically stressed samples.

Miscellany

Need to address short-falls from the EH&S annual lab inspection. Most are minor, easily addressed issues and won’t take much time (e.g. print lab safety signs in color). The various computer tasks still stand: notebook migration from Wikispaces to this notebook, transitioning lab resources from Wikispaces to GitHub, creating/updating “readme” files for directories on our servers, etc. Lab organization goals: improve -80C organization and create an online inventory of the -80C, establish an online inventory of lab supply locations (i.e. thermometers can be found in FTR209, Drawer #02), general clean up.

 

from Sam’s Notebook http://ift.tt/1gcVg6y

Sam’s Notebook: Goals – June 2015

Below is a from Sam’s Lab Notebook and provides some insight into some of our genomic focused ocean acidification studies.

Before we check out this month’s goals, let’s have a quick review of last month’s goals and which, if any, I was able to accomplish.


From May 2015:

Geoduck Reproductive Development Transcriptomics

Goal(s): Isolate RNA from geoduck histology blocks

Status: Accomplished!

BS-Seq Illumina Data Assembly/Mapping

Goal(s): Glean additional info about this data set and our ability/inability to create our own BS-seq libraries.

Status: Still a mystery. Currently reaching out to Doug Turnbull at the Univ. of Oregon Genomics Core Facility to see if he can provide any insight as to why our data looks the way it does, which might help us figure out why we’re having such difficulty mapping our reads to the C.gigas genome…

C.gigas Heat Stress MeDIP/BS-Seq

Goal(s): BS-seq Claire’s samples.

Status: Untouched. Is dependent upon whether or not we can successfully create our own high-throughput sequencing libraries (see above).

Miscellany

Goal(s):

  • Migrate Wikispaces notebook to this notebook
  • Add to GitHub code pages
  • Flesh out/create README files on server(s)
  • Lab cleanup tasks
  • Assist on Jonathan’s Capstone project

Status:

  • Migrate Wikispaces notebook to this notebook – Minimal progress
  • Add to GitHub code pages – Updated “Commercial Protocols”, added wget command for offline notebook backups
  • Flesh out/create README files on server(s) – No progress
  • Lab cleanup tasks – No progress
  • Assist on Jonathan’s Capstone project – Things still not working. Had Jonathan isolate gDNA for proper testing of primers.

 

Geoduck Reproductive Development Transcriptomics

This project is progressing relatively smoothly. Finished RNA isolations from all samples and checked their qualities via Bioanalyzer. Steven and Brent selected samples of males and females to pool for RNA-seq. Goal is to have these two pools sent off to GENEWIZ, Inc. for RNA-seq. Currently awaiting a quote adjustment as well as an answer regarding sample quantity requirements. Hope to have these sent off later today and data back by the end of the month. This data will be used alongside proteomics data that Emma is currently generating.

BS-Seq Illumina Data Assembly/Mapping – C.gigas larvae OA

The troubleshooting for the data from these “homemade” libraries continues. We’ve tried various approaches to trimming the data, but Steven’s mapping attempts are still not yielding great results. I’ve contacted Univ. of Oregon Genomics Core Facility to see if they can provide insight, but haven’t gotten a response. Will hit them up again to see if I can get a response (and some help).

Geoduck & Olympia Oyster Genome Sequencing

We have quotes from BGI Americas for genome sequencing for both of these organisms. Currently, we’re awaiting for funding to be processed, but expect it to be available this month. Hope to send out samples this month.

C.gigas Heat Stress MeDIP/BS-Seq

This is still dependent upon our ability to make our own BS-seq libraries. Until, then, this project will likely be on the back burner for awhile.

Miscellany

I’d like to continue to contribute to our GitHub code repository with various command line tips and tricks. Additionally, I do need to actually spend some time creating/updating README files for our servers. We have a ton of folders that need some sort of descriptor file in them so users know what to expect to find in those folders. Additionally, we have a ton of data that needs descriptions and/or links to the projects from which the data was generated to serve as a means for people to know how/why/from what the data was generated. This has been done for newer data sets, but there’s a tremendous amount of data sets that have no information about them available in the README files. Also along the data management front, I’d like to tackle a bit of a reorganization, particularly re-establishing the go-to resource for lab members to find “stuff.” For example, Jake recently needed to know where/if we had some software and had to ask about it. Better organization on our part would eliminate him wasting time trying to track down this sort of thing. Part of the organizational issue is that we’ve partially transitioned over to using GitHub instead of Wikispaces. However, the transition hasn’t been fully realized/implemented and the result is fragmentation and confusion on where to find lab info. Oh, one last “digital” note. I’ll be teaching the Unix Shell lesson at Software Carpentry on June 25 – 26, so I have to get prepped for that (not on work time, of course).

In the lab, I still need to tackle some lab cleanup tasks that I neglected to deal with last month (autoclaving, glass disposal). Additionally, I need to continue helping Jonathan with his Capstone project, but I need to manage my time with him better.

 

 

 

from Sam’s Notebook http://ift.tt/1SQ9BVs

Sam’s Notebook: Bioinformatics – Trimmomatic/FASTQC on C.gigas Larvae OA NGS Data

Below is a from Sam’s Lab Notebook and provides some insight into some of our genomic focused ocean acidification studies.

Previously trimmed the first 39 bases of sequence from reads from the BS-Seq data in an attempt to improve our ability to map the reads back to the C.gigas genome. However, Mac (and Steven) noticed that the last ~10 bases of all the reads showed a steady increase in the %G, suggesting some sort of bias (maybe adaptor??):

Although I didn’t mention this previously, the figure above also shows an odd “waves” pattern that repeats in all bases except for G. Not sure what to think of that…

Quick summary of actions taken (specifics are available in Jupyter notebook below):

  • Trim first 39 bases from all reads in all raw sequencing files.
  • Trim last 10 bases from all reads in raw sequencing files
  • Concatenate the two sets of reads (400ppm and 1000ppm treatments) into single FASTQ files for Steven to work with.

Raw sequencing files:

Notebook Viewer: 20150521_Cgigas_larvae_OA_Trimmomatic_FASTQC

Jupyter (IPython) notebook: 20150521_Cgigas_larvae_OA_Trimmomatic_FASTQC.ipynb

 

jQuery(function($){
var target_height = $(window).height();
$(“iframe.iframe-class”).height(target_height);
});


http://ift.tt/1IPAeqw

 

Output files

Trimmed, concatenated FASTQ files
20150521_trimmed_2212_lane2_400ppm_GCCAAT.fastq.gz
20150521_trimmed_2212_lane2_1000ppm_CTTGTA.fastq.gz

 

FASTQC files
20150521_trimmed_2212_lane2_400ppm_GCCAAT_fastqc.html
20150521_trimmed_2212_lane2_400ppm_GCCAAT_fastqc.zip

 

Example of FASTQC analysis pre-trim:

 

 

FASTQC post-trim:

 

Trimming has removed the intended bad stuff (inconsistent sequence in the first 39 bases and rise in %G in the last 10 bases). Sequences are ready for further analysis for Steven.

However, we still see the “waves” pattern with the T, A and C. Additionally, we still don’t know what caused the weird inconsistencies, nor what sequence is contained therein that might be leading to that. Will contact the sequencing facility to see if they have any insight.

from Sam’s Notebook http://ift.tt/1IPAeGQ