Plans for today:

[1] Find missing data for high school student
[2] Figure out how to teach RaxML to a high school student (JW slides) COMPLETE
[3] Get Gazey-Staley algorithm set up in R and running. COMPLETE
      -Okay, so we don't actually have to do this step, as its just a measure of cloning success (and since we didn't clone. We don't really need to measure this. What we do need to measure (but not right away, is the # of sequences (reads) v. # genes (unique contigs). This will tell us if we have sampled enough of the genome for the OR genes given our degenerate primers.

Anyways, to download the R code for the Gazey-Staley algorithm along with a heterogeneity test (with very good documentation), visit:
http://batlab.ucd.ie/~spuechmaille/

[4] Figure out most recent problem with babbler paper.
[5] Set up ORA pipeline COMPLETE
____________________________________________
To install ORA (after having install BioPerl, HMMER, and FASTA), download the .zip file from:
http://search.cpan.org/~ceratites/ora-1.9.1/lib/Bio/ORA.pm#DRIVER_SCRIPT

Unzip and navigate to the ORA folder.

#run the perl script to make the "makefile"

perl Makefile.PL

sudo make install

perl Build.PL

WOOOOHOOOO got it working.

There are two main issues (assuming you have everything installed).

1) You must run the perl script in the /ora-*/scripts folder because it must access the .hmm files

2) When you install ORA, it includes the older version of HMMER. The error message you get when you run "or.pl", is: 

...

binary auxfiles are in an outdated HMMER format (3/b); please hmmpress your HMM file again

To fix this, do the following (assuming you have HMMER installed).

-In the /ora-*/scripts folder, there should be several files related to HMMER

Delete the following: or.hmm.h3f     or.hmm.h3i     or.hmm.h3m     or.hmm.h3p

On the command line, type:

hmmpress or.hmm

That should fix things.

To run the ORA:

perl or.pl --sequence ~/Documents/Analyses/Carrollia_OR/GPC-trim.deg.fasta

#still playing with all of the fun parameters

Now I need to trim the data of a certain length.

By the end of the weekend, it would be great to make a tree from the sequences.

I am currently at La Selva biological research station collecting bats for a transcriptomic analysis. The purpose of this post is to record what we did for specimen preparation for future reference:


Lab set up for dissections of tissues:

Checklist:

• isofluorane (anesthetic)
• cotton balls
• RNAlater (lots of bottles!)
• RNAase AWAY
• cryovials (lots!)
• plastic droppers (lots!)
• boxes
• disposable vial rack
• sharpies (small and large)
• ethanol (to clean)
• bleach (to clean)
• plastic petri dishes for dissection
• dissection kit (I especially used sharp and dull forceps, scissors, small scissors, curved forceps)
• dissection scope
• cutting board
• light source for scope
• camera and lens
• tape
• organized binder with data sheets
• calipers/ruler and pesolas for measurements
• sponges
• bottles for sharps
• plastic squeezy bottles for RNA later
• Cooler (for ice)
• Little bucket for ice
• Ice
• Cooler (for dry ice if you want to collect and preserve the whole brain)
• Dry Ice (for brain)
• Wing punches
• scalpels
• liquid nitrogen and dry shipper
• tiny plastic baggies to put cryovials in
• string to hang bat bags on
• RNA protect
• DNA away
• ALL protect
• aluminum foil

Next time, I will get a voice recorder to record observations during dissection (which will also be useful in the field) and transcribe the notes later on. 

1. Make sheet before beginning with labeled tissues in respective order
2. Bleach entire bench and wash down to prevent bleach from contaminating the tissue
3. Wash everything with RNAase AWAY
4. Final solution on top of everything with RNAlater
5. Label cryovials
6. Remove bat from bat bag
7. Euthanize with anesthetic
8. Immediately take measurements and record on data sheet before rigor mortis sets in
9. Place cotton ball in mouth to prevent rigor mortis
10. Separate head from body and have two people working on the dissection simultaneously to remove the different tissues
11. Place tissues in respective labeled tubes
12. Top off the tubes with RNAlater
13. Place tubes on ice as soon as tissue is placed in RNAlater
14. If harvesting the brain, gently scoop the whole brain into a pocket of powdery dry ice and cover with dry ice until the brain hardens. Be careful to not damage the olfactory bulb and keep in mind the strong optic nerve. 
15. Remove brain from dry ice. We had been placing it in RNA later but not sure if this is the best way to preserve the actual brain. 
16. Place all cryovials in tiny plastic baggies and label. Place the entire plastic bag in dry shipper. 
17. Place the remaining specimen in the foil to be frozen at -20 and later prepared in formadehyde
18. Wash and clean bench in between each specimen. 

The most organized way we found to do this was to perform the actions in the following order
To prepare the final specimen:

• Plastic sandwich baggies
• Bottles of formaldehyde 
• WriteInTheRain paper (will not degrade in formaldehyde)
• Sharpies
• Gloves
• Masks