using primer prospector

Objective: select good primers for sequencing a section of the mer operon.

Methods: We continued to use the script check_primer_barcode_dimers.py to test ~1000 barcodes combined with a pair of primers. Abby has list of primers tested.

The overall speed of our testing was greatly increased by using files on the Desktop of the VM running on an internal HDD. This was much faster than using files from a VM stored on an external USB 2.0 drive, or files accessed through the Shared_Folder.

Results: Every primer-barcode combination was eliminated because all pairs produced secondary structures. This seams unrealistic, leading us to believe that we are using the script wrong. This could also happen if there is conflict between our primers and Illumina adapter.

3 hours

pprospector-1.0.1 works!

Objective: get primer prospector to work.

Methods: continuing on from yesterday using Qiime 1.7.0 VM. This includes pprospector-1.0.1 and veinna-1.8.4. Previously, the input script has been reading and writing files in the Shared_Folder which was connected to the windows desktop. On a whim, thumbnails in Nautilus were disabled, all needed files were copied to the Ubuntu desktop, and the following script was run.

check_primer_barcode_dimers.py -p AATGATACGGCGACCACCGAGATCTACACTATGGTAATTGTTCCGCAAGTNGCVACBGTNGG -P ACCATCGTCAGRTARGGRAAVA -b barcodes.txt -e DNA_parameters/dna_DM.par -o out/

Results: For some reason, this worked. Perhaps the delay writing to the Shared_Folder caused a problem or perhaps the NTFS formatting of the host desktop caused permission problems for the script. But now, it works!

The script was stopped after 25 minutes. In that time, +700 files were created in the output folder most with names like Line15_GAATACCAAGTC_primer1V86_primer1V86.px and each around 5.8kB in size. In the 25 minutes it was allowed to run, the script had progressed from Line0 to Line20. The input file, barcodes.txt, contains 1056 lines of barcodes. Back of the napkin, 1000 lines at 25 mins per 20 lines is about 20 hours. Processor usage was about 30% each on 4 cores, so additional parallelization is possible.

3 hours

moving files to the cluster

August 26 for 2 hours

Objective: move files to Juniata's bioinformatics cluster and extract them.

Methods:
Email Chris Walls for an account on the cluster and cc Dr. Lamendella.
Download and install Cyberduck
Compress all the files you want to upload into a .zip archive. You can skip this step if the files are already compressed, like in a .gz or .tgz archive, of if you are uploading a small number of files.
Upload files with Cyberduck. Our server address is 10.39.6.10
Connect to our server over ssh (use putty on windows) and log in.
Extract the files.
For .zip files unzip yourfile.zip
For .tgz files tar xvzf file.tgz

Results: files are on the cluster!

pprospector-1.0.1 does not work

August 26 for 2.5 hours

Objective: Help Abby install primer prospector to test possible primers.

Intro: the program 'pprospector-1.0.1' is bundled with Qiime from 1.5.0 and newer. The program 'RNAfold' is a co-dependency and is included with Vienna RNA. Vienna RNA is included with Qiime 1.7.0 (maybe 1.6.0 as well), so the stock Qiime 1.7.0 VM should be fully capable of running primer prospector.

Methods: Ran
check_primer_barcode_dimers.py

Results: file writing error