Daniel:Notebook/HiResChrPaint/2014-6-16
Probe Design[edit]
Using the Oligopaints scripts by Beliveau, my goal is to design a positive/negative control for the FISH probes. The control is to focus on a region of the X chromosome, yet to be determined. Using male cells (such as PGP1), exactly one region should be painted per cell. This serves as a potential positive and negative control.
Probe Mining[edit]
Scripts:
Setting up the scripts for probe mining. I'm doing this locally on my machine for now, following the instructions in the two pdfs above.
- Download fasta file from ncbi.
- Concatenate .fa files; oligopaints, etc. needs a single reference file
- cat *.fa > hg38.fa
- Download gene information for a specific region
- Selected CASK gene, Xp11.4 (chrX:41514933-41923154); arbitrary
- Generate BLAST database using makeblastdb
- make a directory called BlastDb
- set up a link using ln -s to the .fas file
- makeblastdb -in hg38.fas -out hg38 -dbtype nucl -parse_seqids
- Run OligoArray to generate viable probe sequences
- java -jar /Users/danieljacobsen/Desktop/OligoArray2_1/OligoArray2.jar -i in_caskgene.fas -d BlastDb/hg38 -o oligo_caskgene.txt -r rejected_oligo.fas -R caskgene.log -n 30 -l 60 -L 60 -D 1000 -t 55 -T 65 -s 70 -x 70 -p 35 -P 80 -m "CCCC;GGGG;TTTTT;AAAAA" -g 52
Error: Cannot run program "blastall": error=2, No such file or directory
It would appear that the version of BLAST I downloaded is too new. The old version is known as blast legacy, and contains the command "blastall". The new version, blast+, uses the individual flags in blastall (blastn, blastp, etc) as their own standalone functions, and gets rid of "blastall". However, I examined genome miner, and it turns out genome miner is using the legacy version of blast, containing blastall.
Tomorrow, I'll have to try this again but one genome miner.