Daniel:Notebook/HiResChrPaint/2014-6-16

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Probe Design[edit]

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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.

  1. Download fasta file from ncbi.
    1. hg38 download
  2. Concatenate .fa files; oligopaints, etc. needs a single reference file
    1. cat *.fa > hg38.fa
  3. Download gene information for a specific region
    1. Selected CASK gene, Xp11.4 (chrX:41514933-41923154); arbitrary
  4. Generate BLAST database using makeblastdb
    1. make a directory called BlastDb
    2. set up a link using ln -s to the .fas file
    3. makeblastdb -in hg38.fas -out hg38 -dbtype nucl -parse_seqids
  5. Run OligoArray to generate viable probe sequences
    1. 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.