Matt:LabNotes/2016-4-7: Difference between revisions

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>Mzcai
(Created page with "=Design Padlock Probe Arms for Mouse Embryo Probe Set= *Design probes for genes that reveal cell migration and differentiation into cardiac cells in mouse embryo *Genes provid...")
 
>Mzcai
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   cat outputFile_0gap_chr*.txt > outputFile_0gap.txt
   cat outputFile_0gap_chr*.txt > outputFile_0gap.txt
<!--
 
*2,334 probes
*3,175 probes
*1,425 exons
*1,839 exons
*150 genes
*220 genes
**Only missing Zfpm2
**Only missing Zic5


perl Probes2fasta.pl < outputFile_0gap.txt > outputFile_0gap.fa
perl Probes2fasta.pl < outputFile_0gap.txt > outputFile_0gap.fa
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   /home/kunzhang/softwares/Novocraft/novocraft/novoalign -d /home/mzcai/scratch/Genomes/mm10/novoalign/mm10.ndx -f outputFile_0gap.fa -F FA -r ALL > outputFile_0gap_novoalign_mm10.out &
   /home/kunzhang/softwares/Novocraft/novocraft/novoalign -d /home/mzcai/scratch/Genomes/mm10/novoalign/mm10.ndx -f outputFile_0gap.fa -F FA -r ALL > outputFile_0gap_novoalign_mm10.out &


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perl CleanupProbelist.pl
perl CleanupProbelist.pl
Remove probes that did not align to refMrna or had multiple alignments to mm10
Remove probes that did not align to refMrna or had multiple alignments to mm10

Revision as of 05:48, 8 April 2016

Design Padlock Probe Arms for Mouse Embryo Probe Set

  • Design probes for genes that reveal cell migration and differentiation into cardiac cells in mouse embryo
  • Genes provided by Paola, Jonathan, and Leen from Evans, Chi, and Yeo lab
  • Files: Dropbox\GradZhangLab\CA12k_Apr2016\MouseEmbryo

Gene Selection

  • 221 genes from 222 genes provided: GeneListv1.txt
    • Bapx1 -> Nkx3-2 (already exist)
    • Jarid1b -> Kdm5b (MGI Symbol)
    • MyoD -> MyoD1 (MGI Symbol)
    • Myrf5 (typo)
    • -> Nkx3-1 (added by Paola through email)

Get Transcript Sequences

  • Biomart browser interface
 Dataset
 Mus musculus genes (GRCm38.p4)
 Filters
 with MGI ID(s): Only
 MGI symbol [e.g. Mir1901]: [ID-list specified]
 Status (gene): KNOWN
 Status (transcript): KNOWN
 Attributes
 Ensembl Gene ID
 Ensembl Transcript ID
 Chromosome Name
 Exon Rank in Transcript
 Exon Chr Start (bp)
 Exon Chr End (bp)
 Strand
 Associated Gene Name
  • 221 Unique Ensembl Gene IDs and Associated Gene Names

Create ppDesigner Target Files

  • Files in genome-miner:~/scratch/CA12kApr2016_ProbeDesign/MouseEmbryo
  • Use CreateTargetFile_contig.pl to create target file where targets are contigs of exons
    • Script is modified from here
  • Sort target files into each chromosome and remove 25bp from each end of target and switch strand
    • SortTargetFilesByChr.pl

Run ppDesigner

ppDesignerCommands.sh

 #!/bin/bash
 for indx in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X
 do
 /home/mzcai/scratch/CA12kApr2016_ProbeDesign/MouseEmbryo/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/CA12kApr2016_ProbeDesign/MouseEmbryo/jobFile_chr$indx.pl > /home/mzcai/scratch/CA12kApr2016_ProbeDesign/MouseEmbryo/outputFile_chr$indx.txt &
 wait
 done
  • Add target base to arm with lowest Tm to create zero-gap padlock probe
  • Also filter out any probes targeting soft-masked regions (indicated by lowercase reference sequence)
    • ConvertToZeroGapProbe.pl
 perl /home/mzcai/scratch/CA12kApr2016_ProbeDesign/HumanEmbryo/ConvertToZeroGapProbe.pl /home/mzcai/scratch/CA12kApr2016_ProbeDesign/HumanEmbryo/outputFile_chr$indx.txt > /home/mzcai/scratch/CA12kApr2016_ProbeDesign/HumanEmbryo/outputFile_0gap_chr$indx.txt &
 cat outputFile_0gap_chr*.txt > outputFile_0gap.txt
  • 3,175 probes
  • 1,839 exons
  • 220 genes
    • Only missing Zic5

perl Probes2fasta.pl < outputFile_0gap.txt > outputFile_0gap.fa

 /home/kunzhang/softwares/Novocraft/novocraft/novoalign -d /home/mzcai/scratch/Genomes/mm10/novoalign/mm10_refMrna.ndx -f outputFile_0gap.fa -F FA -r ALL > outputFile_0gap_novoalign_mm10refMrna.out &
 /home/kunzhang/softwares/Novocraft/novocraft/novoalign -d /home/mzcai/scratch/Genomes/mm10/novoalign/mm10.ndx -f outputFile_0gap.fa -F FA -r ALL > outputFile_0gap_novoalign_mm10.out &