Matt:LabNotes/2016-4-7

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Design Padlock Probe Arms for Mouse Embryo Probe Set[edit]

  • 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[edit]

  • 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[edit]

  • 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[edit]

  • 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[edit]

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 &

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

  • 2,469 probes
  • 1,564 exons
  • 220 genes
  • Output: outputFile_0gap_filtered.txt