Matt:LabNotes/2016-10-14

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Revision as of 22:10, 17 October 2016 by >Mzcai (→‎Run ppDesigner)
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New Padlock Probes For RNA Capture with SplintR: CA12k_Oct2016

Human Brain

Mouse Embryo

Mouse Brain

Gene Selection

    • 174 new genes provided by Zizhen
    • 174 new genes: /GradZhangLab/DARTFISH Collaborations/Mouse Brain/Genelist_174genes_part2.txt
      • Gpr133 -> Adgrd1 (MGI Symbol)
      • A730090H04 -> Dlx4os (MGI Symbol)

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
  • 173 Unique Ensembl Gene IDs and Associated Gene Names
    • Missing 5033421B08Rik

Create ppDesigner Target Files

  • Files in genome-miner:~/scratch/CA12kOct2016_ProbeDesign/MouseBrain
  • 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
 ~/scratch/TB12kOct2016_ProbeDesign/MouseBrain/173NewGenes/opt/ppDesigner/src/ppDesigner.pl ~/scratch/TB12kOct2016_ProbeDesign/MouseBrain/173NewGenes/jobFile_chr$indx.pl > ~/scratch/TB12kOct2016_ProbeDesign/MouseBrain/173NewGenes/outputFile_chr$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 ~/scratch/TB12kOct2016_ProbeDesign/MouseBrain/173NewGenes/ConvertToZeroGapProbe.pl ~/scratch/TB12kOct2016_ProbeDesign/MouseBrain/173NewGenes/outputFile_chr$indx.txt > ~/scratch/TB12kOct2016_ProbeDesign/MouseBrain/173NewGenes/outputFile_0gap_chr$indx.txt &
 cat outputFile_0gap_chr*.txt > outputFile_0gap.txt
  • 2,389 probes
  • 1,435 exons
  • 172 genes

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

 /media/Home_Raid1/kunzhang/softwares/Novocraft/novocraft/novoalign -d ~/scratch/Genomes/mm10/novoalign/mm10_refMrna.ndx -f outputFile_0gap.fa -F FA -r ALL > outputFile_0gap_novoalign_mm10refMrna.out &
 /media/Home_Raid1/kunzhang/softwares/Novocraft/novocraft/novoalign -d ~/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

  • Output: outputFile_0gap_filtered.txt
    • 1,794 probes
    • 1,250 exons
    • 171 genes
  • Missing Trbc2
  • RevComp.pl

Add Barcodes to make probes

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