Matt:LabNotes/2016-10-14: Difference between revisions

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>Mzcai
(Created page with "=New Padlock Probes For RNA Capture with SplintR: CA12k_Oct2016= ==Human Brain== *Same as TB12k_Apr2016_V4 but RevComp of annealing arms ==Mouse ...")
 
>Mzcai
Line 51: Line 51:
ppDesignerCommands.sh
ppDesignerCommands.sh
   #!/bin/bash
   #!/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
   for indx in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X
   do
   do
   /home/mzcai/scratch/CA12kApr2016_ProbeDesign/MouseBrain/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/CA12kApr2016_ProbeDesign/MouseBrain/jobFile_chr$indx.pl > /home/mzcai/scratch/CA12kApr2016_ProbeDesign/MouseBrain/outputFile_chr$indx.txt &
   ~/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
   wait
   done
   done
Line 61: Line 62:
**ConvertToZeroGapProbe.pl
**ConvertToZeroGapProbe.pl


   perl /home/mzcai/scratch/CA12kApr2016_ProbeDesign/HumanBrain/ConvertToZeroGapProbe.pl /home/mzcai/scratch/CA12kApr2016_ProbeDesign/HumanBrain/outputFile_chr$indx.txt > /home/mzcai/scratch/CA12kApr2016_ProbeDesign/HumanBrain/outputFile_0gap_chr$indx.txt &
   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
   cat outputFile_0gap_chr*.txt > outputFile_0gap.txt


*2,334 probes
*2,389 probes
*1,425 exons
*1,435 exons
*150 genes
*172 genes
**Only missing Zfpm2
 
*Combine chromosome fasta into single fasta in lexicographical order
**echo "$(ls chr*.fa | sort -V | grep -vP 'chr[^X|Y|\d]'; ls chr*.fa | sort -V | grep -vP 'chr[\d|X|Y]')" | xargs cat > mm10.fa
*Build novoalign index
**/home/kunzhang/softwares/Novocraft/novocraft/novoindex ./novoalign/mm10.ndx ./mm10.fa
**/home/kunzhang/softwares/Novocraft/novocraft/novoindex ./novoalign/mm10_refMrna.ndx ./refMrna.fa


perl Probes2fasta.pl < outputFile_0gap.txt > outputFile_0gap.fa
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 &
   /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 &
   /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 &
   /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
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
*1,808 probes
*1,202 exons
*150 genes


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


[[Matt:LabNotes/2016-4-18|Add Barcodes to make probes]]  
[[Matt:LabNotes/2016-4-18|Add Barcodes to make probes]]


==Lung Cancer Fusion Oncogenes==
==Lung Cancer Fusion Oncogenes==

Revision as of 22:10, 17 October 2016

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

Lung Cancer Fusion Oncogenes