Matt:LabNotes/2016-4-6: Difference between revisions
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>Mzcai (Created page with "=Design Padlock Probe Arms for new Probe Set= *Design probes for genes that reveal heterogeneity in mouse brain *Genes are specific for certain cell types and subtypes determi...") |
>Mzcai m (→Run ppDesigner) |
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(One intermediate revision by the same user not shown) | |||
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==Run ppDesigner== | ==Run ppDesigner== | ||
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 | |||
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 & | |||
wait | |||
done | |||
*Add target base to arm with lowest Tm to create zero-gap padlock probe | *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) | *Also filter out any probes targeting soft-masked regions (indicated by lowercase reference sequence) | ||
**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 /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 & | ||
cat outputFile_0gap_chr*.txt > outputFile_0gap.txt | cat outputFile_0gap_chr*.txt > outputFile_0gap.txt | ||
* | *2,334 probes | ||
* | *1,425 exons | ||
* | *150 genes | ||
**Only missing | **Only missing Zfpm2 | ||
*Combine chromosome fasta into single fasta in lexicographical order | *Combine chromosome fasta into single fasta in lexicographical order | ||
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*Build novoalign index | *Build novoalign index | ||
**/home/kunzhang/softwares/Novocraft/novocraft/novoindex ./novoalign/mm10.ndx ./mm10.fa | **/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/ | |||
/home/kunzhang/softwares/Novocraft/novocraft/novoalign -d /home/ | /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 | perl CleanupProbelist.pl | ||
Remove probes that did not align to refMrna or had multiple alignments to | 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 |
Latest revision as of 18:25, 7 April 2016
Design Padlock Probe Arms for new Probe Set[edit]
- Design probes for genes that reveal heterogeneity in mouse brain
- Genes are specific for certain cell types and subtypes determined by Allen Brain Institute collaborators
- Files: Dropbox\GradZhangLab\CA12k_Apr2016\MouseBrain
Gene Selection[edit]
- 152 genes: GeneListv1.txt
- 2310042E22Rik -> Teddm3 (MGI Symbol)
- 6330527O06Rik -> Lamp5 (MGI Symbol)
- A930038C07Rik -> Ndnf (MGI Symbol)
- Ppapdc1a -> Plpp4 (MGI Symbol)
- Gpr126 -> Adgrg6 (MGI Symbol)
- Fam5c -> Brinp3 (MGI Symbol)
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
- 151 Unique Ensembl Gene IDs and Associated Gene Names
- Missing 9630013A20Rik
Create ppDesigner Target Files[edit]
- Files in genome-miner:~/scratch/CA12kApr2016_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[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/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 & 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/HumanBrain/ConvertToZeroGapProbe.pl /home/mzcai/scratch/CA12kApr2016_ProbeDesign/HumanBrain/outputFile_chr$indx.txt > /home/mzcai/scratch/CA12kApr2016_ProbeDesign/HumanBrain/outputFile_0gap_chr$indx.txt &
cat outputFile_0gap_chr*.txt > outputFile_0gap.txt
- 2,334 probes
- 1,425 exons
- 150 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
/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
- 1,808 probes
- 1,202 exons
- 150 genes
- Output: outputFile_0gap_filtered.txt