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 m (→Run ppDesigner) |
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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 | ||
~/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 / | 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, | *2,389 probes | ||
*1, | *1,435 exons | ||
* | *172 genes | ||
perl Probes2fasta.pl < outputFile_0gap.txt > outputFile_0gap.fa | 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 | 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 | ||
*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
- Same as TB12k_Apr2016_V4 but RevComp of annealing arms
Mouse Embryo
- Same as TB12k_Apr2016_V6 but RevComp of annealing arms
Mouse Brain
- Same as TB12k_Apr2016_V7 but RevComp of annealing arms and add 174 new genes
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