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