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=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 determined by Allen Brain Institute collaborators *Files: Dropbox\GradZhangLab\CA12k_Apr2016\MouseBrain ==Gene Selection== *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== *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== *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 [[Matt:LabNotes/2014-11-10 | 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 /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
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