Matt:LabNotes/2016-4-6: Difference between revisions

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>Mzcai
>Mzcai
 
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   cat outputFile_0gap_chr*.txt > outputFile_0gap.txt
   cat outputFile_0gap_chr*.txt > outputFile_0gap.txt
<!--
 
*6,665 probes
*2,334 probes
*4,132 exons
*1,425 exons
*369 genes
*150 genes
**Only missing TMEM88B, a glial marker gene
**Only missing Zfpm2


*Combine chromosome fasta into single fasta in lexicographical order
*Combine chromosome fasta into single fasta in lexicographical order
Line 73: Line 73:
*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/kunzhang/RNAseq/Data/CommonFiles/refMrna.ndx -f outputFile_0gap.fa -F FA -r ALL > outputFile_0gap_novoalign_refMrna.out &
 
/home/kunzhang/softwares/Novocraft/novocraft/novoalign -d /home/kunzhang/softwares/Novocraft/novocraft/human_g1k_v37 -f outputFile_0gap.fa -F FA -r ALL > outputFile_0gap_novoalign_human_g1k_v37.out &
  /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 human_g1k_v37 (hg19)
Remove probes that did not align to refMrna or had multiple alignments to mm10
*4,978 probes
*1,808 probes
*3,326 exons
*1,202 exons
*368 genes
*150 genes
**Missing SHISA8, an Interneuron3 subtype marker gene


*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