Matt:LabNotes/2017-2-8

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New Padlock Probes for Agi15k_Feb2017[edit]

Human Brain[edit]

  • Add GFAP

Get Transcript Sequences[edit]

  • Use Biomart like always

Create ppDesigner Target Files[edit]

  • Files in genome-miner:~/scratch/Agi15kFeb2017_ProbeDesign/
  • Use CreateTargetFile_contig.pl to create target file where targets are contigs of exons
  • Sort target files into each chromosome and remove 25bp from each end of target
    • SortTargetFilesByChr.pl

Run ppDesigner[edit]

 ~/scratch/Agi15kFeb2017_ProbeDesign/opt/ppDesigner/src/ppDesigner.pl ~/scratch/Agi15kFeb2017_ProbeDesign/jobFile_chr17.pl > ~/scratch/Agi15kFeb2017_ProbeDesign/outputFile_chr17.txt &
  • 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/Agi15kFeb2017_ProbeDesign/ConvertToZeroGapProbe.pl ~/scratch/Agi15kFeb2017_ProbeDesign/outputFile_chr17.txt > ~/scratch/Agi15kFeb2017_ProbeDesign/outputFile_0gap.txt &


  • 20 probes
  • 14 exons
 perl Probes2fasta.pl < outputFile_0gap.txt > outputFile_0gap.fa
 /media/Home_Raid1/kunzhang/softwares/Novocraft/novocraft/novoalign -d /media/12TB_ext/GenomeDB/HsGenome/1KG.ref/NovoAlign/human_g1k_v37 -f outputFile_0gap.fa -F FA -r ALL > outputFile_0gap_novoalign_human_g1k_v37.out &
  • No need to filter because all unique alignments

Combine with Human Brain CA12kOct2016_V4 Probes[edit]

  • Copy GradZhangLab\CA12k_Oct2016\HumanBrain_V4\outputFile_0gap_mod.txt to GradZhangLab\CA12k_Oct2016\Agi15k_Feb2017\HumanBrain_V4
  • Add GFAP probes from outputFile_0gap.txt to outputFile_0gap_mod.txt -> outputFile_0gap_combined.txt
  • RevComp_Probes.pl
 perl Probe2padlockFISSEQ_Oct2016_HumanBrain_6Cycle_V4 > padlockFile_0gap_HumanBrain_V4.txt

Mouse Brain[edit]

Gene Selection[edit]

  • 349 genes from Long Cai Neuron paper
    • 317 genes we do not already have in CA12kOct2016_V7 Mouse Brain set
    • After trying with biomart ended up with 315 genes after clearing up nomenclature

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


Create ppDesigner Target Files[edit]

  • Files in genome-miner:~/scratch/Agi15kFeb2017_ProbeDesign/
  • 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
 ~/scratch/Agi15kFeb2017_ProbeDesign/opt/ppDesigner/src/ppDesigner.pl ~/scratch/Agi15kFeb2017_ProbeDesign/jobFile_chr$indx.pl > ~/scratch/Agi15kFeb2017_ProbeDesign/outputFile_chr$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/Agi15kFeb2017_ProbeDesign/ConvertToZeroGapProbe.pl ~/scratch/Agi15kFeb2017_ProbeDesign/outputFile_chr$indx.txt > ~/scratch/Agi15kFeb2017_ProbeDesign/outputFile_0gap_chr$indx.txt &
 cat outputFile_0gap_chr*.txt > outputFile_0gap.txt
  • 5,439 probes
  • 3,087 exons
  • 313 genes
    • Missing Rbm31y and Myh14

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
    • 4,383 probes
    • 2,664 exons
    • 311 genes
  • Missing Slc6a16, Gm15688

Combine Part 3 (315 New Genes) with Previous 150 Genes[edit]

  • In GradZhangLab\CA12k_Oct2016\Agi15k_Feb2017\MouseBrain_V7
    • Add GradZhangLab\CA12k_Oct2016\MouseBrain_V7\outputFile_0gap_combined.txt with outputFile_0gap_filtered.txt -> outputFile_0gap_Agicombined.txt
  • RevComp_Probes.pl
  • Need to cut down 537 probes to hit 15k oligos
    • Looked at Long Cai Neuron paper at 249 gene experiment and chose genes with lowest count per cell and not mentioned in any clustering analysis (Fig S6)
      • Atr
      • Pml
      • Sall1
      • Nr2f2
      • Gmeb2
      • Srebf1
      • Foxd3
      • Rorc
      • Esrrb
      • Nfkbiz
      • Egf
      • Notch3
      • Hoxa1
      • Zfp64
      • Sox13
      • Nhlh1
      • Lhx1
      • Ciita
      • Foxa1
      • Hoxd13
      • Nfkb2
      • Dmbx1
      • Trp73
      • E2f7
      • Pknox2
      • Tbx2
      • Taf6l
      • Vav1
      • Nr2e1
      • Mybl1
      • Wt1
      • Rest
      • Phox2b
      • Ctss
      • Lmx1a
      • Ikzf1
      • Mtf2
      • Pax6
      • Pax2
  • outputFile_0gap_Agicombinedfinal_RevComp.txt
  • outputFile_0gap_Agicombined_removed_RevComp.txt

Add Barcode[edit]

 perl Probe2padlockFISSEQ_Feb2017_MouseBrain_6Cycle_V7.pl > padlockFile_0gap_MouseBrain_V7.txt
  • Since added GFAP to HumanBrain_V4 probeset that had 20 probes, need to remove 19+ probes from MouseBrain
    • Removed 19 Abca9 probes since there were 30+ Abca9 probes

Summary[edit]

  • 15,000 Oligos
Probeset Amplification # Probes # Barcodes # Genes
Human Brain V4 4,998 392 369
Mouse Brain V7 7,428 614 591
Mouse Embryo V6 2,469 251 220
Lung Cancer V8 105 105 85