Matt:LabNotes/2017-2-8
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New Padlock Probes for Agi15k_Feb2017[edit]
- Previous probe set was a 12k order with 11,154 unique oligos
- So there is 3,846 oligos that can be designed
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
- 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)
- 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 |