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=New Padlock Probes for Agi15k_Feb2017= *[[Matt:LabNotes/2016-10-14 | Previous probe set was a 12k order with 11,154 unique oligos]] **So there is 3,846 oligos that can be designed ==Human Brain== *Add GFAP ===Get Transcript Sequences=== *Use Biomart like always ===Create ppDesigner Target Files=== *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=== ~/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=== *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== ===Gene Selection=== *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=== *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=== *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 [[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 ~/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=== *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=== 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= *15,000 Oligos {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:11pt" valign="bottom" | width="51" height="14" | Probeset | width="51" | Amplification | width="51" | # Probes | width="51" | # Barcodes | width="51" | # Genes |- style="font-size:11pt" valign="bottom" | height="14" | Human Brain | V4 | align="right" | 4,998 | align="right" | 392 | align="right" | 369 |- style="font-size:11pt" valign="bottom" | height="14" | Mouse Brain | V7 | align="right" | 7,428 | align="right" | 614 | align="right" | 591 |- style="font-size:11pt" valign="bottom" | height="14" | Mouse Embryo | V6 | align="right" | 2,469 | align="right" | 251 | align="right" | 220 |- style="font-size:11pt" valign="bottom" | height="14" | Lung Cancer | V8 | align="right" | 105 | align="right" | 105 | align="right" | 85 |}
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