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=Design Padlock Probe Arms for new Probe Set= *Design probes for genes that reveal heterogeneity in human brain *Genes are specific for certain cell types and subtypes *Files: Dropbox\GradZhangLab\CA12k_Apr2016\HumanBrain ==Gene Selection== *FinalListv1.txt **Made from GenesFromBA8Analyses_Suppl_Tables_v2.xlsx ***Neuronal and glial markers (Table S3) ***Neuronal subtype-specific genes (Table S6) ***Allen Human Brain Atlas ISH (Table S12) ***Blue's requested genes, 12 unique added ***Interneuron genes, 6 unique added ***Projection neuron genes, 4 unique added ***OLFM1 (RNAscope probe data) **TMEM90A -> SYNDIG1L (Official HGNC) *370 genes total ==Get Transcript Sequences== *Biomart browser interface Dataset Homo sapiens genes (GRCh38.p5) Filters with HGNC ID(s): Only HGNC symbol(s) [e.g. NTN3]: [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 *Remove coordinates on PATCH (fixes and novel), HSCHR (haplotype variants), and chromosomes **370 Unique Ensembl Gene IDs and 370 Unique HGNC Symbols ==Create ppDesigner Target Files== *Files in genome-miner ~/scratch/CA12kApr2016_ProbeDesign/HumanBrain *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 20 21 22 X do /home/mzcai/scratch/CA12kApr2016_ProbeDesign/HumanBrain/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/CA12kApr2016_ProbeDesign/HumanBrain/jobFile_chr$indx.pl > /home/mzcai/scratch/CA12kApr2016_ProbeDesign/HumanBrain/outputFile_chr$indx.txt & wait done ==Transform from 1base gap to 0base gap and Filter Softmasked== *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 *6,665 probes *4,132 exons *369 genes **Only missing TMEM88B, a glial marker gene ==Align to Ref mRNA and Genome== *Remove probes that did not align to refMrna or had multiple alignments to human_g1k_v37 (hg19) 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 & perl CleanupProbelist.pl *4,978 probes *3,326 exons *368 genes **Missing SHISA8, an Interneuron3 subtype marker gene *Output: outputFile_0gap_filtered.txt
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