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==Design FISSEQ Padlock Probeset for in situ cDNA Capture (New Genelist + Contigs)== *[[Matt:LabNotes/2014-11-1 | Previously]] designed padlock probe set for cDNA that only capture constitutive exons (target doesn't span any exon boundaries) only had ~2,000 probes (+2,000 for RevComp RNA capture) *Here I want to relax parameters so we can meet the minimum 12,000 oligo requirement for ordering from CustomArray **This will be done by targeting ALL exons of the genes (i.e. make exons into contigs and use those as targets) ===Build Target File for ppDesigner=== *Since the 409 genes are the same as [[Matt:LabNotes/2014-11-1#Get_Genes]] can use the same Biomart exon output (mart_export_newGenes.txt) *Use [[Media:CreateTargetFile_contig.txt | CreateTargetFile_contig.pl]] to create target file where targets are contigs of exons CreateTargetFile_contig.pl > target_file_contigs_newGenes.txt *Fix Gene names **For unknown reason perl script missed ~100 gene names so had to manually insert them into target file *Sort target files into each chromosome and remove 25bp from each end of target and switch strand [[Media:SortTargetFilesByChr.txt | sortTargetFilesByChr.pl]] ===Run ppDesigner=== ppDesignerCommands.sh cat outputFile_chr*.txt > outputFile_1gap.txt *Convert to 0gap perl [[Media:ConvertToZeroGapProbe.txt | ConvertToZeroGapProbe.pl]] *Probecount is 7,727 *Exons is 4,353 *Genes is 406 ===Filter Out Bad Probes=== *Same as [[Matt:LabNotes/2014-11-1#Filter_Out_Bad_Probes]] perl [[Media:Probes2fasta.txt | 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 [[Media:CleanupProbelist.txt | CleanupProbelist.pl]] *Probecount is 5,251 *Exons is 3,193 *Genes is 402 ====Filter probes with low coverage based on Brain RNA-seq Data from Rui/Blue ==== perl [[Media:ConvertProbesHg38_2_BedHg19.txt | ConvertProbesHg38_2_BedHg19.pl]] coverageBed -abam BrainBulkTissueRNA_mappedhg19.sorted.bam -b outputFile_0gap_hg19.bed > outputFile_BrainBulkTissueRNA.cov *Filter out probes with < 10 reads perl [[Media:FilterProbelist_LowReadCounts.txt | FilterProbelist_LowReadCounts.pl]] > outputFile_0gap_10reads_contig.txt *Rank Order plot of Probes and the number of reads that overlap with each probe target region **859 of 5,251 probes had 0 overlap [[File:RankOrderProbesOverlapReads_contig.JPG]] ===RevComp Probes for potential RNA capture=== perl [[Media:RevComp_Probes.txt | RevComp_Probes.pl]] > outputFile_0gap_10reads_RevComp_contig.txt [[File:20141111_ProbesPerGeneHistogram.JPG | 650px]] *Probecount is 3,095 *Exons is 1,848 *Genes is 378
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