Matt:LabNotes/2014-11-10
Jump to navigation
Jump to search
Design FISSEQ Padlock Probeset for in situ cDNA Capture (New Genelist + Contigs)[edit]
- 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[edit]
- 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 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
sortTargetFilesByChr.pl
Run ppDesigner[edit]
ppDesignerCommands.sh cat outputFile_chr*.txt > outputFile_1gap.txt
- Convert to 0gap
perl ConvertToZeroGapProbe.pl
- Probecount is 7,727
- Exons is 4,353
- Genes is 406
Filter Out Bad Probes[edit]
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
- 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[edit]
perl 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 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[edit]
perl RevComp_Probes.pl > outputFile_0gap_10reads_RevComp_contig.txt
File:20141111 ProbesPerGeneHistogram.JPG
- Probecount is 3,095
- Exons is 1,848
- Genes is 378