Matt:LabNotes/2014-11-10

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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]

 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