Matt:LabNotes/2014-11-19

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Design FISSEQ Padlock Probeset for in situ cDNA Capture (newGenes, constitutive exons & contigs, variable capture arm length)[edit]

  • Previous designs (Matt:LabNotes/2014-11-1 & Matt:LabNotes/2014-11-10) had exactly 25bp capture arms due to some different code in ppDesigner
    • In jobFiles, H1_plus_H2_Len was the exact length the two arms had to equal
    • Changed ppDesigner so H1_plus_H2_Len is the maximum length the two arms can equal
      • Each arm can now vary between 20-25bp as was originally intended

Changes to ppDesigner Code[edit]

get_probes.pl[edit]

  • Lines 563 and 677
    • Commented out: next if(! (length($a_oligo)+length($b_oligo) == $H1_plus_H2_Len) );
    • Added: next if( length($a_oligo)+length($b_oligo) > $H1_plus_H2_Len );

output_text.pl[edit]

  • Error of dividing by 0 caused by $searchEnd being <= $searchStart (due to sortTargetFilesByChr.pl removing 25bp from each end even if total target < 50bp)
  • Inserted in lines 26-28
 if($searchEnd <= $searchStart) {
      $searchEnd = $searchStart + 1;
 }

Constitutive Exon Targets[edit]

 run ppDesigner.sh
 cat outputFile_chr*.txt > outputFile_1gap.txt
 perl ConvertToZeroGapProbe.pl
  • 7,027 probes successfully designed
  • 5,845 exons
  • 407 genes
 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
  • 5,629 probes
  • 4,735 exons
  • 406 genes
 perl  ConvertProbesHg38_2_BedHg19.pl
 coverageBed -abam BrainBulkTissueRNA_mappedhg19.sorted.bam -b outputFile_0gap_hg19.bed > outputFile_BrainBulkTissueRNA.cov

File:20141119 Constitutive RankOrderProbesOverlapReads.JPG

 perl FilterProbelist_LowReadCounts.pl > outputFile_0gap_10reads.txt
 perl RevComp_Probes.pl
  • 3,126 probes
  • 2,655 exons
  • 378 genes
 perl FilterProbelist_LowReadCounts.pl > outputFile_0gap_8reads.txt
 perl RevComp_Probes.pl
  • 3,403 probes
  • 2,879 exons
  • 390 genes

File:20141119 Constitutive8reads ProbesperGene.JPG

 perl FilterProbelist_LowProbesPerGene.pl
  • Result: 2,542 probes for 165 genes
 mv outputFile* newGenes_variableArms_ppDesignerFiles

Contiguous Exon Targets[edit]

 run ppDesigner.sh
 cat outputFile_chr*.txt > outputFile_1gap.txt
 perl ConvertToZeroGapProbe.pl
  • 10,429 probes successfully designed
  • 6,435 exons
  • 406 genes
 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
  • 7,390 probes
  • 4,969 exons
  • 406 genes
 perl  ConvertProbesHg38_2_BedHg19.pl
 coverageBed -abam BrainBulkTissueRNA_mappedhg19.sorted.bam -b outputFile_0gap_hg19.bed > outputFile_BrainBulkTissueRNA.cov

File:20141119 Contig RankOrderProbesOverlapReads.JPG

 perl FilterProbelist_LowReadCounts.pl > outputFile_0gap_10reads.txt
 perl RevComp_Probes.pl
  • 4,250 probes
  • 2,830 exons
  • 390 genes
 perl FilterProbelist_LowReadCounts.pl > outputFile_0gap_8reads.txt
 perl RevComp_Probes.pl
  • 4,598 probes
  • 3,073 exons
  • 394 genes

File:20141119 Contig8reads ProbesperGene.JPG

 perl FilterProbelist_LowProbesPerGene.pl
  • Result: 3,938 probes each; 240 genes
 mv outputFile* newGenes_variableArms_contigs_ppDesignerFiles

Final Probes to Order: Trim to 12,000 probes[edit]

  • Manually remove probes from genes with most probes per gene on outputFile_0gap_8reads_8pergene.txt and outputFile_0gap_contig_8reads_8pergene.txt to reach 12,000 total probes
    • For outputFile_0gap_contig_8reads_8pergene.txt removed probes so that max probes per gene = 25
      • If there were >25 exons I removed probes from the last exons
      • If there were many probes for the same exon, I removed probes from the bottom of the list leaving ~2-4 probes
    • For outputFile_0gap_8reads_8pergene.txt removed probes so that max probes per gene = 40
 perl RevComp_Probes.pl

Probe Set Statistics[edit]

  • For outputFile_0gap_final.txt and outFile_0gap_final_RevComp.txt
    • 2,486 probes
    • 2,096 exons
    • 165 genes
  • # probes for each gene

File:20141119 ProbesPerGene final.JPG

  • For outputFile_0gap_contig_final.txt and outFile_0gap_contig_final_RevComp.txt
    • 3,514 probes
    • 2,432 exons
    • 240 genes
  • # probes for each gene

File:20141119 ProbesPerGene contig final.JPG


  • Of the 20 Glial Marker Genes 11 had probes successfully designed
Glial Marker Gene Constitutive: # Probes Contig: # Probes
ATP1A2 20 22
BCAN 11 14
CNP #N/A 14
GFAP #N/A 12
GJA1 8 8
GPR56 9 17
GSN 11 10
MBP #N/A 16
MOBP #N/A 13
SLC1A2 9 14
SLC1A3 10 16

Assembly of oligo pool for ordering[edit]

 ./probe2padlockFISSEQ_Nov2014_20bp_v4.pl > Media:OutputFile_0gap_contig_final.full_info.V4.txt 
 ./probe2padlockFISSEQ_Nov2014_20bp_v6.pl > Media:OutputFile_0gap_contig_final_RevComp.full_info.v6.txt 
 ./probe2padlockFISSEQ_Nov2014_20bp_v7.pl > Media:OutputFile_0gap_final.full_info.V7.txt 
 ./probe2padlockFISSEQ_Nov2014_20bp_v8.pl > Media:OutputFile_0gap_final_RevComp.full_info.V8.txt
 A full list of 12,000 oligos to order: Media:Fisga_Nov2014_seq.txt