Matt:LabNotes/2014-11-19: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Mzcai
>Mzcai
mNo edit summary
Line 113: Line 113:


   mv outputFile* newGenes_variableArms_contigs_ppDesignerFiles
   mv outputFile* newGenes_variableArms_contigs_ppDesignerFiles
===Final Probes to Order: Trim to 12,000 probes===
*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
*[[Media:OutputFile_0gap_final.txt | outputFile_0gap_final.txt]] (trimmed out probes: [[Media:OutputFile_0gap_final_trimmedprobes.txt | outputFile_0gap_final_trimmedprobes.txt]])
*[[Media:OutputFile_0gap_contig_final.txt | outputFile_0gap_contig_final.txt]] (trimmed out probes: [[Media:OutputFile_0gap_contig_final_trimmedprobes.txt | outputFile_0gap_contig_final_trimmedprobes.txt]])
  perl RevComp_Probes.pl
*[[Media:OutputFile_0gap_final_RevComp.txt | outputFile_0gap_final_RevComp.txt]]
*[[Media:OutputFile_0gap_contig_final_RevComp.txt | outputFile_0gap_contig_final_RevComp.txt]]
====Probe Set Statistics====
*For outputFile_0gap_final.txt and outFile_0gap_final_RevComp.txt
**2,486 probes
**2,096 exons
**165 genes
*[[Media:CountProbesPerGene_final.txt |# 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
*[[Media:CountProbesPerGene_contig_final.txt |# probes for each gene]]
[[File:20141119_ProbesPerGene_contig_final.JPG]]

Revision as of 06:24, 20 November 2014

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

  • 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

get_probes.pl

  • 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

  • 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

 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

 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

  • 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

  • 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