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]
- Use TargetFiles from Matt:LabNotes/2014-11-1
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
- Remove probes belonging to genes with <8 probes per gene
- Result: 2,542 probes for 165 genes
mv outputFile* newGenes_variableArms_ppDesignerFiles
Contiguous Exon Targets[edit]
- Use TargetFiles from Matt:LabNotes/2014-11-10
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
- Remove probes belonging to genes with <8 probes per gene
- 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
- For outputFile_0gap_contig_8reads_8pergene.txt removed probes so that max probes per gene = 25
- outputFile_0gap_final.txt (trimmed out probes: outputFile_0gap_final_trimmedprobes.txt)
- outputFile_0gap_contig_final.txt (trimmed out probes: outputFile_0gap_contig_final_trimmedprobes.txt)
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