Editing
Matt:LabNotes/2014-11-19
Jump to navigation
Jump to search
Warning:
You are not logged in. Your IP address will be publicly visible if you make any edits. If you
log in
or
create an account
, your edits will be attributed to your username, along with other benefits.
Anti-spam check. Do
not
fill this in!
==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=== *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 **Outputs: [[Media:OutputFile_0gap_8reads_8pergene.txt | outputFile_0gap_8reads_8pergene.txt]] & [[Media:OutputFile_0gap_8reads_8pergene_RevComp.txt | outputFile_0gap_8reads_8pergene_RevComp.txt]] *Result: 2,542 probes for 165 genes mv outputFile* newGenes_variableArms_ppDesignerFiles ===Contiguous Exon Targets=== *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 **Outputs: [[Media:OutputFile_0gap_contig_8reads_8pergene.txt | outputFile_0gap_contig_8reads_8pergene.txt]] & [[Media:OutputFile_0gap_contig_8reads_8pergene_RevComp.txt | outputFile_0gap_contig_8reads_8pergene_RevComp.txt]] *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 *[[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 | 650px]] *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 | 650px]] *Of the 20 Glial Marker Genes 11 had probes successfully designed {| {{table}} | align="center" style="background:#f0f0f0;"|'''Glial Marker Gene''' | align="center" style="background:#f0f0f0;"|'''Constitutive: # Probes''' | align="center" style="background:#f0f0f0;"|'''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==== ./[[Media:probe2padlockFISSEQ_Nov2014_20bp_v4.txt|probe2padlockFISSEQ_Nov2014_20bp_v4.pl]] > [[Media:OutputFile_0gap_contig_final.full_info.V4.txt]] ./[[Media:probe2padlockFISSEQ_Nov2014_20bp_v6.txt|probe2padlockFISSEQ_Nov2014_20bp_v6.pl]] > [[Media:OutputFile_0gap_contig_final_RevComp.full_info.v6.txt]] ./[[Media:probe2padlockFISSEQ_Nov2014_20bp_v7.txt|probe2padlockFISSEQ_Nov2014_20bp_v7.pl]] > [[Media:OutputFile_0gap_final.full_info.V7.txt]] ./[[Media:probe2padlockFISSEQ_Nov2014_20bp_v8.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]]
Summary:
Please note that all contributions to ZhangLabWiki may be edited, altered, or removed by other contributors. If you do not want your writing to be edited mercilessly, then do not submit it here.
You are also promising us that you wrote this yourself, or copied it from a public domain or similar free resource (see
ZhangLabWiki:Copyrights
for details).
Do not submit copyrighted work without permission!
Cancel
Editing help
(opens in new window)
Template used on this page:
Template:Table
(
edit
)
Navigation menu
Personal tools
Not logged in
Talk
Contributions
Create account
Log in
Namespaces
Page
Discussion
English
Views
Read
Edit
View history
More
Search
Navigation
Main Page
Current events
Recent changes
Random page
Investigators
Matt Cai
Song Chen
Eric Chu
Dinh Diep
Elizabeth Duong
Shicheng Guo
Alan Fung
Daniel Jacobsen
Blue Lake
Huy Lam
Alice Li
Andrew Richards
Brandon Sos
Chris Wei
Yan Wu
Kun Zhang
Tools
What links here
Related changes
Special pages
Page information