Editing
Matt:LabNotes/2014-10-30
(section)
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== ===Get Genes=== *Took all HUGO gene names from [[media:TopDiffExpGenes_1000hNucDataset_09252014.xlsx | TopDiffExpGenes_1000hNucDataset_09252014.xlsx]] and removed duplicates **Result: 542 Genes ***378 Duplicates removed *Removed all genes with names that had: "-", "AC###.#", "AS", "RP", "RNA", "MT" ... **[[Media:FISSEQ_cDNA_removedgenes.txt | 92 genes removed]] **[[Media:FISSEQ_cDNA_genelist.txt | Result: 450 genes]] ===Build Target File for ppDesigner=== ====Biomart: Get Exon Locations for Genes on Reference Genome==== *Use 450 HUGO names as input *GRCh38/hg38 reference genome *Unfortunately when try to output 'Associated Gene Name' (aka HUGO Official Gene Symbol) Biomart gave error so had to output Ensembl Gene IDs **Some LRG (locus reference genome) gene names and HSCHR (haplotype variants) outputted so will filter those out *Output parameters: **Ensembl Gene ID **Ensembl Transcript ID **Chromosome **Exon Rank **Exon Start (bp) **Exon End (bp) **Strand ====Create Hash to convert Ensembl to HUGO==== *Using [[media:TopDiffExpGenes_1000hNucDataset_09252014.xlsx]] created hash table for converting Ensembl IDs to HUGO since Biomart output had to be ENSG **Hash naturally removes duplicates but also checked to see if there was gene names that didn't match 1-to-1 ***ENSG000000236922 matched to LINC01378 and AC092661.1 but since these were removed from the final 450 genes can ignore **Hash size is 541 which agrees with the 542 Genes counted when duplicates removed plus taking into considering the ambiguity of ENSG000000236922 *This code can be seen in top block of [[Media:CreateTargetFile.txt | CreateTargetFile.pl]] ====Create Target File: Target is contigs of overlapping exons==== *Wrote perl script to convert biomart output to target file **input: mart_export.txt **output: target_file.txt **General idea is to create intervals or contigs of exons based on overlapping start/end position foreach exon if (start position > current end position) save current start/end position as an interval/contig start new interval/contig with this start/end position elsif (end position > current end position) set this end position as current end position *Tested with one gene and two genes consisting of multiple overlapping exons **Runs with warnings but behaves correctly **Hash for converting Ensembl to HUGO missed a few genes due to multiple Ensembl Gene IDs for same HUGO IDs so had to do them manually ***NEFL ***KCNMB2 ***KLRD1 *[[Media:SortTargetFilesByChr.txt | sortTargetFilesByChr.pl]] **Removes 25bp from each end of target **Switch strand from + to - and - to + **Also runs with warnings that 'given' and 'when' are experimental ====Create Target File v2: Target consitutive exons (no crossing exon boundaries)==== *Changed the algorithm to only select intervals that don't contain boundaries of exons (like constitutive exons but doesn't have to be present in all transcripts) **Ordered all boundaries from least to great regardless if start or stop ***In another array of equal size kept track of whether it was start or stop boundary **Selected intervals that on the left have a start boundary and on the right has a stop boundary with no boundaries in the middle *[[Media:CreateTargetFile_v2.txt | CreateTargetFile_v2.pl]] **input: biomart_export.txt **output: target_file_v2.txt *Tested with one gene and two genes consisting of multiple overlapping exons **Runs with warnings but behaves correctly **Hash for converting Ensembl to HUGO missed a few genes due to multiple Ensembl Gene IDs for same HUGO IDs so had to do them manually ***CCDC144A ***KCNMB2 ***KLRD1 ***NBPF15 ***NEFL *[[Media:SortTargetFilesByChr.txt | sortTargetFilesByChr.pl]] **Removes 25bp from each end of target **Switch strand from + to - and - to + **Also runs with warnings that 'given' and 'when' are experimental ===Run ppDesigner=== *Commands nohup /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/jobFile_chr1.pl > /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/outputFile_chr1.txt & nohup /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/jobFile_chr2.pl > /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/outputFile_chr2.txt & nohup /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/jobFile_chr3.pl > /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/outputFile_chr3.txt & nohup /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/jobFile_chr4.pl > /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/outputFile_chr4.txt & nohup /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/jobFile_chr5.pl > /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/outputFile_chr5.txt & nohup /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/jobFile_chr6.pl > /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/outputFile_chr6.txt & nohup /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/jobFile_chr7.pl > /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/outputFile_chr7.txt & nohup /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/jobFile_chr8.pl > /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/outputFile_chr8.txt & nohup /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/jobFile_chr9.pl > /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/outputFile_chr9.txt & nohup /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/jobFile_chr10.pl > /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/outputFile_chr10.txt & nohup /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/jobFile_chr11.pl > /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/outputFile_chr11.txt & nohup /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/jobFile_chr12.pl > /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/outputFile_chr12.txt & nohup /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/jobFile_chr13.pl > /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/outputFile_chr13.txt & nohup /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/jobFile_chr14.pl > /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/outputFile_chr14.txt & nohup /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/jobFile_chr15.pl > /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/outputFile_chr15.txt & nohup /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/jobFile_chr16.pl > /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/outputFile_chr16.txt & nohup /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/jobFile_chr17.pl > /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/outputFile_chr17.txt & nohup /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/jobFile_chr18.pl > /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/outputFile_chr18.txt & nohup /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/jobFile_chr19.pl > /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/outputFile_chr19.txt & nohup /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/jobFile_chr20.pl > /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/outputFile_chr20.txt & nohup /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/jobFile_chr21.pl > /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/outputFile_chr21.txt & nohup /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/jobFile_chr22.pl > /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/outputFile_chr22.txt & nohup /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/jobFile_chrX.pl > /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/outputFile_chrX.txt & ====Errors that had to be dealt with==== Name "main::exon_info_file" used only once: possible typo at /home/mzcai/scratch/FISSEQ_cDNA_ProbeDesign/opt/ppDesigner/src/ppDesigner.pl line 23. *This is just a warning Very small chromosome detected. FASTA file chr1 sequence most likely not correctly loaded! *Had to change probe_sequences.pl to search for files that ended in ".fa.masked" or wtv the filename suffix of reference sequence file substr outside of string at (eval 12) line 44. Use of uninitialized value $gSeq in length at (eval 12) line 49. Use of uninitialized value $target_sequence in length at (eval 13) line 14. Use of uninitialized value $target_sequence in length at (eval 13) line 20. Illegal division by zero at (eval 13) line 20. *Got this error at end of output for chr17 and chr20 **Turns out the reference genome I was using was hg19 when Biomart export was based on hg38 ***hg38 has longer chr17 and chr20 so the exon locations were out of bounds for hg19 reference **Downloaded hg38 and used that ====Consolidate OutputFiles and Convert to 0gap==== *Concatenate output files into 1 file: outputFile_1gap.txt cat outputFile_chr*.txt > outputFile_1gap.txt *[[Media:ConvertToZeroGapProbe.txt | ConvertToZeroGapProbe.pl]] **5,174 probes designed **443 genes **4,055 exons ===Filter Out Bad Probes=== *Align probes to reference genome and mRNA perl [[Media:Probes2fasta.txt | 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 & *Filter out probes that map to multiple locations in genome or don't map to refMrna perl [[Media:CleanupProbelist.txt | CleanupProbelist.pl]] *3,933 probes **431 genes **3,117 exons *Must still filter out probes that target regions with low coverage based on RNA-seq data **Sequencing data for transcripts from bulk tissue Brodmann Area 8,10,17,21,22,41 /media/LTS_33T/RL_LTS33T/201404_201405_7Samples_BulkNucleiBatch1-20140623_Expt146/STAR/
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)
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