Editing
Matt:LabNotes/2014-11-1
(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 (New Genelist)== ===Get Genes=== *Took all HUGO gene names from new genelist: [[media:TopDiffExpGenes_1000hNucDataset_10282014.xlsx | TopDiffExpGenes_1000hNucDataset_10282014.xlsx]] **Removed all genes with names that had: "-", "AC###.#", "AS", "RP", "RNA", "MT", "LINC" ... *[[Media:FISSEQ_cDNA_genelist_newGenes.txt | Result: 409 genes]] ===Build Target File for ppDesigner=== ====Biomart: Get Exon Locations for Genes on Reference Genome==== *Use 409 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_10282014.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 ***ENSG000000228918 matched to LINC01344 and GS1-122H1.2 ====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_newGenes.txt **output: target_file_newGenes.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 ***KCNMB2 ***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=== ppDesignerCommands.sh ====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]] **4,593 probes designed **402 genes **3,599 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]] *3,553 probes **389 genes **2,813 exons ====Filter Probes with Low Coverage based on Brain RNA-seq Data from Rui/Blue==== *Must still filter out probes that target regions with low coverage based on RNA-seq data **Sequencing data for transcripts from tissue Brodmann Area 8,10,17,21,22,41 /media/LTS_33T/RL_LTS33T/201404_201405_7Samples_BulkNucleiBatch1-20140623_Expt146/STAR/ samtools merge ~/scratch/FISSEQ_cDNA_ProbeDesign/BrainBulkTissueRNA_mappedhg19.bam RL-BA10-t-N707-15May14_S7_mapped/RL-BA10-t-N707-15May14_S7_Aligned.sorted.bam RL-BA17-t-N703-15May14_S3_mapped/RL-BA17-t-N703-15May14_S3_Aligned.sorted.bam RL-BA21-t-N705-15May14_S5_mapped/RL-BA21-t-N705-15May14_S5_Aligned.sorted.bam RL-BA22-t-N709-15May14_S2_mapped/RL-BA22-t-N709-15May14_S2_Aligned.sorted.bam RL-BA41-t-N711-15May14_S4_mapped/RL-BA41-t-N711-15May14_S4_Aligned.sorted.bam RL-BA8-sec9-t-N701-15May14_S1_mapped/RL-BA8-sec9-t-N701-15May14_S1_Aligned_chr12_16.sorted.bam samtools sort BrainBulkTissueRNA_mappedhg19.bam BrainBulkTissueRNA_mappedhg19.sorted samtools index BrainBulkTissueRNA_mappedhg19.sorted.bam BrainBulkTissueRNA_mappedhg19.sorted.bam.bai *Convert probe outputFile_0gap_filtered.txt (from CleanupProbelist.pl) to BED file: [[Media:ConvertProbesHg38_2_BedHg19.txt | ConvertProbesHg38_2_BedHg19.pl]] **Also converts hg38 positions to hg19 to match RNA-seq data ***Uses position alignments to human_g1k_v37 to get hg19 coordinates ***Checked new coordinates with UCSC Genome Browser for genes on forward and reverse strands - both were correct coverageBed -abam BrainBulkTissueRNA_mappedhg19.sorted.bam -b outputFile_0gap_hg19.bed > outputFile_BrainBulkTissueRNA.cov *Rank Order plot of Probes and the number of reads that overlap with each probe target region **621 of 3,553 probes had 0 overlap [[File:RankOrderProbesOverlapReads.JPG]] *Filter out probes with <10 reads overlapping target region [[Media:FilterProbelist_LowReadCounts.txt | FilterProbelist_LowReadCounts.pl]] **Result: [[Media:OutputFile_0gap_10reads.txt | outputFile_0gap_10reads.txt]] ===RevComp Probes for potential RNA capture=== *RevComp() each arm and then swap arms (columns 5 and 7) *RevComp() target region (column 11) [[Media:RevComp_Probes.txt | RevComp_Probes.pl]] *Result: [[Media:OutputFile_0gap_10reads_RevComp.txt | outputFile_0gap_10reads_RevComp.txt]] *For each outputFile_0gap_10reads.txt and outputFile_0gap_10reads_RevComp.txt **1,993 probes **1,570 exons **348 genes [[File:20141110_ProbesPerGeneHistogram.JPG | 650px]]
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