Matt:LabNotes/2014-11-1: Difference between revisions

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>Mzcai
(Created page with "==Design FISSEQ Padlock Probeset for in situ cDNA Capture (New Genelist)== ===Get Genes=== *Took all HUGO gene names from new genelist: [[media:TopDiffExpGenes_1000hNucDatase...")
 
>Mzcai
mNo edit summary
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===Run ppDesigner===
===Run ppDesigner===
   ppDesignerCommands.sh
   ppDesignerCommands.sh
<!--
 
====Consolidate OutputFiles and Convert to 0gap====
====Consolidate OutputFiles and Convert to 0gap====
*Concatenate output files into 1 file: outputFile_1gap.txt
*Concatenate output files into 1 file: outputFile_1gap.txt
   cat outputFile_chr*.txt > outputFile_1gap.txt
   cat outputFile_chr*.txt > outputFile_1gap.txt
*[[Media:ConvertToZeroGapProbe.txt | ConvertToZeroGapProbe.pl]]
*[[Media:ConvertToZeroGapProbe.txt | ConvertToZeroGapProbe.pl]]
**probes designed
**4,593 probes designed
**genes
**402 genes
**exons
**3,599 exons


===Filter Out Bad Probes===
===Filter Out Bad Probes===
Line 67: Line 67:
*Filter out probes that map to multiple locations in genome or don't map to refMrna
*Filter out probes that map to multiple locations in genome or don't map to refMrna
   perl [[Media:CleanupProbelist.txt | CleanupProbelist.pl]]
   perl [[Media:CleanupProbelist.txt | CleanupProbelist.pl]]
*3,933 probes
*3,553 probes
**431 genes
**389 genes
**3,117 exons
**2,813 exons


<!--
*Must still filter out probes that target regions with low coverage based on RNA-seq data
*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
**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/-->
   /media/LTS_33T/RL_LTS33T/201404_201405_7Samples_BulkNucleiBatch1-20140623_Expt146/STAR/-->

Revision as of 11:17, 1 November 2014

Design FISSEQ Padlock Probeset for in situ cDNA Capture (New Genelist)

Get 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
  • 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
  • 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

Filter Out Bad Probes

  • Align probes to reference genome and mRNA
 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 &
 
  • Filter out probes that map to multiple locations in genome or don't map to refMrna
 perl  CleanupProbelist.pl
  • 3,553 probes
    • 389 genes
    • 2,813 exons