Matt:LabNotes/2016-4-1

From ZhangLabWiki
Jump to navigation Jump to search

Design Padlock Probe Arms for new Probe Set[edit]

  • Design probes for genes that reveal heterogeneity in human brain
  • Genes are specific for certain cell types and subtypes
  • Files: Dropbox\GradZhangLab\CA12k_Apr2016\HumanBrain

Gene Selection[edit]

  • FinalListv1.txt
    • Made from GenesFromBA8Analyses_Suppl_Tables_v2.xlsx
      • Neuronal and glial markers (Table S3)
      • Neuronal subtype-specific genes (Table S6)
      • Allen Human Brain Atlas ISH (Table S12)
      • Blue's requested genes, 12 unique added
      • Interneuron genes, 6 unique added
      • Projection neuron genes, 4 unique added
      • OLFM1 (RNAscope probe data)
    • TMEM90A -> SYNDIG1L (Official HGNC)
  • 370 genes total

Get Transcript Sequences[edit]

  • Biomart browser interface
 Dataset
 Homo sapiens genes (GRCh38.p5)
 Filters
 with HGNC ID(s): Only
 HGNC symbol(s) [e.g. NTN3]: [ID-list specified]
 Status (gene): KNOWN
 Status (transcript): KNOWN
 Attributes
 Ensembl Gene ID
 Ensembl Transcript ID
 Chromosome Name
 Exon Rank in Transcript
 Exon Chr Start (bp)
 Exon Chr End (bp)
 Strand
 Associated Gene Name
  • Remove coordinates on PATCH (fixes and novel), HSCHR (haplotype variants), and chromosomes
    • 370 Unique Ensembl Gene IDs and 370 Unique HGNC Symbols

Create ppDesigner Target Files[edit]

  • Files in genome-miner ~/scratch/CA12kApr2016_ProbeDesign/HumanBrain
  • Use CreateTargetFile_contig.pl to create target file where targets are contigs of exons
    • Script is modified from here
  • Sort target files into each chromosome and remove 25bp from each end of target and switch strand
    • SortTargetFilesByChr.pl

Run ppDesigner[edit]

ppDesignerCommands.sh

  1. !/bin/bash

for indx in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X do /home/mzcai/scratch/CA12kApr2016_ProbeDesign/HumanBrain/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/CA12kApr2016_ProbeDesign/HumanBrain/jobFile_chr$indx.pl > /home/mzcai/scratch/CA12kApr2016_ProbeDesign/HumanBrain/outputFile_chr$indx.txt & wait done

Transform from 1base gap to 0base gap and Filter Softmasked[edit]

  • Add target base to arm with lowest Tm to create zero-gap padlock probe
  • Also filter out any probes targeting soft-masked regions (indicated by lowercase reference sequence)
    • ConvertToZeroGapProbe.pl
 perl /home/mzcai/scratch/CA12kApr2016_ProbeDesign/HumanBrain/ConvertToZeroGapProbe.pl /home/mzcai/scratch/CA12kApr2016_ProbeDesign/HumanBrain/outputFile_chr$indx.txt > /home/mzcai/scratch/CA12kApr2016_ProbeDesign/HumanBrain/outputFile_0gap_chr$indx.txt &
 cat outputFile_0gap_chr*.txt > outputFile_0gap.txt
  • 6,665 probes
  • 4,132 exons
  • 369 genes
    • Only missing TMEM88B, a glial marker gene

Align to Ref mRNA and Genome[edit]

  • Remove probes that did not align to refMrna or had multiple alignments to human_g1k_v37 (hg19)
 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
  • 4,978 probes
  • 3,326 exons
  • 368 genes
    • Missing SHISA8, an Interneuron3 subtype marker gene
  • Output: outputFile_0gap_filtered.txt