Matt:LabNotes/2016-4-8: Difference between revisions

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*For DARTFISH in human and mouse tissues, we are most interested in seeing the heterogeneity and variation in gene expression
*For DARTFISH in human and mouse tissues, we are most interested in seeing the heterogeneity and variation in gene expression
*However, to distinguish biological variation from technical noise it would be nice to have some probes for genes that are uniformly detected in each tissue
*However, to distinguish biological variation from technical noise it would be nice to have some probes for genes that are uniformly detected in each tissue


*Files: Dropbox\GradZhangLab\CA12k_Apr2016\ControlGenes\MouseEmbryo and Dropbox\GradZhangLab\CA12k_Apr2016\ControlGenes\HumanBrain
*Files: Dropbox\GradZhangLab\CA12k_Apr2016\ControlGenes\MouseEmbryo and Dropbox\GradZhangLab\CA12k_Apr2016\ControlGenes\HumanBrain
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*Selected all genes '''not''' mitochondrial (mt-)
*Selected all genes '''not''' mitochondrial (mt-)
**42 genes left: "42genes.txt"
**42 genes left: "42genes.txt"
***Changed Gm5506 -> Eno1b (MGI symbol)


==Get Transcript Sequences==
==Get Transcript Sequences==
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   Associated Gene Name
   Associated Gene Name


*25 Unique Ensembl Gene IDs and Associated Gene Names
*42 Unique Ensembl Gene IDs and Associated Gene Names


==Create ppDesigner Target Files==
==Create ppDesigner Target Files==
Line 50: Line 49:
ppDesignerCommands.sh
ppDesignerCommands.sh
   #!/bin/bash
   #!/bin/bash
   for indx in 1 2 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
   for indx in 1 2 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X
   do
   do
   /home/mzcai/scratch/CA12kApr2016_ProbeDesign/ControlGenes/MouseEmbryo/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/CA12kApr2016_ProbeDesign/ControlGenes/MouseEmbryo/jobFile_chr$indx.pl > /home/mzcai/scratch/CA12kApr2016_ProbeDesign/ControlGenes/MouseEmbryo/outputFile_chr$indx.txt &
   /home/mzcai/scratch/CA12kApr2016_ProbeDesign/ControlGenes/MouseEmbryo/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/CA12kApr2016_ProbeDesign/ControlGenes/MouseEmbryo/jobFile_chr$indx.pl > /home/mzcai/scratch/CA12kApr2016_ProbeDesign/ControlGenes/MouseEmbryo/outputFile_chr$indx.txt &
Line 64: Line 63:
   cat outputFile_0gap_chr*.txt > outputFile_0gap.txt
   cat outputFile_0gap_chr*.txt > outputFile_0gap.txt


*140 probes
*258 probes
*100 exons
*191 exons
*25 genes
*41 genes
**Missing Ubc


perl Probes2fasta.pl < outputFile_0gap.txt > outputFile_0gap.fa
perl Probes2fasta.pl < outputFile_0gap.txt > outputFile_0gap.fa
Line 75: Line 75:
perl CleanupProbelist.pl
perl CleanupProbelist.pl
Remove probes that did not align to refMrna or had multiple alignments to mm10
Remove probes that did not align to refMrna or had multiple alignments to mm10
*47 probes
*85 probes
*42 exons
*76 exons
*18 genes
*33 genes


*Output: outputFile_0gap_filtered.txt
*Output: outputFile_0gap_filtered.txt
Line 92: Line 92:
**I recognize these in my current CA12kNov2014_V4 probe set
**I recognize these in my current CA12kNov2014_V4 probe set
*Take Top 200 genes with lowest variance "200genes.txt"
*Take Top 200 genes with lowest variance "200genes.txt"
**Change LPHN1 -> ADGRL1 (updated HGNC symbol)


==Get Transcript Sequences==
==Get Transcript Sequences==
Line 97: Line 98:


   Dataset
   Dataset
   Mus musculus genes (GRCm38.p4)
   Homo sapiens genes (GRCh38.p5)
   Filters
   Filters
   with MGI ID(s): Only
   with HGNC ID(s): Only
   MGI symbol [e.g. Mir1901]: [ID-list specified]
   HGNC symbol(s) [e.g. NTN3]: [ID-list specified]
   Status (gene): KNOWN
   Status (gene): KNOWN
   Status (transcript): KNOWN
   Status (transcript): KNOWN
Line 112: Line 113:
   Strand
   Strand
   Associated Gene Name
   Associated Gene Name
<!--
 
*221 Unique Ensembl Gene IDs and Associated Gene Names
*Remove coordinates on PATCH (fixes and novel), HSCHR (haplotype variants), and  chromosomes
**198 Unique Ensembl Gene IDs and 198 Unique HGNC Symbols
***Could not find LZTS3 and RP11-217O12.1


==Create ppDesigner Target Files==
==Create ppDesigner Target Files==
*Files in genome-miner:~/scratch/CA12kApr2016_ProbeDesign/MouseEmbryo
*Files in genome-miner:~/scratch/CA12kApr2016_ProbeDesign/HumanBrain


*Use CreateTargetFile_contig.pl to create target file where targets are contigs of exons
*Use CreateTargetFile_contig.pl to create target file where targets are contigs of exons
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   cat outputFile_0gap_chr*.txt > outputFile_0gap.txt
   cat outputFile_0gap_chr*.txt > outputFile_0gap.txt


*3,175 probes
*7,743 probes
*1,839 exons
*4,756 exons
*220 genes
*196 genes
**Only missing Zic5
**Removed MT- gene and missing RN7SL2


perl Probes2fasta.pl < outputFile_0gap.txt > outputFile_0gap.fa
perl Probes2fasta.pl < outputFile_0gap.txt > outputFile_0gap.fa


   /home/kunzhang/softwares/Novocraft/novocraft/novoalign -d /home/mzcai/scratch/Genomes/mm10/novoalign/mm10_refMrna.ndx -f outputFile_0gap.fa -F FA -r ALL > outputFile_0gap_novoalign_mm10refMrna.out &
   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/mzcai/scratch/Genomes/mm10/novoalign/mm10.ndx -f outputFile_0gap.fa -F FA -r ALL > outputFile_0gap_novoalign_mm10.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
perl CleanupProbelist.pl
Remove probes that did not align to refMrna or had multiple alignments to mm10
Remove probes that did not align to refMrna or had multiple alignments to hg38
*2,469 probes
*5,569 probes
*1,564 exons
*3,772 exons
*220 genes
*191 genes
 
**Missing PWAR6, SNORD116-8, SNORD116-15, SNORD116-16, SNORD116-24
*Output: outputFile_0gap_filtered.txt-->
*Output: outputFile_0gap_filtered.txt

Latest revision as of 04:53, 18 April 2016

Design Padlock Probe Arms for Mouse and Human Housekeeping Gene Probe Set[edit]

  • For DARTFISH in human and mouse tissues, we are most interested in seeing the heterogeneity and variation in gene expression
  • However, to distinguish biological variation from technical noise it would be nice to have some probes for genes that are uniformly detected in each tissue
  • Files: Dropbox\GradZhangLab\CA12k_Apr2016\ControlGenes\MouseEmbryo and Dropbox\GradZhangLab\CA12k_Apr2016\ControlGenes\HumanBrain

Mouse Embryo[edit]

Gene Selection[edit]

  • "high_genes_mouse_project.xls" contains 54 high expression genes with lowest variance (variance included)
  • Selected top 25 lowest variance genes "Top25_LowVarGenes.txt"
    • Includes Gapdh and Actg
    • After going through process resulted in <200 probes so expanded genes
  • Selected all genes not mitochondrial (mt-)
    • 42 genes left: "42genes.txt"
      • Changed Gm5506 -> Eno1b (MGI symbol)

Get Transcript Sequences[edit]

  • Biomart browser interface
 Dataset
 Mus musculus genes (GRCm38.p4)
 Filters
 with MGI ID(s): Only
 MGI symbol [e.g. Mir1901]: [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
  • 42 Unique Ensembl Gene IDs and Associated Gene Names

Create ppDesigner Target Files[edit]

  • Files in genome-miner:~/scratch/CA12kApr2016_ProbeDesign/ControlGenes/MouseEmbryo
  • 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

 #!/bin/bash
 for indx in 1 2 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X
 do
 /home/mzcai/scratch/CA12kApr2016_ProbeDesign/ControlGenes/MouseEmbryo/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/CA12kApr2016_ProbeDesign/ControlGenes/MouseEmbryo/jobFile_chr$indx.pl > /home/mzcai/scratch/CA12kApr2016_ProbeDesign/ControlGenes/MouseEmbryo/outputFile_chr$indx.txt &
 wait
 done
  • 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/HumanEmbryo/ConvertToZeroGapProbe.pl /home/mzcai/scratch/CA12kApr2016_ProbeDesign/HumanEmbryo/outputFile_chr$indx.txt > /home/mzcai/scratch/CA12kApr2016_ProbeDesign/HumanEmbryo/outputFile_0gap_chr$indx.txt &
 cat outputFile_0gap_chr*.txt > outputFile_0gap.txt
  • 258 probes
  • 191 exons
  • 41 genes
    • Missing Ubc

perl Probes2fasta.pl < outputFile_0gap.txt > outputFile_0gap.fa

 /home/kunzhang/softwares/Novocraft/novocraft/novoalign -d /home/mzcai/scratch/Genomes/mm10/novoalign/mm10_refMrna.ndx -f outputFile_0gap.fa -F FA -r ALL > outputFile_0gap_novoalign_mm10refMrna.out &
 /home/kunzhang/softwares/Novocraft/novocraft/novoalign -d /home/mzcai/scratch/Genomes/mm10/novoalign/mm10.ndx -f outputFile_0gap.fa -F FA -r ALL > outputFile_0gap_novoalign_mm10.out &

perl CleanupProbelist.pl Remove probes that did not align to refMrna or had multiple alignments to mm10

  • 85 probes
  • 76 exons
  • 33 genes
  • Output: outputFile_0gap_filtered.txt

Human Brain[edit]

Gene Selection[edit]

  • Got TPM table of Blue's single neurons including unclassified ones (4,053 cells)
    • Google Drive: BA8 Analyses/TPM Matrices/"09.anno_exon_log2tpm.dat_nc"
  • Calculated variance and mean for each gene (Dropbox/PythonScripts/CalcTPMmatrix.py) -> "neurons_variance.txt"
  • Filter only genes with mean TPM >= 5
  • Sort by variance low to high
      1. 1 gene is MALAT1
    • Other top genes are KIF5C and SNAP25
    • I recognize these in my current CA12kNov2014_V4 probe set
  • Take Top 200 genes with lowest variance "200genes.txt"
    • Change LPHN1 -> ADGRL1 (updated HGNC symbol)

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
    • 198 Unique Ensembl Gene IDs and 198 Unique HGNC Symbols
      • Could not find LZTS3 and RP11-217O12.1

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

 #!/bin/bash
 for indx in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X
 do
 /home/mzcai/scratch/CA12kApr2016_ProbeDesign/MouseEmbryo/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/CA12kApr2016_ProbeDesign/MouseEmbryo/jobFile_chr$indx.pl > /home/mzcai/scratch/CA12kApr2016_ProbeDesign/MouseEmbryo/outputFile_chr$indx.txt &
 wait
 done
  • 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/HumanEmbryo/ConvertToZeroGapProbe.pl /home/mzcai/scratch/CA12kApr2016_ProbeDesign/HumanEmbryo/outputFile_chr$indx.txt > /home/mzcai/scratch/CA12kApr2016_ProbeDesign/HumanEmbryo/outputFile_0gap_chr$indx.txt &
 cat outputFile_0gap_chr*.txt > outputFile_0gap.txt
  • 7,743 probes
  • 4,756 exons
  • 196 genes
    • Removed MT- gene and missing RN7SL2

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 Remove probes that did not align to refMrna or had multiple alignments to hg38

  • 5,569 probes
  • 3,772 exons
  • 191 genes
    • Missing PWAR6, SNORD116-8, SNORD116-15, SNORD116-16, SNORD116-24
  • Output: outputFile_0gap_filtered.txt