Matt:LabNotes/2016-4-8: Difference between revisions
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>Mzcai m (→Gene Selection) |
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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== | ||
Line 36: | Line 35: | ||
Associated Gene Name | Associated Gene Name | ||
* | *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 & | ||
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cat outputFile_0gap_chr*.txt > outputFile_0gap.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 | 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 | ||
* | *85 probes | ||
* | *76 exons | ||
* | *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 | ||
Homo sapiens genes (GRCh38.p5) | |||
Filters | Filters | ||
with | with HGNC ID(s): Only | ||
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 | ||
* | *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/ | *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 | ||
Line 141: | Line 144: | ||
cat outputFile_0gap_chr*.txt > outputFile_0gap.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 | 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/ | /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 | 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 | *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)
- 42 genes left: "42genes.txt"
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 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
- 198 Unique Ensembl Gene IDs and 198 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
#!/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