Matt:LabNotes/2016-4-1
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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)
- Made from GenesFromBA8Analyses_Suppl_Tables_v2.xlsx
- 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
- !/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