Matt:LabNotes/2016-10-14
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New Padlock Probes For RNA Capture with SplintR: CA12k_Oct2016[edit]
Human Brain[edit]
- Same as TB12k_Apr2016_V4 but RevComp of annealing arms
Probe2padlockFISSEQ_Oct2016_HumanBrain_6Cycle_V4.pl > padlockFile_0gap_HumanBrain_V4.txt
Mouse Embryo[edit]
- Same as TB12k_Apr2016_V6 but RevComp of annealing arms
perl Probe2padlockFISSEQ_Oct2016_MouseEmbryo_6Cycle_V6.pl > padlockFile_0gap_MouseEmbryo_V6.txt
Mouse Brain[edit]
- Same as TB12k_Apr2016_V7 but RevComp of annealing arms and add 174 new genes
Gene Selection[edit]
- 174 new genes provided by Zizhen
- 174 new genes: /GradZhangLab/DARTFISH Collaborations/Mouse Brain/Genelist_174genes_part2.txt
- Gpr133 -> Adgrd1 (MGI Symbol)
- A730090H04 -> Dlx4os (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
- 173 Unique Ensembl Gene IDs and Associated Gene Names
- Missing 5033421B08Rik
Create ppDesigner Target Files[edit]
- Files in genome-miner:~/scratch/CA12kOct2016_ProbeDesign/MouseBrain
- 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 ~/scratch/TB12kOct2016_ProbeDesign/MouseBrain/173NewGenes/opt/ppDesigner/src/ppDesigner.pl ~/scratch/TB12kOct2016_ProbeDesign/MouseBrain/173NewGenes/jobFile_chr$indx.pl > ~/scratch/TB12kOct2016_ProbeDesign/MouseBrain/173NewGenes/outputFile_chr$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 ~/scratch/TB12kOct2016_ProbeDesign/MouseBrain/173NewGenes/ConvertToZeroGapProbe.pl ~/scratch/TB12kOct2016_ProbeDesign/MouseBrain/173NewGenes/outputFile_chr$indx.txt > ~/scratch/TB12kOct2016_ProbeDesign/MouseBrain/173NewGenes/outputFile_0gap_chr$indx.txt &
cat outputFile_0gap_chr*.txt > outputFile_0gap.txt
- 2,389 probes
- 1,435 exons
- 172 genes
perl Probes2fasta.pl < outputFile_0gap.txt > outputFile_0gap.fa
/media/Home_Raid1/kunzhang/softwares/Novocraft/novocraft/novoalign -d ~/scratch/Genomes/mm10/novoalign/mm10_refMrna.ndx -f outputFile_0gap.fa -F FA -r ALL > outputFile_0gap_novoalign_mm10refMrna.out & /media/Home_Raid1/kunzhang/softwares/Novocraft/novocraft/novoalign -d ~/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
- Output: outputFile_0gap_filtered.txt
- 1,794 probes
- 1,250 exons
- 171 genes
- Missing Trbc2
Combine Part 2 (173 New Genes) with Original 150 Genes[edit]
- In GradZhangLab\TB12k_Oct2016\MouseBrain_V7
- Add \173NewGenes_part2\outputFile_0gap_filtered.txt to outputFile_0gap_mod.txt -> outputFile_0gap_combined.txt
- RevComp.pl
Add Barcode[edit]
perl Probe2padlockFISSEQ_Oct2016_MouseBrain_6Cycle_V7.pl > padlockFile_0gap_MouseBrain_V7.txt
Lung Cancer Fusion Oncogenes[edit]
- In GradZhangLab/TB12k_Oct2016/LungCancer_V8
- Reference file: AmpliSeq_lung_fusion_panel_info.pdf
- Sequence file provided by Dr. Zhang: Oncomine_Solid_Tumour_Fusion_REFERENCE_MAN0013544_RevA.FASTA
- Identified junction positions by Blat-ing with UCSC genome browser and manually finding where the transcript aligns to two different genes
- For imbalance genes identified the sequence that contained the exons contained in the name
- Result: LungCancerPanel.xlsx
Fusion Transcripts[edit]
- Files in GradZhangLab\TB12k_Oct2016\LungCancer_V8\Fusion_Transcripts
- FusionCancerGene_ppDesigner.py in Dropbox\PythonScripts
- Take LungCancerPanel_ForFusionCancerGene_ppDesigner.xlsx and run FusionCancerGene_ppDesigner.py
- Uses Bio.SeqUtils MeltingTemp module to calculate Tm of RNA/DNA hybrid sequences
- Tm calculated with salt concentrations of 1X SplintR Ligase Buffer
- Searches for optimal annealing arms that are adjacent to junctions with length 18-28bp and Tm 60-65C
- Optimal arm length = 22bp
- Uses Bio.SeqUtils MeltingTemp module to calculate Tm of RNA/DNA hybrid sequences
- Final output version: outputFile_1XSplintR_Tm_18-28bp_LungCancerFusionGenes.xlsx
Control and Imbalance Genes[edit]
- In genomeMiner ~/scratch/TB12kOct2016_ProbeDesign/LungCancer/
- Sequences: Ctrl_and_Imbalance.fa
- Split into 13 separate FASTA files
- Create jobFile.pl
- Create targetFile.txt
- Target Start and End are 25bp away from the beginning and end of sequences
./opt/ppDesigner/src/ppDesigner.pl ./jobFile.pl > ./outputFile.txt & perl ConvertToZeroGapProbe.pl outputFile.txt > outputFile_0gap.txt perl Probes2fasta.pl < outputFile_0gap.txt > outputFile_0gap.fa
/media/Home_Raid1/kunzhang/softwares/Novocraft/novocraft/novoalign -d /media/12TB_ext/GenomeDB/HsGenome/1KG.ref/NovoAlign/human_g1k_v37 -f outputFile_0gap.fa -F FA -r ALL > outputFile_0gap_novoalign_human_g1k_v37.out & perl CleanupProbelist.pl
- outputFile_0gap_filtered.txt
- All 13 have probes
- 37 probes total
Combine[edit]
- Convert outputFile_1XSplintR_Tm_18-28bp_LungCancerFusionGenes.xlsx to outputFile.txt format by adding column that concatenates the two arms and removing header
- outputFile_1XSplintR_Tm_18-28bp_LungCancerFusionGenes.txt
- Add outputFile_1XSplintR_Tm_18-28bp_LungCancerFusionGenes.txt to outputFile_0gap_filtered.txt -> outputFile_0gap_combined.txt
- For any genes with multiple probes in outputFile_0gap_filtered changed names by adding "_#" so each is uniquely named
- RevComp.pl -> outputFile_0gap_combined_RevComp.txt
Add Barcodes[edit]
perl Probe2padlockFISSEQ_Oct2016_LungCancer_5or6cycle_V8.pl > padlockFile_0gap_LungCancer_V8.txt
- 4 fusion transcripts could not have padlock probes designed because the total length of arms > 51bp needed to fit in 195bp order
- EZR-ROS1.E10R34.COSF1267
- TPM3-ROS1.T7R35.COSF1273
- LRIG3-ROS1.L16R35.COSF1269
- GOPC-ROS1.G8R35.COSF1139
- If we really need these they can be ordered individually from IDT
- Not the same 6 cycle (4 'on' 2 'off') barcodes
- Instead, this barcode can be fully decoded in 5 cycles and the 6th cycle is a redundant 'on' value
- How to make this barcode?
- Take 5 cycle (3 'on' 2'off') barcodes from decoding_linkers_5-cycles.txt
- For each 5 cycle barcode add a '1', '2', or '3' to the end and alternate which one is added
- Find the matching 6 cycle barcode in decoding_linkers_6-cycles_4-on.txt and use it to replace
- New barcode file: decoding_linkers_5or6-cycles.txt
- Each target has unique barcode
Combining into One Order[edit]
- In GradZhangLab\TB12k_Oct2016
perl padlock2oligo_order.pl > CA12k_Oct2016_Oligos2Order.txt
- Total # of probes = 11,154 so copied LungCancer_V8 probes 8X plus a little remainder to bring up to 12k probes -> CA12k_Oct2016_Oligos2Order_FINAL.txt
- Double checked probes for all 4 sets
- Made sure they were 0 gap
- Made sure they were reverse complementary to mRNA
- Made sure barcodes were same as decoding probes (so that rolony would be reverse complementary to decoding probes)
Probeset | Amplification | # Probes | # Barcodes | # Genes |
Human Brain | V4 | 4,978 | 391 | 368 |
Mouse Brain | V7 | 3,602 | 342 | 319 |
Mouse Embryo | V6 | 2,469 | 251 | 220 |
Lung Cancer | V8 | 105 | 105 | 85 |