Matt:LabNotes/2016-10-14: Difference between revisions

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(Created page with "=New Padlock Probes For RNA Capture with SplintR: CA12k_Oct2016= ==Human Brain== *Same as TB12k_Apr2016_V4 but RevComp of annealing arms ==Mouse ...")
 
>Mzcai
 
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==Human Brain==
==Human Brain==
*Same as [[Matt:LabNotes/2016-4-18|TB12k_Apr2016_V4]] but RevComp of annealing arms
*Same as [[Matt:LabNotes/2016-4-18|TB12k_Apr2016_V4]] but RevComp of annealing arms
  Probe2padlockFISSEQ_Oct2016_HumanBrain_6Cycle_V4.pl > padlockFile_0gap_HumanBrain_V4.txt


==Mouse Embryo==
==Mouse Embryo==
*Same as [[Matt:LabNotes/2016-4-18|TB12k_Apr2016_V6]] but RevComp of annealing arms
*Same as [[Matt:LabNotes/2016-4-18|TB12k_Apr2016_V6]] but RevComp of annealing arms
  perl Probe2padlockFISSEQ_Oct2016_MouseEmbryo_6Cycle_V6.pl > padlockFile_0gap_MouseEmbryo_V6.txt


==Mouse Brain==
==Mouse Brain==
Line 51: Line 55:
ppDesignerCommands.sh
ppDesignerCommands.sh
   #!/bin/bash
   #!/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
   for indx in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X
   do
   do
   /home/mzcai/scratch/CA12kApr2016_ProbeDesign/MouseBrain/opt/ppDesigner/src/ppDesigner.pl /home/mzcai/scratch/CA12kApr2016_ProbeDesign/MouseBrain/jobFile_chr$indx.pl > /home/mzcai/scratch/CA12kApr2016_ProbeDesign/MouseBrain/outputFile_chr$indx.txt &
   ~/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
   wait
   done
   done
Line 61: Line 66:
**ConvertToZeroGapProbe.pl
**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 &
   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
   cat outputFile_0gap_chr*.txt > outputFile_0gap.txt


*2,334 probes
*2,389 probes
*1,425 exons
*1,435 exons
*150 genes
*172 genes
**Only missing Zfpm2
 
*Combine chromosome fasta into single fasta in lexicographical order
**echo "$(ls chr*.fa | sort -V | grep -vP 'chr[^X|Y|\d]'; ls chr*.fa | sort -V | grep -vP 'chr[\d|X|Y]')" | xargs cat > mm10.fa
*Build novoalign index
**/home/kunzhang/softwares/Novocraft/novocraft/novoindex ./novoalign/mm10.ndx ./mm10.fa
**/home/kunzhang/softwares/Novocraft/novocraft/novoindex ./novoalign/mm10_refMrna.ndx ./refMrna.fa


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 &
   /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 &
   /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 &
   /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
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
*1,808 probes
*1,202 exons
*150 genes


*Output: outputFile_0gap_filtered.txt
*Output: outputFile_0gap_filtered.txt
**1,794 probes
**1,250 exons
**171 genes
*Missing Trbc2


[[Matt:LabNotes/2016-4-18|Add Barcodes to make probes]]
===Combine Part 2 (173 New Genes) with Original 150 Genes===
*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===
  perl Probe2padlockFISSEQ_Oct2016_MouseBrain_6Cycle_V7.pl > padlockFile_0gap_MouseBrain_V7.txt


==Lung Cancer Fusion Oncogenes==
==Lung Cancer Fusion Oncogenes==
*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===
*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
*Final output version: outputFile_1XSplintR_Tm_18-28bp_LungCancerFusionGenes.xlsx
===Control and Imbalance Genes===
*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===
*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===
  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==
*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)
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="font-size:11pt"  valign="bottom"
| width="51" height="14" | Probeset
| width="51" | Amplification
| width="51" | # Probes
| width="51" | # Barcodes
| width="51" | # Genes
|- style="font-size:11pt"  valign="bottom"
| height="14" | Human Brain
| V4
| align="right" | 4,978
| align="right" | 391
| align="right" | 368
|- style="font-size:11pt"  valign="bottom"
| height="14" | Mouse Brain
| V7
| align="right" | 3,602
| align="right" | 342
| align="right" | 319
|- style="font-size:11pt"  valign="bottom"
| height="14" | Mouse Embryo
| V6
| align="right" | 2,469
| align="right" | 251
| align="right" | 220
|- style="font-size:11pt"  valign="bottom"
| height="14" | Lung Cancer
| V8
| align="right" | 105
| align="right" | 105
| align="right" | 85
|}

Latest revision as of 18:10, 20 October 2016

New Padlock Probes For RNA Capture with SplintR: CA12k_Oct2016[edit]

Human Brain[edit]

 Probe2padlockFISSEQ_Oct2016_HumanBrain_6Cycle_V4.pl > padlockFile_0gap_HumanBrain_V4.txt

Mouse Embryo[edit]

 perl Probe2padlockFISSEQ_Oct2016_MouseEmbryo_6Cycle_V6.pl > padlockFile_0gap_MouseEmbryo_V6.txt

Mouse Brain[edit]

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
  • 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