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=New Padlock Probes For RNA Capture with SplintR: CA12k_Oct2016= ==Human Brain== *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== *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== *Same as [[Matt:LabNotes/2016-4-18|TB12k_Apr2016_V7]] but RevComp of annealing arms and add 174 new genes ===Gene Selection=== **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=== *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=== *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 [[Matt:LabNotes/2014-11-10 | here]] *Sort target files into each chromosome and remove 25bp from each end of target and switch strand **SortTargetFilesByChr.pl ===Run ppDesigner=== 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=== *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== *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 |}
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