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==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
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