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=Probe Mining-Zhang= [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] I'm still trying to design some new FISH probes using different spacing than the original set. However, the [[Daniel:Experiments-HRCP|OligoArray probe mining]] is really tedious and slow. So I'm going to pursue alternate methods. First, I'll try the method Dr. Zhang used to design the original set. ==High Density Probes== *Dr. Zhang's [[Kun:LabNotes/HiResChrPaint|original protocol and scripts]] Beliveau's publication lists several probe sets, but they commonly fall around 10 probes/kb. Similarly, the RNAseq method published by Long Cai uses 20-48 probes per transcript, with average mRNA transcripts being ~10kb (2-5 probes/kb). Therefore, I'll design the new probe set to include higher density. I'll aim for 10 probes/kb, which should be sufficient. I'll have to be careful, however, as this density means 600/1000 bases (60%) will be probe hybridized using the new scheme. I don't think this should be a big problem, but its worth noting. ===5kb region, 10kb spacing=== #Run probes2digits, which groups probe sequences into digits of specified size, spacing, and probe number ##./DJprobes2digits.pl 5000 10000 50 < /home/kunzhang/chromosomePainting/chrX_probe_seq.txt > chrX_probes2digits_5k_10k_50.txt & #Group digits into 7 digit barcodes ##./DJdigits2barcodes.pl 7 50000 < chrX_probes2digits_5k_10k_50.txt > chrX_7digitbarcodes_sp50k.txt #Allocate barcodes to genome ##./DJallocatebarcodespace.pl 7 5000 10000 > HsGenomeBarcodeAllocation.txt #Assign allocated barcodes to digits and probes ##./DJassignBarcodeColor.pl HsGenomeBarcodeAllocation.txt chrX_7digitbarcodes_sp50k.txt chrX_7digitbarcodes_sp50k_final #Add adaptors and group based on fluorophores ##./DJprobes2oligopools.pl < chrX_7digitbarcodes_sp50k_final_probe_seq_color.txt > chrX_oligoPools.txt The output of this was 0 bytes (whoops). Time for some debugging. I'm guessing there is a file called in one of the scripts that is not in the directory I'm working with (to counteract permissions, I'm working in my own directory). ===10kb region, 20kb spacing=== #Run probes2digits, which groups probe sequences into digits of specified size, spacing, and probe number ##./DJprobes2digits.pl 10000 20000 50 < /home/kunzhang/chromosomePainting/chrX_probe_seq.txt > chrX_probes2digits_10k_20k_50.txt & #Group digits into 7 digit barcodes ##./DJdigits2barcodes.pl 7 50000 < chrX_probes2digits_10k_20k_50.txt > chrX_7digitbarcodes_10kdigit_sp50k.txt #Allocate barcodes to genome ##./DJallocatebarcodespace.pl 7 10000 20000 > HsGenomeBarcodeAllocation.txt #Assign allocated barcodes to digits and probes ##./DJassignBarcodeColor.pl HsGenomeBarcodeAllocation.txt chrX_7digitbarcodes_10kdigit_sp50k.txt chrX_7digitbarcodes_10kdigit_sp50k_final Also didn't work. I cut it short. I'll have to rework things a little to get the probes out of this. ===50bp Oligos=== Going to run the first script to make 50bp oligos. #First step, download blat. ##cd /home/kunzhang/softwares/blat_34 ##./gfServer start localhost 17778 /home/kunzhang/HsGenome/hg19.masked/HsGenome19.2bit & [[Category:HRCP]] [[Category:Probe Mining]] [[Category:ZhangProbeDesign]]
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