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=Convert Probes to Oligos to Order= *Probes: [[Matt:LabNotes/2016-4-1|Human Brain]], [[Matt:LabNotes/2016-4-6|Mouse Brain]], [[Matt:LabNotes/2016-4-7|Mouse Embryo]], [[Matt:LabNotes/2016-4-8|Control Genes]] *Add Barcodes and Amplification Primers *Files in Dropbox/GradZhangLab/CA12k_Apr2016 *dcProbe_Sequences.txt **header: "Cycle \t Color \t 20nt Sequence" **21 (7x3) total ==Make Barcode Sequence File== *eg. B01220 ATCGGGAT... *Use MakeBarcodeSequenceFile.py in Dropbox/PythonScripts/ to make 3 'on' barcodes **decoding_linkers_5-cycles.txt **decoding_linkers_6-cycles.txt *Use Make4onBarcodeSequenceFile.py to make 4 'on' barcodes **decoding_linkers_6-cycles_4-on.txt ==Combine Human and Mouse Control/Housekeeping Genes== *CA12k_Apr2016_ControlGenes_OutputFile.py ***First need to make alter gene names so human and mouse are separate '''and''' barcodes are assigned appropriately **Mouse ***Since only 85 probes each probe gets own barcode by adding #1-85 to end of gene name (eg. m-Rpl8-85_0) ***Add "m-" in front of each gene name **Human ***Since 5,569 probes too many for unique barcodes ****Instead, each gene gets 2 barcodes. One barcode is for highest score probe for that gene. Second barcode is for the other probes of that gene. ****191 genes x 2 = 382 barcodes ***Add "h-" in front of each gene name ==Add 3 'on' barcodes to probes== *Mouse Brain has only 2 'off' there 5 cycles is all that is needed to decode *Others have 3 'off' and require 6 cycles of decoding perl Probe2padlockFISSEQ_Apr2016_HumanBrain_V4.pl > outputFile_0gap_FULL_HumanBrain_V4.txt perl Probe2padlockFISSEQ_Apr2016_MouseBrain_V7.pl > outputFile_0gap_FULL_MouseBrain_V7.txt perl Probe2padlockFISSEQ_Apr2016_MouseEmbryo_V6.pl > outputFile_0gap_FULL_MouseEmbryo_V6.txt perl Probe2padlockFISSEQ_Apr2016_ControlGenes_V8.pl > outputFile_0gap_FULL_ControlGenes_V8.txt ==Add 4 'on barcodes to probes== *All probesets are 6 cycle: 4 'on' 2 'off' barcodes perl Probe2padlockFISSEQ_Apr2016_HumanBrain_6cycle_V4.pl > outputFile_0gap_FULL200_HumanBrain_V4.txt perl Probe2padlockFISSEQ_Apr2016_MouseBrain_6cycle_V7.pl > outputFile_0gap_FULL200_MouseBrain_V7.txt perl Probe2padlockFISSEQ_Apr2016_MouseEmbryo_6cycle_V6.pl > outputFile_0gap_FULL200_MouseEmbryo_V6.txt perl Probe2padlockFISSEQ_Apr2016_ControlGenes_6cycle_V8.pl > outputFile_0gap_FULL200_ControlGenes_V8.txt perl padlock2oligo_order.pl ("CA12k_Apr2016_OligosOrder.txt") ==3 'on' 180bp oligos vs 4 'on' 200bp oligos== *Going with 180bp will increase % perfect oligos by 10% **CustomArray Error Rate per Base = 0.5-1.0% **% perfect for 180bp = 0.995^180 = 40.6% **% perfect for 200bp = 0.995^200 = 36.7% *Going with 180bp will decrease risk of too many truncated oligos such that the pool will not amplify *Going with 200bp will leave much more unused barcodes **HumanBrain: 23% to 69% unused **MouseBrain: 38% (5cycles) to 87% unused **MouseEmbryo: 55% to 81% *180bp will improve rolony generation **Possibly more accurate padlock probes ***Base substitution is much less likely than deletion **Possibly more rolonies *200bp will improve decoding accuracy **Less likely for incorrectly decoded rolony to be assigned to used barcode *Conclusions: Go with 200bp ==Modify Certain Genes so Every Probe Unique Barcode== *Need to modify outputFile_0gap_filtered gene names so that some probes have unique barcodes so decoding can be traced back to probe instead of gene **outputFile_0gap_filtered.txt -> outputFile_0gap_mod.txt ***Select certain genes to modify with "-p#" eg."GAD1-p5" **HumanBrain genes modified: SLC17A7, GNG4, PDE9A **MouseBrain genes modified: Gad1, Vip, Pvalb **MouseEmbryo genes modified: Myf5, Col1a1, Foxa1 *6 cycle: 4 'on' 2 'off' barcodes perl Probe2padlockFISSEQ_Apr2016_HumanBrain_6cycle_V4.pl > outputFile_0gap_FULL200_HumanBrain_V4.txt perl Probe2padlockFISSEQ_Apr2016_MouseBrain_6cycle_V7.pl > outputFile_0gap_FULL200_MouseBrain_V7.txt perl Probe2padlockFISSEQ_Apr2016_MouseEmbryo_6cycle_V6.pl > outputFile_0gap_FULL200_MouseEmbryo_V6.txt perl Probe2padlockFISSEQ_Apr2016_ControlGenes_6cycle_V8.pl > outputFile_0gap_FULL200_ControlGenes_V8.txt *Remove 2,909 probes from Human Control Genes to get to 12k oligos perl padlock2oligo_order.pl ("CA12k_Apr2016_OligosOrder.txt") ==Final Summary== {| 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" | 1,808 | align="right" | 173 | align="right" | 150 |- 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" | Control Genes | V8 | 85 mouse, 2660 human | 85 mouse, 245 human | 33 mouse, 171 human |} *Note: Added 1,000 more human control gene probes to make 13,000 probes for Agilent order "Agi13k_Apr2016_DARTFISHOligosOrder.txt"
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