Matt:LabNotes/2016-4-18

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Convert Probes to Oligos to Order[edit]

  • 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[edit]

  • 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[edit]

  • 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[edit]

  • 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[edit]

  • 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[edit]

  • 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[edit]

  • 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[edit]

Probeset Amplification # Probes # Barcodes # Genes
Human Brain V4 4,978 391 368
Mouse Brain V7 1,808 173 150
Mouse Embryo V6 2,469 251 220
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"