Matt:LabNotes/2016-4-18

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Revision as of 02:50, 21 April 2016 by >Mzcai
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Convert Probes to Oligos to Order

  • Add barcodes
  • Files in Dropbox/GradZhangLab/CA12k_Apr2016
  • dcProbe_Sequences.txt
    • Cycle \t Color \t 20nt Sequence
    • 21 (7x3) total
  • 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
  • CA12k_Apr2016_ControlGenes_OutputFile.py
    • Combine human and mouse control genes
      • 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
  • 3 'on' barcodes

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

  • 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")

  • Need to modify outputFile_0gap_FULL_* so that some genes, exons, and probes have multiple barcodes
    • Modify outputFile_0gap_FULL_MouseEmbryo_V6.txt ->outputFile_0gap_FULLmod_MouseEmbryo_V6.txt
      • Acta2: each probe has different barcode
 Acta2	Acta2_0	chr19:34241431-34241432	AATCACTTCAACAGAGGAAGGTCCA	64	TATATTCCCAGGCATGGATGGCATC	64	2.715838	C	TATATTCCCAGGCATGGATGGCATCAATCACTTCAACAGAGGAAGGTCCA	B010303	GTCATATCGGTCACTGTTAATCACTTCAACAGAGGAAGGTCCACTTCAGCTTCCCGATATCCGACGGTAGTGT*TCTTGCGTGCGATACGGAGTAACTGCGACCGTCGGCTTACCATGCGATTAACCGCGACTG*AAATATATTCCCAGGCATGGATGGCATCGATCAGGATACACACTACCC
 Acta2	Acta2_0	chr19:34241664-34241665	GCCTCCAGTTCCTTTCCAAATCAT	64	CAACTGTGAACGTTTTGTGGATCAGC	63	2.761038	C	CAACTGTGAACGTTTTGTGGATCAGCGCCTCCAGTTCCTTTCCAAATCAT	B020023	GTCATATCGGTCACTGTTGCCTCCAGTTCCTTTCCAAATCATCTTCAGCTTCCCGATATCCGACGGTAGTGT*AACGGTATTCGGTCGTCATCTACCATTCGCGTGCGATTCCCATGCGATTAACCGCGACTG*AAACAACTGTGAACGTTTTGTGGATCAGCGATCAGGATACACACTACCC
 Acta2	Acta2_1	chr19:34243507-34243508	CATCATGAAGTGTGATATTGACATCA	59	GCATCCACGAAACCACCTATAACAG	64	2.768782	C	GCATCCACGAAACCACCTATAACAGCATCATGAAGTGTGATATTGACATCA	B030031	GTCATATCGGTCACTGTTCATCATGAAGTGTGATATTGACATCACTTCAGCTTCCCGATATCCGACGGTAGTGT*CTGGTTCGGGCGTACCTAACCAGGGATCGGTCGAGTACGCGAGTGTCGCGCAACTTAGCG*AAGCATCCACGAAACCACCTATAACAGGATCAGGATACACACTACCC
 Acta2	Acta2_2	chr19:34245117-34245118	CTGTGCTATGTAGCTCTGGACTTTG	63	GATTGTCCGTGACATCAAGGAGAAG	64	2.760554	C	GATTGTCCGTGACATCAAGGAGAAGCTGTGCTATGTAGCTCTGGACTTTG	B300032	GTCATATCGGTCACTGTTCTGTGCTATGTAGCTCTGGACTTTGCTTCAGCTTCCCGATATCCGACGGTAGTGT*ACGCTACGGAGTACGCCACTCAGGGATCGGTCGAGTACGCACGTCTGCGTACCGGCTTAG*AAAGATTGTCCGTGACATCAAGGAGAAGGATCAGGATACACACTACCC
 Acta2	Acta2_3	chr19:34246264-34246265	TGTGACTCACAACGTGCCTATCTAT	64	GTATTGTGCTGGACTCTGGAGATGG	63	2.744643	C	GTATTGTGCTGGACTCTGGAGATGGTGTGACTCACAACGTGCCTATCTAT	B210020	GTCATATCGGTCACTGTTTGTGACTCACAACGTGCCTATCTATCTTCAGCTTCCCGATATCCGACGGTAGTGT*CGTATCGGTAGTCGCAACGCTCTTGCGTGCGATACGGAGTTACCATTCGCGTGCGATTCC*AAAGTATTGTGCTGGACTCTGGAGATGGGATCAGGATACACACTACCC
 Acta2	Acta2_4	chr19:34246758-34246759	CCATGTATGTGGCTATTCAGGCTG	64	TATGTTTGAGACCTTCAATGTCCCCG	63	2.740051	C	TATGTTTGAGACCTTCAATGTCCCCGCCATGTATGTGGCTATTCAGGCTG	B110020	GTCATATCGGTCACTGTTCCATGTATGTGGCTATTCAGGCTGCTTCAGCTTCCCGATATCCGACGGTAGTGT*TGTATCGCGCTCGATTGGCATCTTGCGTGCGATACGGAGTTACCATTCGCGTGCGATTCC*AAATATGTTTGAGACCTTCAATGTCCCCGGATCAGGATACACACTACCC
 Acta2	Acta2_5	chr19:34248545-34248546	CCCTGAAGAGCATCCGACACTG	63	CTTTCTATAACGAGCTTCGTGTGGC	64	2.661623	C	CTTTCTATAACGAGCTTCGTGTGGCCCCTGAAGAGCATCCGACACTG	B023100	GTCATATCGGTCACTGTTCCCTGAAGAGCATCCGACACTGCTTCAGCTTCCCGATATCCGACGGTAGTGT*AACGGTATTCGGTCGTCATCGACGAACGGTCGAGATTTACGAATTGTCCGCGCTCTACGA*AAAAAACTTTCTATAACGAGCTTCGTGTGGCGATCAGGATACACACTACCC
 Acta2	Acta2_6	chr19:34251822-34251823	GATAGAACACGGCATCATCACCAAC	64	AGGGATCCTGACGCTGAAGTATCC	64	2.693671	C	AGGGATCCTGACGCTGAAGTATCCGATAGAACACGGCATCATCACCAAC	B110030	GTCATATCGGTCACTGTTGATAGAACACGGCATCATCACCAACCTTCAGCTTCCCGATATCCGACGGTAGTGT*TGTATCGCGCTCGATTGGCATCTTGCGTGCGATACGGAGTCAGGGATCGGTCGAGTACGC*AAAAAGGGATCCTGACGCTGAAGTATCCGATCAGGATACACACTACCC
 Acta2	Acta2_7	chr19:34252601-34252602	GTGTGAAGAGGAAGACAGCACAG	64	CTGAGACGCTGCTCCAGCTAT	64	2.541243	C	CTGAGACGCTGCTCCAGCTATGTGTGAAGAGGAAGACAGCACAG	B210002	GTCATATCGGTCACTGTTGTGTGAAGAGGAAGACAGCACAGCTTCAGCTTCCCGATATCCGACGGTAGTGT*CGTATCGGTAGTCGCAACGCTCTTGCGTGCGATACGGAGTACGTCTGCGTACCGGCTTAG*AAAAAAAAACTGAGACGCTGCTCCAGCTATGATCAGGATACACACTACCC

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


  • 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")