Hosuk:LabNotes/2014-8-29

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Decoding PGP1F with Agi26k0gap Padlock Probe – 2nd full decoding[edit]

Sample[edit]

  • This PGP1F MatTek dish was
    • S2, 1st rolonies made at 2014-04-10
    • 2nd rolonies finished at 2014-04-14
    • 2 color dye + 5 cycle decoding done at 2014-04-14
  • Matt run 7 cycle decoding - 2014-08-28
  • Data stored 2014-08-28 folder


Procedure[edit]

    1. Align all images based on BF
    2. Shift and crop images of every steps, make MIP image with all steps of aligned and cropped images
    3. Count 1st Rolony by PISA
    4. Count 1st Rolony and make BW image from MIP of all step images by PISA
      • PISA param test and apply
      • PISA parameter
      • PISAParam.log_radius = 3;
      • PISAParam.log_upper = -2e-4;
      • PISAParam.area_upper = 100;
      • PISAParam.area_lower = 10;
      • PISAParam.axratio_lower = 0.5;
      • PISAParam.circ_upper = 1.6;
      • PISAParam.circ_lower = 0.8;
      • PISAParam.pconn = 8;
      • PISAParam.bkgmult_lower = 3;
      • PISAParam.FilterOption = 1;
      • PISAParam.Deconvoluted = 0;
      • PISAParam.DoMedflt = 1;
      • PISAParam.nofindbkg = 0;
    5. Run Script (will be specified later...)



Result[edit]

  • Result
  • Number of cells : Pos1 ~ 120, Pos2 : 100
  • 1st Rolony :
    • Pos1 : 31371, 261.43/cell
    • Pos2 : 27354, 273.54/cell
  • MIP of all steps :
  • Pos1 : 6621, 55.18/cell
  • Pos2 : 4739, 47.39/cell


  • Even if 2nd full decoding sample has more 1st rolonies, it has less number of decoded features…
  • To DO : Decode this with 63x!!!, and compare RNA expression data from Jay’s or Zhang Lab’s


Comparison of 1st rolonies between 1st decoding and 2nd decoding[edit]


Decoded counts and gene mapping table : position 1[edit]

File:DecodeResult MarkBarcode Pos1 2014-08-29.png


Decoded counts and gene mapping table : position 2[edit]

File:DecodeResult MarkBarcode Pos2 2014-08-29.png


Comparison of color distribution between 1st decoding and 2nd decoding(position 1)[edit]