Hosuk:LabNotes/2014-2-9

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NCIH1975 : Mutation Detection : 2nd Try, Data Analysis

  • Data from 2/3
  • Took pictures at 3 Positions on the sample in MatTek dish

Procedure

    1. Aligned BW images of all steps at each positions, and shift and crop the original images
    2. Maximum projection image with all images of 8 steps
    3. Run PISA to make BW images
    4. Use the BW image as mask image file and do masking an each image (=image file .* mask image)
    5. Run PISA with masked image, and obtain a number of rolonies, centroid coordinate information of each rolony
    6. Decode the rolony to extract decoding information along steps


Major Parameters

Decode Binary table of each target
EGFR_T790M_WT EGFR_T790M_MU EGFR_L858R_WT EGFR_L858R_MU
Step1 (1FAM) 0 1 0 1
Step2 (1Cy3) 1 0 0 0
Step3 (2FAM) 0 0 1 0
Step4 (2Cy3) 0 0 0 1
Step5 (3FAM) 1 0 1 0
Step6 (3Cy3) 0 1 0 0
Step7 (4FAM) 1 0 0 1
Step8 (4Cy3) 0 1 1 0


Decode Binary String of each target
  • EGFR_T790M_WT = '01010010'
  • EGFR_T790M_MU = '10100001'
  • EGFR_L858R_WT = '10010100'
  • EGFR_L858R_MU = '01001001'


Allowing 1 bit Error cases of Decode Binary String
  • EGFR_T790M_WT
    • Normal : '01010010'
    • Error 1 : '01010000'
    • Error 2 : '01000010'
    • Error 3 : '00010010'


  • EGFR_T790M_MU
    • Normal : '10100001'
    • Error 1 : '10100000'
    • Error 2 : '10000001'
    • Error 3 : '00100001'


  • EGFR_L858R_WT
    • Normal : '10010100'
    • Error 1 : '10010000'
    • Error 2 : '10000100'
    • Error 3 : '00010100'


  • EGFR_L858R_MU
    • Normal : '01001001'
    • Error 1 : '01001000'
    • Error 2 : '01000001'
    • Error 3 : '00001001'


Result

Rolony Counts
  • After masking and running PISA, before decoding
  • Counts per each step were too big, I think the counts include many false positive signals.
  • However decoding step might filter out those false positive.

File:RolonyCountTable.png File:RolonyCount Bar.png


Target Counts
Target Pos 1 Pos 2 Pos 3 Avg Std
EGFR_T790M_WT
01010010 4 2 6 4 2
01010000 5 14 16 11.67 5.86
01000010 3 2 1 2 1
00010010 4 12 10 8.67 4.16
EGFR_T790M_MU
10100001 0 0 2 0.67 1.15
10100000 1 1 2 1.33 0.58
10000001 1 1 0 0.67 0.58
00100001 0 0 0 0 0
EGFR_L858R_WT
10010100 3 6 8 5.67 2.52
10010000 5 11 18 11.33 6.51
10000100 4 7 5 5.33 1.53
00010100 2 28 22 17.33 13.61
EGFR_L858R_MU
01001001 0 0 0 0 0
01001000 0 0 1 0.33 0.58
01000001 17 12 6 11.67 5.51
00001001 1 0 1 0.67 0.58


Data Files


  • Position 1 (Red : L858R-MU, Green : L858R-WT, Blue : T790M-MU, Cyan : T790M-WT, Gray : All Targets (Masked MIP images of all Steps))
    • Each target includes its 4 binary variations.

File:Composite Red-L858RMU Green-L858RWT Blue-T790MMU Cyan-T790MWT Gray-MSKD-DCDATA Pos1.png


  • Position 2 (Red : L858R-MU, Green : L858R-WT, Blue : T790M-MU, Cyan : T790M-WT, Gray : All Targets (Masked MIP images of all Steps))
    • Each target includes its 4 binary variations.

File:Composite Red-L858RMU Green-L858RWT Blue-T790MMU Cyan-T790MWT Gray-MSKD-DCDATA Pos2.png


  • Position 3 (Red : L858R-MU, Green : L858R-WT, Blue : T790M-MU, Cyan : T790M-WT, Gray : All Targets (Masked MIP images of all Steps))
    • Each target includes its 4 binary variations.

File:Composite Red-L858RMU Green-L858RWT Blue-T790MMU Cyan-T790MWT Gray-MSKD-DCDATA Pos3.png