Hosuk:LabNotes/2014-2-9: Difference between revisions

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====Procedure====
====Procedure====
*#Aligned BW images of all steps at each positions, and shift and crop the original images
*#Maximum projection image with all images of 8 steps
*#Maximum projection image with all images of 8 steps
*#Run PISA to make BW images
*#Run PISA to make BW images
Line 49: Line 50:




=====1 bit Error cases of Decode Binary String=====
=====Allowing 1 bit Error cases of Decode Binary String=====
*EGFR_T790M_WT
*EGFR_T790M_WT
**Normal : '01010010'
**Normal : '01010010'
Line 80: Line 81:


====Result====
====Result====
[[File:EGFR_DecodingInfo_ResultTable.png|450px]]
=====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|250px]]
[[File:RolonyCount_Bar.png|600px]]
 
 
=====Target Counts=====
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Target'''
| align="center" style="background:#f0f0f0;"|'''Pos 1'''
| align="center" style="background:#f0f0f0;"|'''Pos 2'''
| align="center" style="background:#f0f0f0;"|'''Pos 3'''
| align="center" style="background:#f0f0f0;"|'''Avg'''
| align="center" style="background:#f0f0f0;"|'''Std'''
|-
| align="center" | EGFR_T790M_WT||align="center" |  || align="center" | || align="center" |  || align="center" |  || align="center" | 
|-
| align="center" | 01010010||align="center" | 4 || align="center" | 2 || align="center" |  6 || align="center" |  4 || align="center" |  2
|-
| align="center" | 01010000||align="center" | 5 || align="center" |  14 || align="center" |  16 || align="center" |  11.67 || align="center" | 5.86
|-
| align="center" | 01000010||align="center" |  3 || align="center" |  2 || align="center" |  1 || align="center" |  2 || align="center" |  1
|-
| align="center" | 00010010||align="center" |  4 || align="center" |  12 || align="center" |  10 || align="center" |  8.67 || align="center" | 4.16
|-
| align="center" | EGFR_T790M_MU||align="center" |  || align="center" | || align="center" |  || align="center" |  || align="center" | 
|-
| align="center" | 10100001||align="center" |  0 || align="center" | 0 || align="center" |  2 || align="center" |  0.67 || align="center" | 1.15
|-
| align="center" | 10100000||align="center" |  1 || align="center" | 1 || align="center" |  2 || align="center" |  1.33 || align="center" | 0.58
|-
| align="center" | 10000001||align="center" |  1 || align="center" | 1 || align="center" |  0 || align="center" |  0.67 || align="center" | 0.58
|-
| align="center" | 00100001||align="center" |  0 || align="center" | 0 || align="center" |  0 || align="center" |  0 || align="center" | 0
|-
| align="center" | EGFR_L858R_WT||align="center" |  || align="center" | || align="center" |  || align="center" |  || align="center" | 
|-
| align="center" | 10010100||align="center" | 3 || align="center" | 6 || align="center" |  8 || align="center" |  5.67 || align="center" |  2.52
|-
| align="center" | 10010000||align="center" | 5 || align="center" |  11 || align="center" |  18 || align="center" |  11.33 || align="center" | 6.51
|-
| align="center" | 10000100||align="center" |  4 || align="center" |  7 || align="center" |  5 || align="center" | 5.33 || align="center" |  1.53
|-
| align="center" | 00010100||align="center" |  2 || align="center" |  28 || align="center" |  22 || align="center" |  17.33 || align="center" | 13.61
|-
| align="center" | EGFR_L858R_MU||align="center" |  || align="center" | || align="center" |  || align="center" |  || align="center" | 
|-
| align="center" | 01001001||align="center" |  0 || align="center" | 0 || align="center" |  0 || align="center" |  0 || align="center" | 0
|-
| align="center" | 01001000||align="center" |  0 || align="center" | 0 || align="center" |  1 || align="center" |  0.33 || align="center" | 0.58
|-
| align="center" | 01000001||align="center" |  17 || align="center" | 12 || align="center" |  6 || align="center" |  11.67 || align="center" | 5.51
|-
| align="center" | 00001001||align="center" |  1 || align="center" | 0 || align="center" | 1 || align="center" |  0.67 || align="center" | 0.58
|}
 
 
=====Data Files=====
*[[Media:Datafile_AfterMasking_2014-02-09.zip|'''Data Files after Masking''']]
*[[Media:DecodedEachTargetEachBinaryVariationEachPos_2014-02-09.zip|'''Dilated Images of each target, each binary variation''' ]]
*[[Media:DecodingInfo_4.xlsx|'''Analysis Excel file''']]
*[[Media:Composite_Red-L858RMU_Green-L858RWT_Blue-T790MMU_Cyan-T790MWT_Gray-MaskedMIPofSteps.zip|'''Overlay images''']] : (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.
 
 
 
*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|700px]]
 
 
*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|700px]]
 
 
*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|700px]]
 
 
*Matlab code : [[Media:Rolony_Decoding_v8.m|Rolony_Decoding_v8.m]]

Latest revision as of 19:21, 14 February 2014

NCIH1975 : Mutation Detection : 2nd Try, Data Analysis[edit]

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

Procedure[edit]

    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[edit]

Decode Binary table of each target[edit]
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[edit]
  • 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[edit]
  • 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[edit]

Rolony Counts[edit]
  • 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[edit]
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[edit]


  • 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