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*[[Hosuk:Lab_Notes|LabNote]] ===NCIH1975 : Mutation Detection : 2nd Try, Data Analysis=== *Data from [[Hosuk:LabNotes/2014-2-3|2/3]] *Took pictures at 3 Positions on the sample in MatTek dish ====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 *#Run PISA to make BW images *#Use the BW image as mask image file and do masking an each image (=image file .* mask image) *#Run PISA with masked image, and obtain a number of rolonies, centroid coordinate information of each rolony *#Decode the rolony to extract decoding information along steps ====Major Parameters==== =====Decode Binary table of each target===== {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|''' ''' | align="center" style="background:#f0f0f0;"|'''EGFR_T790M_WT''' | align="center" style="background:#f0f0f0;"|'''EGFR_T790M_MU''' | align="center" style="background:#f0f0f0;"|'''EGFR_L858R_WT''' | align="center" style="background:#f0f0f0;"|'''EGFR_L858R_MU''' |- | align="center" | Step1 (1FAM)|| align="center" | '''0'''|| align="center" | '''1'''|| align="center" | '''0'''|| align="center" | '''1''' |- | align="center" | Step2 (1Cy3)||align="center" | '''1'''|| align="center" | '''0'''|| align="center" | '''0'''|| align="center" | '''0''' |- | align="center" | Step3 (2FAM)||align="center" | '''0'''|| align="center" | '''0'''|| align="center" | '''1'''|| align="center" | '''0''' |- | align="center" | Step4 (2Cy3)||align="center" | '''0'''|| align="center" | '''0'''|| align="center" | '''0'''|| align="center" | '''1''' |- | align="center" | Step5 (3FAM)||align="center" | '''1'''|| align="center" | '''0'''|| align="center" | '''1'''|| align="center" | '''0''' |- | align="center" | Step6 (3Cy3)||align="center" | '''0'''|| align="center" | '''1'''|| align="center" | '''0'''|| align="center" | '''0''' |- | align="center" | Step7 (4FAM)||align="center" | '''1'''|| align="center" | '''0'''|| align="center" | '''0'''|| align="center" | '''1''' |- | align="center" | Step8 (4Cy3)||align="center" | '''0'''|| align="center" | '''1'''|| align="center" | '''1'''|| align="center" | '''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|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]]
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