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*[[Hosuk:Lab_Notes|LabNote]] ===Decoding with Agi26k0gap Padlock Probe=== *Image resolution : 20x obj, 4096 x 4086, z step = 1.04um **Since how many features would be detected or how much dense rolonies in this sample, so image in wide field of view **eventually 63x obj. *Data is in '2014-08-13' ====Analysis==== *1. Arrange data *#File name (decided at imaging) : [Sample]_[Resolution]_[Step#]_[Pos#]_z##_ch##.tif *#*ex) PGP1F : PGP1F_Agi26k0gap_Decode_P20_S2_2014-08-09_20x_4k_Step1_Pos2_z01_ch01.tif *#*ex) Tissue section : HBTissue_2014-08-12_RCAagain_20x_4k_Pos1_z00_ch00.tif *#Step# after confocal imaging *#*Step0 : 1st Rolony --> ch00 : Cy3, ch01 : BF *#*Step1~7 : ch00 : 488, ch01 : Cy3, ch02 : Cy5, ch03 : BF *#Maximum Intensity Projection(MIP) at each channel *#*Re-format the Step1~Step7 --> Step1~21 *#*Step1 --> Step1:488, Step2:Cy3, Step3:Cy5 *#*Step2 --> Step4:488, Step5:Cy3, Step6:Cy5 *#*...Step7 --> Step19:488, Step20:Cy3, Step21:Cy5 *#Image aligning *#*Step# in one cycle is an exact position, no shift happened --> Cycle N = Step[3(N-1)+1], Step[3(N-1)+2], Step[3(N-1)+3] *#*Get offset using Bright field images (ch03) of each Cycle : Step1, Step4, Step7, ... Step19 *#**I've done by manually, but eventually need automation *#Shift, Crop of ch00, ch01, ch02 images(Image_Aligning_Crop_v5.m) *#*Name : Crop_[file name] *#*ex)Offset {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Cycle''' | align="center" style="background:#f0f0f0;"|'''X''' | align="center" style="background:#f0f0f0;"|'''Y''' |- | align="center" | 1 || align="center" | 0 || align="center" | 0 |- | align="center" | 2 || align="center" | -13 || align="center" | +8 |- | align="center" | 3 || align="center" | -9 || align="center" | +8 |- | align="center" | 4 || align="center" | -19 || align="center" | +4 |- | align="center" | 5 || align="center" | -16 || align="center" | +4 |- | align="center" | 5 || align="center" | -15 || align="center" | +10 |- | align="center" | 5 || align="center" | -6 || align="center" | -9 |- | align="center" | Average Offset || align="center" | -11.14 || align="center" | +0.71 |- | align="center" | Round || align="center" | -11 || align="center" | +1 |- | align="center" | New Offset || align="center" | || align="center" | |- | align="center" | 1 || align="center" | -11 || align="center" | +1 |- | align="center" | 2 || align="center" | -2 || align="center" | +7 |- | align="center" | 3 || align="center" | +2 || align="center" | +7 |- | align="center" | 4 || align="center" | -8 || align="center" | +3 |- | align="center" | 5 || align="center" | -5 || align="center" | +3 |- | align="center" | 5 || align="center" | -4 || align="center" | -11 |- | align="center" | 5 || align="center" | +5 || align="center" | -10 |- | align="center" | Crop Size || align="center" | 12 || align="center" | 12 |} *2. Run Script for detected barcodes and counts(RolonyDataInfo_v12_7StageDecoding.m, Rolony_Decoding_v18c_7StageDecoding.m) *#Run PISA with every image (Step1~21) *#*PISA parameter *#*PISAParam.log_radius = 2; *#*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; *#*eventually masking each image with MIP image will be used *#Result gemerated : Barcodes, Counts *3. Mapping detected barcode string to gene *#Matt made master key table : barcode and gene name *#Allow 1 bit error (--> but these barcodes of 1 bit error were found in other genes...) *#Map detected barcode to gene name using VLOOKUP(E#, Sheet0Ex!$A$2:$B$24000, 2, 0) ====Result==== *Saved at 2014-08-18 **Total num : 2471 **Num detected only one dye at each digit : 1100 **Num detected 488 and Cy3 at each digit : 775 **Num detected 488 and Cy5 at each digit : 785 **Num detected Cy3 and Cy5 at each digit : 275 **Num detected 488, Cy3 and Cy5 at each digit : 99 *Mapped to gene : 348 *Need to consider features with two or three dyes to be mapped to gene --> need more to polish the data [[File:DecodeResultSummary_2014-08-18.png|700px]]
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