lieberinstitute / dotdotdot Goto Github PK

Hello, Kindly help me with this. I am getting the same error for my ROIs - has three channels in total (DAPI, FITC and Cy3)

Error using save
Variable 'excel_dots_of_ROI' not found.

Error in rnascope_mouse (line 159)
save([filename(1:end-4),'_dots_of_ROI.mat'],'excel_dots_of_ROI')

Dear Dr. Tippani,

Thank you for this fantastic piece of work. You have made my life much more relaxed and will be cited for sure as long as our RNAScope validation works.

With that said, I cannot find where you list what all the .csv column head abbreviations mean! Unless I am mistaken, they appear to be described differently or worded differently across this body of work.

For my example, I have,
|PO_Cy5 | DO_Cy5 | IO_Cy5 | MI_Cy5 |
| PO_Cy3 | DO_Cy3 | IO_Cy3 | MI_Cy3 |
|PO_DAPI | DO_DAPI | IO_DAPI | MI_DAPI

Thank you again.

Sincerely,
Josh

Hi,

I am trying to run the Mouse RNAscope workflow as described in mouse_vignette.md.

I managed to run the entire script and have now the two files: long_data.csv and man.csv but have problems understanding the downstream analyses.

A dataframe from long_data.csv for channel 1 (BdnfEx4) is shown in the guide but I am confused were to find these values in the long_data.csv file.

Is no_of_dots_c1 = D_c1 in long_data.csv, and
avg_dot_size_c1 = V_c1 in long_data.csv, and
avg_dot_Intensity_c1 = MI_D_c1 in long_data.csv?

Best regards,

Sofie

There's an error at the above-mentioned place in rnascope_mouse.m using the default settings. Line 66 is:

[cellbw1,~,~,~] = cellsegm.segmct(ch,(0.038),((4/3)*(pi)*(Z/2*0.4)^3),'prm',prm);

The 2nd and 3rd inputs into cellsegm.segmct are the minimum minN and maximum maxN nucleus thresholds, respectively.

minN is explicitly stated as 0.038.
maxN is calculated according to the equation ((4/3)*(pi)*(Z/2*0.4)^3), which calculates to 0.0335.

Rather said, maxN is less than minN. This leads to exclusion of every nucleus in cellsegm, and creates errors downstream in the pipeline.

For example, I was getting an error at line 162 of rnascope_mouse.m:

Error using save
Variable 'excel_dots_of_ROI' not found.
Error in rnascope_mouse (line 162)
save([filename(1:end-4),'_dots_of_ROI.mat'],'excel_dots_of_ROI')

When I changed the maxN threshold to 100, the full pipeline ran fine without error.

I'm posting it as an issue instead of a pull request because 1) I don't know the appropriate maxN for all cases; each user should set it themselves and 2) so other users will see it.

Hi, I am trying to run the rnascope_human script on a 3 channel z-stack czi file which contains a gene labeled in Opal 690 in Ch1, a gene labeled in Opal 570 in Ch2, and DAPI in Ch3. Every channel contains 37 z stacks. The script can read all 111 frames but gives 2 error messages at the end.

filename='/Users/samirrahman/Documents/MATLAB/dotdotdot-master/images/553_NEUROD1_CRISPRi_scrambled_63x_1.czi';
toolbox='/Users/samirrahman/Documents/MATLAB/dotdotdot-master/toolbox';
DAPI='Ch3';
LIP='';
DROP='';
addpath(genpath(toolbox))
rnascope_human(filename,toolbox,DAPI,LIP,DROP)
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extracted
{'ODye1'}
{'ODye2'}
{'ODye3'}

Elapsed time is 162.937864 seconds.
Undefined function 'std2' for input arguments of type 'double'.

Error in rnascope_human (line 75)
channel = imhmin(eval(channe_i),std2(eval(channe_i)));% suppress background noise in RNA scope channels.

What do these errors mean? Looking forward to your assistance.

Does the input file have to be in .czi?
Will any of the following formats work: .tiff , .jpg , .gci?

Hi,
I am trying to use this code to analyze the .tiff images that I got from mouse. I have 4 channels and an overlay and my 1st channel is the DAPI channel. I entered the following command:
rnascope_human_tif('/Users/youseralrawi/Documents/MATLAB projects/Keyence trial1 imgs/PV_Per1_Per2_CA1/PV_Per1_Per2_CA1__Z001','/Users/youseralrawi/Documents/MATLAB projects/Keyence trial1 imgs/toolbox','CH1','','Overlay')

I got the following error:

_Index exceeds the number of array elements (6).

Error in rnascope_human_tif>@(x)x{7}(1:end-4) (line 8)
channels = cellfun(@(x) x{7}(1:end-4) , cellfun(@(x) strsplit(x,'_'),{myfiles.name}',
'UniformOutput', false), 'UniformOutput', false);

Error in rnascope_human_tif (line 8)
channels = cellfun(@(x) x{7}(1:end-4) , cellfun(@(x) strsplit(x,''),{myfiles.name}',
'UniformOutput', false), 'UniformOutput', false);

Is there anything I can change to fix this issue?
Thanks.

Hi,
I am currently working through the tutorial for implementing Dotdotdot for human RNAscope data. However, after assigning the MATLAB and czi files to variables (as shown in the tutorial), I try to run the rnascope_human function, but it says the function is not recognized.

So instead, I assigned rnascope_human to the pathway leading to the rnascope_human.m file within my toolbox folder, but now when I run rnascope_human(filename, toolbox, DAPI, LIP, DROP), I am getting an error that says "Index in position 1 exceeds array bounds (must not exceed 1)." Because I am relatively new to MATLAB, I am not sure how to resolve this issue. Please let me know next steps for how to fix this issue and continue using Dotdotdot. This is a great software that I think really helps scientists everywhere.

Attached is an image depicting the problems above; please look at the code from the 1/3 from the top of the command window:

Any help is much appreciated! I really want to use this software and learn more about this cutting-edge technology.

Regards,
Balaji

Hi,
I am new to MATLAB and I have been trying to use the rnascope_mouse script. I tried using the raw image that was posted over here "Mouse2.czi" but I was still unable to run the code. What I did was that I downloaded the toolbox and put .czi image and associated .jpg images that I found in over here in the same folder as the toolbox. Then, I opened the rnascope_mouse.m script and changed the filename definition to the path that I was using and I did the same thing for the toolbox. Then I tried running the code but I kept getting errors with the input or with some parts of the script as shown in the screenshot.

I am not sure whether there are some errors in the code or I am not doing things correctly.

Currently, I am using the mouse vignette, and the first problem I am having is the correct thresholding of my DAPI signal. I’ve attached the PNG generated from your program of my image. How can I improve the thresholding of DAPI? It is missing the majority of that signal. I know the 488 signal (second column) is poor, but I don’t necessarily need that channel. So far, I have attempted to modify the filter size for thresholding, thresholding, and minN and maxN, but with no success. I’m guessing there is something that needs to change in the threshold settings to improve the signal-to-noise ratio, but I don’t know how to proceed. Any help would be greatly appreciated, and I am really excited about using your software.

I am getting an error running the rnascope_mouse function at line 56. Another labmate of mine downloaded this 3 weeks ago and is not having this error. I noticed the latest commit was 6 days ago- has something changed with the code or do you have suggestions on why I am receiving this error when running it? The error says the statement is incomplete.

I am new to MatLab and experiencing some issues when trying to run the code. I am trying to run it on the provided data and the following warning shows up:

I also run into the following error:

Is there a way to fix these issues? My apologies if these questions are naive.

Hi,

I have an issue running the rnascope_human_tif function with my tif files. I have 4 channels and an overlay assigned as follows:
DAPI = 'CH1'; %
LIP = 'CH2';
DROP = 'Overlay';
addpath(genpath(toolbox)) %adding toolbox path to current working directory
rnascope_human_tif(filename, toolbox, DAPI, LIP, DROP)

I changed the rnascope_human_tif fn code here from 7 to 4, because 7 did not run at all.
channels = cellfun(@(x) x{4}(1:end-4) , cellfun(@(x) strsplit(x,'_'),{myfiles.name}', 'UniformOutput', false), 'UniformOutput', false);

What I got
extracted
{'DAPI' }
{'Lipofuscin'}
{'CH3' }
{'CH4' }

segmented DAPI: 1216 threshold:0.11176
segmented Lipofuscin: 2532 threshold:0.33922
segmented CH3: 1750 threshold:0.49216
segmented CH4: 1200 threshold:0.56667
Completed Masking DAPI
Completed Masking Lipofuscin
Completed Masking CH3
Completed Masking CH4
Started Lipofuscin
Error using cellfun
Input #2 expected to be a cell array, was double instead.

Error in rnascope_human_tif (line 147)
dots_m=cellfun(@(x) intersect(x,cel{i}),
statsc_m.VoxelIdxList,'UniformOutput',
false);

When running the final mouse table command (screenshot below) - the output files are created but they are blank. Any ideas?

Hi, I have a CZI file with 4 channels (Cy5, Cy3, GFP, DAPI), and each channel has 37 z-stacks. I am trying to read this file with the rnascope_human script in the dotdotdot Toolbox directory. I received an error message about not having enough input arguments:

filename = '/Users/samirrahman/Documents/MATLAB/dotdotdot-master/images/NEUROD1_CUX2_SATB2_63x_200_ms_1.czi';
toolbox = '/Users/samirrahman/Documents/MATLAB/dotdotdot-master/toolbox';
rnascope_human(filename, toolbox)
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Not enough input arguments.

Error in rnascope_human (line 29)
if strcmp(O(i),DAPI)

Do I specify the name of every channel. For example, in my case DAPI is channel 4, the gene CUX2 is in channel 1, NEUROD1 is in channel 2, SATB2 is in channel 3. Should I load maximum projections of every channel or can the script read in 3D?

Hi,

I ran your script using the script on vignette. I have 3 .tif images, 3 channels: DAPI, GFP and TRITC. The files are like this cropped_WT_20X_00100_CH1.tif, cropped_WT_20X_00100_CH2.tif, cropped_WT_20X_00100_CH3.tif containing DAPI, GFP, and TRICT in order.

then I run the following code:

filename` = 'output/cropped_WT_20X_00100'

toolbox = '~/dotdotdot-master/toolbox'

addpath(genpath(toolbox))

DAPI = 'CH1'
LIP = ''
DROP = 'Overlay'
rnascope_human_tif(filename_wt, toolbox, DAPI, LIP, DROP)

sofar, so good. I have the following output files:

• cropped_WT_20X_00100.png
• cropped_WT_20X_00100_dots_of_ROI.mat
• cropped_WT_20X_00100_img.mat
• cropped_WT_20X_00100_segmentation.mat
• cropped_WT_20X_00100_thresholds.mat
• cropped_WT_20X_00100_totaldots.mat

Now, I'd like to create a final table, using the final_table function as described.

How do I proceed?

So far I tried the following:

path1 = 'output'
ext = 'cropped_WT_20X_00100*.tif'
toolbox = '~/dotdotdot-master/toolbox'
channels = {'DAPI', 'CH2', 'CH3'}

final_table(path1,ext,toolbox,channels)

however this fails... In the vignette ext expect a .czi image, but I have .tif, could that be a problem?