No positive phenotypes found

[AbbyKimball]

Hello!

I'm having an issue with the consolidation step. I keep getting the following error: Error in split_phenotypes: No positive phenotypes found. do I need to edit the names of my phenotypes in my merge file?

Thank you!

[AbbyKimball]

Update: I changed all of my phenotypes to have a + (ex: B cells, to B220+), but now I am getting another error message when I try to upload it to the "analyze consolidated data" step:

This analysis requires a data file with "Slide ID" and "Tissue Category" columns.

There is a slide ID column, but it contains the same characters for all events. Also, I did not do tissue segmentation in my experiment as it is all virally infected lung, so I do not have a "Tissue Category" column.

I am thinking a potential solution could be: 1) Modifying the slide ID's to be unique. For example, I have CD4KO lungs and 4 ROIs, so I could label events from that particular ROI to be "CD4KO #1" 2) Add a "tissue category" column and simply write "all" for all of the events?

I can email you my merged file in case you want to look it over (it's too large to upload here) :)

Thanks!

[AB-Kent]

You may not have to change Slide ID. phenoptrReports now can aggregate by Sample Name so if that meets your needs you can use it. Otherwise, yes, modify the Slide IDs to reflect the aggregation you want.

For Tissue Category, yes, the workaround is to add a dummy "Tissue Category" column with a single value. Note that capitalization counts! The column should be "Tissue Category", not "tissue category".

[AbbyKimball]

Thanks for the reply! I did those modifications and I got a consolidated output file, however, I also got this message... I just want to make sure this won't affect downstream analysis...

Writing report for "Merge_cell_seg_data". Quitting from lines 74-76 (Cell_seg_summary_report.Rmd) Unable to write summary report invalid argument type Writing consolidated data. Writing report for consolidated data. Quitting from lines 74-76 (Cell_seg_summary_report.Rmd) Unable to write summary report invalid argument type Done!

I'm guessing the lines it quit were related to the slide ID aggregation?

Thanks!

[AB-Kent]

That line is trying to report the number of fields in each slide. It is expecting a Sample Name or Annotation ID column in the data and will fail if that is missing.

[AbbyKimball]

I was able to get results that look correct despite getting the above message, I assume it means that I satisfied the program with the modifications that I did?

Just a quick question about the analysis: 1) Why does it ask you to define a "phenotype" (see screenshot below)? Is it not using the phenotypes assigned by inForm? Is it using these phenotype definitions/expression to assign new phenotypes? I just want to ensure that the resulting distance calculations are based on the inForm defined phenotypes and not new ones being generated by expression data. 2) I got multiple lines of code saying: Warning: Removed 2 rows containing missing values (position_stack).... I'm assuming that these were removed because they were 0 values? For example, I have slides from CD4KO mice, so there are no CD4 T cells in the slide and thus the "distance" calcs related to CD4 T cells could not be performed?

Please let me know if you'd like to discuss this over email/do a screen share session. I would love to include Phenoptr to a publication before sending it off to review, but I want to make sure my data are accurate :) thanks!

[AB-Kent]

1. This allows you to define multiply-positive phenotypes for analysis, for example CD8+/PDL1+. If you want to use the existing single phenotypes, re-enter them each in their own Phenotype line. In your case, click "Add another phenotype" three times, then fill in the four "Phenotype definition" fields with CD4+, etc.
2. These warnings are from the plotting code and you can safely ignore them.

On Tue, Jun 11, 2019 at 4:00 PM Abby Kimball notifications@github.com wrote:

I was able to get results that look correct despite getting the above message, I assume it means that I satisfied the program with the modifications that I did?

Just a quick question about the analysis:

1. Why does it ask you to define a "phenotype" (see screenshot below)? Is it not using the phenotypes assigned by inForm? Is it using these phenotype definitions/expression to assign new phenotypes? I just want to ensure that the resulting distance calculations are based on the inForm defined phenotypes and not new ones being generated by expression data. [image: Screen Shot 2019-06-11 at 1 52 06 PM] https://user-images.githubusercontent.com/24797260/59302003-2d7dc400-8c50-11e9-8318-89dafc4f6605.png
2. I got multiple lines of code saying: Warning: Removed 2 rows containing missing values (position_stack).... I'm assuming that these were removed because they were 0 values? For example, I have slides from CD4KO mice, so there are no CD4 T cells in the slide and thus the "distance" calcs related to CD4 T cells could not be performed?

Please let me know if you'd like to discuss this over email/do a screen share session. I would love to include Phenoptr to a publication before sending it off to review, but I want to make sure my data are accurate :) thanks!

— You are receiving this because you commented. Reply to this email directly, view it on GitHub https://github.com/akoyabio/phenoptrReports/issues/14?email_source=notifications&email_token=ADQWXDLARHZHTRM44HYOJNDPZ775TA5CNFSM4HVZZUY2YY3PNVWWK3TUL52HS4DFVREXG43VMVBW63LNMVXHJKTDN5WW2ZLOORPWSZGODXOKRRY#issuecomment-501000391, or mute the thread https://github.com/notifications/unsubscribe-auth/ADQWXDJWESWKUDHXGRZAHSLPZ775TANCNFSM4HVZZUYQ .

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Kent Johnson | Principal Software Developer

[AbbyKimball]

1. So then for the expression drop-down menu would I select "none" or "entire CD4 expression" for instance?

Thanks for your help!

[AB-Kent]

The expression dropdown lets you compute mean expression within a phenotype. It is usually used to show expression of a different marker than the one corresponding to the phenotype, for example to show mean PDL1 expression for CD8+ cells. You don't have to use it if you don't want mean expression values in your reports.

On Tue, Jun 11, 2019 at 4:08 PM Abby Kimball notifications@github.com wrote:

1. So then for the expression drop-down menu would I select "none" or "entire CD4 expression" for instance?

Thanks for your help!

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Kent Johnson | Principal Software Developer

[AbbyKimball]

Ah, I see. So regardless of what expression dropdown I choose, or phenotypes I add, the resulting distance calculations are done using inForm defined phenotypes. Thank you for your quick responses!

[AB-Kent]

Distance calculations are done between all pairs of phenotypes defined in the "Phenotype definition" fields.

On Tue, Jun 11, 2019 at 4:19 PM Abby Kimball notifications@github.com wrote:

Ah, I see. So regardless of what expression dropdown I choose, or phenotypes I add, the resulting distance calculations are done using inForm defined phenotypes. Thank you for your quick responses!

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Kent Johnson | Principal Software Developer