confused about results

[nick-webster]

Hi, I have a question about interpreting individual AS results.

For SE it seems straight forward. The SE exon is colored orange in the PSI plot and in the transcript plot, so no problem. The A3SS plot has the orange exon downstream in both PSI and transcript plots, so again no problem.

But with AFE, the schematic of the AFE event shows the downstream first exon in orange so I interpret the PSI plot to indicate that the icon with the orange exon included indicates use of the downstream first exon, i.e. downstream exon is the alternative. But if I look at the transcript information, the upstream first exon is shaded orange and the downstream one blue, i.e. the upstream exon is the alternative exon. Which is correct? I think this also causes problems with the A5SS interpretation. The upstream SS is labeled orange in the schematic, but it is the downstream SS labeled orange in the transcript plot. This is confusing as it completely reverses the conclusions.

Thanks, Nick

[nuno-agostinho]

Hi Nick! I am very sorry for this inconsistency and thank you for pointing it out.

The event ID in the top right (e.g. A5SS_1_-_1274742_1274667_1274033_DVL1) has the event coordinates: one for each exon needed to calculate PSI. The coordinate of the exon used as reference for PSI quantification is always in the middle (in the given example, the exon that ends in position 1274667). That should always be the orange exon in both plots:

After some quick testing, the transcript plot colours in orange the right exon, whereas the schematic is colouring the wrong exon in AFE and A5SS events.

I will update psichomics with this corrected after doing some more tests. In the meantime, please disregard the schematic for these event types.

If you have any further questions, please ask me away.

Once again, I am sorry for the inconvenience. Thank you!

[nick-webster]

Hi Nuno, Thanks for the clarification. On another topic, did you ever develop a splicing annotation for mouse? Thanks, Nick Nick Webster Sent from my iPhone. On Aug 23, 2021, at 2:25 AM, Nuno Agostinho ***@***.***> wrote:  Hi Nick! I am very sorry for this inconsistency and thank you for pointing it out. The event ID in the top right (e.g. A5SS_1_-_1274742_1274667_1274033_DVL1) has the event coordinates: one for each exon needed to calculate PSI. The coordinate of the exon used as reference for PSI quantification is always in the middle (in the given example, the exon that ends in position 1274667). That should always be the orange exon in both plots: [image]<https://urldefense.com/v3/__https://user-images.githubusercontent.com/3199157/130422272-1360d816-c9a9-47b3-917a-f95bd51f852e.png__;!!LLK065n_VXAQ!1GmWfUU-VKuU62seG0miiziRSpdMmgEL7lVvERxGBYtgifitPPKIcm55LVhUg86RjA$> [image]<https://urldefense.com/v3/__https://user-images.githubusercontent.com/3199157/130422288-58cca91c-72ef-403f-bd84-b086e99057e4.png__;!!LLK065n_VXAQ!1GmWfUU-VKuU62seG0miiziRSpdMmgEL7lVvERxGBYtgifitPPKIcm55LViWZG2iNA$> After some quick testing, the transcript plot colours in orange the right exon, whereas the schematic is colouring the wrong exon in AFE and A5SS events. I will update psichomics with this corrected after doing some more tests. In the meantime, please disregard the schematic for these event types. If you have any further questions, please ask me away. Once again, I am sorry for the inconvenience. Thank you! — You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub<https://urldefense.com/v3/__https://github.com/nuno-agostinho/psichomics/issues/429*issuecomment-903596345__;Iw!!LLK065n_VXAQ!1GmWfUU-VKuU62seG0miiziRSpdMmgEL7lVvERxGBYtgifitPPKIcm55LVglePT3rA$>, or unsubscribe<https://urldefense.com/v3/__https://github.com/notifications/unsubscribe-auth/AD3AGXCKOJMMWMLCD56273LT6IHX5ANCNFSM5CRGJSUQ__;!!LLK065n_VXAQ!1GmWfUU-VKuU62seG0miiziRSpdMmgEL7lVvERxGBYtgifitPPKIcm55LVipFBPZ-A$>. Triage notifications on the go with GitHub Mobile for iOS<https://urldefense.com/v3/__https://apps.apple.com/app/apple-store/id1477376905?ct=notification-email&mt=8&pt=524675__;!!LLK065n_VXAQ!1GmWfUU-VKuU62seG0miiziRSpdMmgEL7lVvERxGBYtgifitPPKIcm55LVhEx8sWLA$> or Android<https://urldefense.com/v3/__https://play.google.com/store/apps/details?id=com.github.android&utm_campaign=notification-email__;!!LLK065n_VXAQ!1GmWfUU-VKuU62seG0miiziRSpdMmgEL7lVvERxGBYtgifitPPKIcm55LVgL6hsXsw$>.

[nuno-agostinho]

Hey Nick,

On another topic, did you ever develop a splicing annotation for mouse?

Yes, there is already a mouse AS annotation for mm9 and mm10 based on VAST-TOOLS annotation. This will be available in the next release of Bioconductor (probably released in mid-October).

In the meanwhile, feel free to download the mm9 and mm10 annotation from here: https://we.tl/t-3mHlwEOzNJ

Best,
Nuno

[nick-webster]

Hi Nuno, I tried your mouse annotations using the browser interface but could not load the annotations. I get a message “Maximum upload size exceeded”. I haven’t tried on the CLI yet. Is there a upload limit that can be changed? Thanks, Nick **************************************************************** Nicholas Webster, Ph.D., M.A. Professor of Medicine Chief, Division of Endocrinology and Metabolism Associate Director for Shared Resources, Moores Cancer Center University of California, San Diego 9500 Gilman Drive La Jolla CA 92093 (858) 534-6275 On Aug 23, 2021, at 11:24 AM, Nuno Agostinho ***@***.******@***.***>> wrote: Hey Nick, On another topic, did you ever develop a splicing annotation for mouse? Yes, there is already a mouse AS annotation for mm9 and mm10 based on VAST-TOOLS annotation. This will be available in the next release of Bioconductor (probably released in mid-October). In the meanwhile, feel free to download the mm9 and mm10 annotation from here: https://we.tl/t-3mHlwEOzNJ<https://urldefense.com/v3/__https://we.tl/t-3mHlwEOzNJ__;!!LLK065n_VXAQ!xbW8hXk3wTS0b0yygJ4c0iqZd3kC3TvAc4hd227sFsZQr5hk2w4XmOSDu2GwhWKsIw$> Best, Nuno — You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub<https://urldefense.com/v3/__https://github.com/nuno-agostinho/psichomics/issues/429*issuecomment-904007705__;Iw!!LLK065n_VXAQ!xbW8hXk3wTS0b0yygJ4c0iqZd3kC3TvAc4hd227sFsZQr5hk2w4XmOSDu2HkxwoDkQ$>, or unsubscribe<https://urldefense.com/v3/__https://github.com/notifications/unsubscribe-auth/AD3AGXEFQG2YS744YBUXV6DT6KG7JANCNFSM5CRGJSUQ__;!!LLK065n_VXAQ!xbW8hXk3wTS0b0yygJ4c0iqZd3kC3TvAc4hd227sFsZQr5hk2w4XmOSDu2FNMFPDeg$>. Triage notifications on the go with GitHub Mobile for iOS<https://urldefense.com/v3/__https://apps.apple.com/app/apple-store/id1477376905?ct=notification-email&mt=8&pt=524675__;!!LLK065n_VXAQ!xbW8hXk3wTS0b0yygJ4c0iqZd3kC3TvAc4hd227sFsZQr5hk2w4XmOSDu2GOnAJdcA$> or Android<https://urldefense.com/v3/__https://play.google.com/store/apps/details?id=com.github.android&utm_campaign=notification-email__;!!LLK065n_VXAQ!xbW8hXk3wTS0b0yygJ4c0iqZd3kC3TvAc4hd227sFsZQr5hk2w4XmOSDu2Gl3AYXtg$>.

[nuno-agostinho]

Hey @nick-webster,

That is a limit of Shiny (that I changed, but it seems it isn't working in your case). You can change the limit by running the following command yourself before opening psichomics:

options(shiny.maxRequestSize = 50 * 1024^2) 

This allows to upload a file with up to 50 MB, if you need more just increase the number.

Cheers,
Nuno

[nick-webster]

That fixed it. Thanks, Nick **************************************************************** Nicholas Webster, Ph.D., M.A. Professor of Medicine Chief, Division of Endocrinology and Metabolism Associate Director for Shared Resources, Moores Cancer Center University of California, San Diego 9500 Gilman Drive La Jolla CA 92093 (858) 534-6275 On Sep 2, 2021, at 9:51 AM, Nuno Agostinho ***@***.******@***.***>> wrote: Hey @nick-webster<https://urldefense.com/v3/__https://github.com/nick-webster__;!!LLK065n_VXAQ!wFkC2_1pQx97vTAPM3TglmqIarmBOmAKYMguync2qgOV_CtVypQpDzhGl_Mmh_mxIg$>, That is a limit of Shiny (that I changed, but it seems it isn't working in your case). You can change the limit by running the following command yourself before opening psichomics: options(shiny.maxRequestSize = 50 * 1024^2) This allows to upload a file with up to 50 MB, if you need more just increase the number. Cheers, Nuno — You are receiving this because you were mentioned. Reply to this email directly, view it on GitHub<https://urldefense.com/v3/__https://github.com/nuno-agostinho/psichomics/issues/429*issuecomment-911876159__;Iw!!LLK065n_VXAQ!wFkC2_1pQx97vTAPM3TglmqIarmBOmAKYMguync2qgOV_CtVypQpDzhGl_N5_0Cdbw$>, or unsubscribe<https://urldefense.com/v3/__https://github.com/notifications/unsubscribe-auth/AD3AGXGFC3AY62CUFLYO4RDT76TQLANCNFSM5CRGJSUQ__;!!LLK065n_VXAQ!wFkC2_1pQx97vTAPM3TglmqIarmBOmAKYMguync2qgOV_CtVypQpDzhGl_PgeZYJcw$>. Triage notifications on the go with GitHub Mobile for iOS<https://urldefense.com/v3/__https://apps.apple.com/app/apple-store/id1477376905?ct=notification-email&mt=8&pt=524675__;!!LLK065n_VXAQ!wFkC2_1pQx97vTAPM3TglmqIarmBOmAKYMguync2qgOV_CtVypQpDzhGl_NkkoENXg$> or Android<https://urldefense.com/v3/__https://play.google.com/store/apps/details?id=com.github.android&referrer=utm_campaign*3Dnotification-email*26utm_medium*3Demail*26utm_source*3Dgithub__;JSUlJSU!!LLK065n_VXAQ!wFkC2_1pQx97vTAPM3TglmqIarmBOmAKYMguync2qgOV_CtVypQpDzhGl_NMrehFsg$>.