Cannot export intensities without a reference channel

[asalt]

I am trying to use TMTintegrator (as part of the philosopher pipeline), but I do not have a reference channel. However, TMTintegrater seems to assume 126 is the reference channel (or some other strange behavior?).

Compare my annotation.txt file to the output table I obtain. Notice that '293T-1', which is 127N, is strangely annotated as "RefInt_293T-1', and "Unnamed: 14" (no column header) must be the 126 channel, WHIM12-1, as this matches the "ReferenceIntensity" column exactly and is the only sample missing..

See below for my config file:

[asalt]

In another situation, I had to specify my ref_pool as "131N" as this is the channel with the pooled samples. Specifying ref_tag=RefMix produced empty files.

Additionally, I still obtain reporter ion values for channel 126 (all NA) even though I intentionally left it out of the annotation file as that channel was not used.

Also in this case the columns were not named correctly - (perhaps that has something to do with my specifying the ref_tag as 131N?)

[huiyinc]

Hi

Can you please check if the channel names in your PSM.tsv file are replaced by the labels in the annotation.txt? It looks like the channels are still named by 126, 127N, 127C etc... Thanks.

Huiyin

asalt notifications@github.com 於 2020年8月25日 上午12:42 寫道：



In another situation, I had to specify my ref_pool as "131N" as this is the channel with the pooled samples. Specifying ref_tag=RefMix produced empty files.

[image: Screenshot from 2020-08-24 11-36-49] https://user-images.githubusercontent.com/11843940/91071954-6a886900-e5fe-11ea-98ea-fed6ae6786ae.png

Additionally, I still obtain reporter ion values for channel 126 (all NA) even though I intentionally left it out of the annotation file as that channel was not used.

[image: Screenshot from 2020-08-24 11-37-35] https://user-images.githubusercontent.com/11843940/91071884-55133f00-e5fe-11ea-90c1-8998d2a61dc8.png

[image: Screenshot from 2020-08-24 11-40-52] https://user-images.githubusercontent.com/11843940/91072147-b5a27c00-e5fe-11ea-996d-72d9beab2393.png

Also in this case the columns were not named correctly - (perhaps that has something to do with my specifying the ref_tag as 131N?)

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[asalt]

Correct, in that PSM.tsv file the channels are still named by the reporter ion. I did fiddle around with the annotations.txt file a bit and may have messed something up there.

However, in my other run, the psm.tsv file had the columns named correctly.

In this experiment there is no reference channel and I wish to obtain the reporter ion intensities at the phosphosite level. It looks like I can obtain this info from the "abundance" reports from TMTintegrator if I set print_RefInt: true. As seen below, I have all 10 channels. Somewhat inconveniently, the 126 channel has been renamed to RefInt_WHIM12-1.

Note that ref_tag: WHIM12-1 has been set - If I leave the pool blank I get a weird header output:

[huiyinc]

Hi,

You will need to (1) leave a blank space for ref_tag (i.e.g, ref_tag: # unique tag for identifying the...) and (2) use a virtual reference (add_Ref: 1), if there is no reference channel in your experiments. Thanks.

Huiyin

asalt notifications@github.com 於 2020年8月25日 下午10:36 寫道：



Correct, in that PSM.tsv file the channels are still named by the reporter ion. I did fiddle around with the annotations.txt file a bit and may have messed something up there.

However, in my other run, the psm.tsv file had the columns named correctly.

[image: Screenshot from 2020-08-25 09-24-39] https://user-images.githubusercontent.com/11843940/91186905-2d32e280-e6b5-11ea-8de3-a4732b8be3b6.png

In this experiment there is no reference channel and I wish to obtain the reporter ion intensities at the phosphosite level. It looks like I can obtain this info from the "abundance" reports from TMTintegrator if I set print_RefInt: true. As seen below, I have all 10 channels. Somewhat inconveniently, the 126 channel has been renamed to RefInt_WHIM12-1.

[image: Screenshot from 2020-08-25 09-29-11] https://user-images.githubusercontent.com/11843940/91187207-86027b00-e6b5-11ea-97f4-21509924cf0f.png

Note that ref_tag: WHIM12-1 has been set - If I leave the pool blank I get a weird header output:

[image: Screenshot from 2020-08-25 09-35-18] https://user-images.githubusercontent.com/11843940/91188016-733c7600-e6b6-11ea-9d30-fd53811808c8.png

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[asalt]

Hm not sure what I am doing wrong here:

My two annotation.txt files:

---

Apologies if I've overlooked it, but I couldn't find add_Ref: 1 in documentation anywhere. Is there a comprehensive list of all options for TMTIntegrator? I simply have the sample yml file provided by the philosopher pipeline.

Thanks

[huiyinc]

Hi,

I guess Philosopher's yml file doesn't include add_Ref:1. Maybe you could check TMT-Integrator's yml file: https://github.com/Nesvilab/TMT-Integrator/releases Thanks.

Huiyin

asalt notifications@github.com 於 2020年8月27日 週四 下午11:04寫道：

Hm not sure what I am doing wrong here:

[image: Screenshot from 2020-08-27 09-59-44] https://user-images.githubusercontent.com/11843940/91459376-34402900-e84c-11ea-8778-92cecc88dee2.png

[image: Screenshot from 2020-08-26 21-00-24] https://user-images.githubusercontent.com/11843940/91459542-5cc82300-e84c-11ea-896f-43743d5dee6e.png

Apologies if I've overlooked it, but I couldn't find add_Ref: 1 in documentation anywhere. Is there a comprehensive list of all options for TMTIntegrator? I simply have the sample yml file provided by the philosopher pipeline.

Thanks

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-- Hui-Yin Chang, 張彙音 Assistant Professor, 助理教授 Department of Biomedical Sciences and Engineering, 生醫科學與工程學系 National Central University (Taiwan), 國立中央大學 (台灣)