input data problem

paolo.d...@gmail.com

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Feb 10, 2020, 11:58:07 AM2/10/20

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hello,

thank you for developing this app but i have some problems with it.

I am stuck in the "data generation" tab, i upload my annotation csv, my fasta file used to build salmon index and my salmon output in zip format. i've checked them many times and i am pretty sure that anything is ok with my data but when i click on "run" (lenghtScaledTPM) the web page suddenly becomes grey and stucks.

Any idea?

Best wishes

Screenshot from 2020-02-10 17-56-16.png

paolo.d...@gmail.com

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Feb 10, 2020, 12:05:46 PM2/10/20

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Screenshot from 2020-02-10 18-03-12.png

This message appeared after 30 min i launched analysis

Runxuan zhang

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Feb 11, 2020, 4:22:58 AM2/11/20

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Dear Paolo,

Can you share your input files somewhere, e.g. google drives etc so that we can have a look?

Thanks,

Runxuan

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paolo.d...@gmail.com

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Feb 11, 2020, 5:10:46 AM2/11/20

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Dear Runxuan,

Here is:

https://drive.google.com/open?id=1K6JQm3M8Nx8ussdeqlLLQLnFpsO8ZDVE

Thank you and best wishes.

Paolo

Il giorno martedì 11 febbraio 2020 10:22:58 UTC+1, Runxuan zhang ha scritto:

Dear Paolo,

Can you share your input files somewhere, e.g. google drives etc so that we can have a look?

Thanks,

Runxuan

On Mon, Feb 10, 2020 at 4:58 PM <paolo....@gmail.com> wrote:

hello,

thank you for developing this app but i have some problems with it.

I am stuck in the "data generation" tab, i upload my annotation csv, my fasta file used to build salmon index and my salmon output in zip format. i've checked them many times and i am pretty sure that anything is ok with my data but when i click on "run" (lenghtScaledTPM) the web page suddenly becomes grey and stucks.

Any idea?

Best wishes

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To unsubscribe from this group and stop receiving emails from it, send an email to 3d-rna-seq-app-user-group+unsub...@googlegroups.com.

wenbin guo

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Feb 11, 2020, 11:12:12 AM2/11/20

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Hi Paolo,

It seems that your metatable is not corret. As you can see, you coniditon column and brep column didn't distinguish the sampe grouping and replicates labels.E.g. the sample from the same condition should have the same condition labels and the brep from different biological replicates should have different brep labels. You can take a look of the user manual (Figure 1 in https://github.com/wyguo/ThreeDRNAseq/blob/master/vignettes/user_manuals/3D_RNA-seq_App_manual.md#input-files) for details of to prepare the input files. If you still have problem, please let me know.

Best

paolo.d...@gmail.com

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Feb 11, 2020, 12:26:09 PM2/11/20

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Hi Runxuan,

I've modified my metatable in order to have only 2 condition labels (Pz and CTRL) and brep as each sample was a biological replicate of its label but i still have the same problem.

I've attached my modified metatable below.

condition	brep	srep	quant.files
PZ	brep1	srep1	1740R-121-01_quant
PZ	brep2	srep1	1740R-121-02_quant
PZ	brep3	srep1	1740R-121-03_quant
PZ	brep4	srep1	1740R-121-04_quant
PZ	brep5	srep1	1740R-121-05_quant
PZ	brep6	srep1	1740R-121-06_quant
CTRL_DT	brep1	srep1	1740R-121-07_quant
CTRL_DT	brep2	srep1	1740R-121-08_quant
CTRL_DT	brep3	srep1	1740R-121-09_quant

Thank you again and best wishes.

Paolo

Il giorno martedì 11 febbraio 2020 17:12:12 UTC+1, wenbin guo ha scritto:

Hi Paolo,
It seems that your metatable is not corret. As you can see, you coniditon column and brep column didn't distinguish the sampe grouping and replicates labels.E.g. the sample from the same condition should have the same condition labels and the brep from different biological replicates should have different brep labels. You can take a look of the user manual (Figure 1 in https://github.com/wyguo/ThreeDRNAseq/blob/master/vignettes/user_manuals/3D_RNA-seq_App_manual.md#input-files) for details of to prepare the input files. If you still have problem, please let me know.

Best

wenbin guo

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Feb 11, 2020, 1:31:14 PM2/11/20

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Hi Paolo,

After detailed exploring, I find the problem. The tximport R package which I used for generating expression matrix from the quantification files quant.sf will automatically read the bootstrap information of the quantification from the folders. But it seems that there isa problem of the bootstrap files in your datasets. The solution is in your quantfication folder, please remove all the redundant files, only keep the quant.sf file in each folder. Then zip the quantification again as the input for 3D analysis. I have tested, it works fine in this way. If you have any problem, please let me know.

Best

Wenbin

paolo.d...@gmail.com

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Feb 12, 2020, 10:17:58 AM2/12/20

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Hi Wenbin,

Thank you for the hint.

Keeping only quant.sf files in each folder worked fine for the experimental design, in which there was only 2 condition labels and all samples in each label were biological replicates, and i managed to complete the whole analysis, but when i try to give multiple labels with no biological replicates it stucks in the second tab (data pre-processing) during step 1 (Filter low expressed transcripts and genes) where it fails to generate the "Mean variance trend plot" and fails to generate the PCA groups and plots.

I've attached this metatable below

Thank you again and best wishes.

Paolo

condition,brep,srep,quant.files PZ1,brep1,srep1,1740R-121-01_quant PZ2,brep1,srep1,1740R-121-02_quant PZ3,brep1,srep1,1740R-121-03_quant PZ4,brep1,srep1,1740R-121-04_quant PZ5,brep1,srep1,1740R-121-05_quant PZ6,brep1,srep1,1740R-121-06_quant CTRL_DT,brep1,srep1,1740R-121-07_quant CTRL_DT,brep2,srep1,1740R-121-08_quant CTRL_DT,brep3,srep1,1740R-121-09_quant

Il giorno martedì 11 febbraio 2020 19:31:14 UTC+1, wenbin guo ha scritto:

Hi Paolo,

wenbin guo

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Feb 14, 2020, 4:58:36 AM2/14/20

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Hi Paolo,

3D App doesn't work for "no replicates" cases, because it needs replicates to estimate the expression variance. In your experiment, do the label PZ1, PZ2, PZ3, ... refer to the same condition? If so, you should make the labels to PZ, brep1; PZ, brep2; ... The srep column is not neccessary if your data doesn't have sequening replicates.

Best,

Wenbin

montazerinezhad

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May 20, 2021, 6:13:16 PM5/20/21

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hello

I encountered the following problems in entering my data.

If possible, guide me on what to do.

Thankful

The compressed quantification file name or its sub-file names include special symbols. Please uncompressed it and then compressed again to "zip" format. (click "x" to close) (2021-05-20 21:56:31)

×

The quantification file names in the sample meta-table do not match to the quantification files. Please double check whether (1) the quantification file names in the metadata table match to the files in the quantification folder or (2) you have selected the correct quantification method in Step 1: (3). (click "x" to close) (2021-05-20 21:57:05)

paolo.d...@gmail.com در تاریخ دوشنبه ۱۰ فوریهٔ ۲۰۲۰ ساعت ۲۰:۲۸:۰۷ (UTC+3:30) نوشت:

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