Question about AQUAS pipeline ( the version of AQUAS pipeline is download at 2017/02/17)

[洪彧丞]

Hello:

       

       I had tried to use AQUAS pipeline. However, I suffered a trouble:

       

       My data had 3 replicates and each of them had a chip and an input, so, there are "marked_SOC1_chip_rep1.bam", "marked_SOC1_input_rep1.bam", "marked_SOC1_chip_rep2.bam", "marked_SOC1_input_rep2.bam",

       "marked_SOC1_chip_rep3.bam" and "marked_SOC1_input_rep3.bam".

     

       But, when I put my data into AQUAS pipeline, it seems that the AQUAS pipeline couldn't get the "marked_SOC1_input_rep3.bam"(the input of replicate 3).

       

       This is my command:

       bds chipseq.bds -species ath10 -se -bam1 /home/rommelhong/Chip-seq/picard/GSE45846_SOC1/marked_SOC1_chip_rep1.bam -bam2 /home/rommelhong/Chip-seq/picard/GSE45846_SOC1/marked_SOC1_chip_rep2.bam -bam3  /home/rommelhong/Chip-seq/picard/GSE45846_SOC1/marked_SOC1_chip_rep3.bam -ctl_bam1 /home/rommelhong/Chip-seq/picard/GSE45846_SOC1/marked_SOC1_input_rep1.bam -ctl_bam2 /home/rommelhong/Chip-seq/picard/GSE45846_SOC1/marked_SOC1_input_rep2.bam -ctl_bam3 /home/rommelhong/Chip-seq/picard/GSE45846_SOC1/marked_SOC1_input_rep3.bam -out_dir ../GSE45846_SOC1 -idr_thresh 0.05 

       I couldn't upload my final log file because Google will stop it, thank you for your help. 

[洪彧丞]

Attach is my final log file, I had changed the Filename Extension to .txt , please modify .txt to .js and .html when you want to check it.

洪彧丞於 2017年9月13日星期三 UTC+8下午1時48分32秒寫道：

[Jin]

Hello,

Number of controls exceeded number of chip replicates. Please reduce number of controls by merging some of them.

For example, you can merge 2nd and 3rd controls with:

-ctl_bam1 /home/rommelhong/Chip-seq/picard/GSE45846_SOC1/marked_SOC1_input_rep1.bam -ctl_bam2:1 /home/rommelhong/Chip-seq/picard/GSE45846_SOC1/marked_SOC1_input_rep2.bam -ctl_bam2:2 /home/rommelhong/Chip-seq/picard/GSE45846_SOC1/marked_SOC1_input_rep3.bam

Or merge all controls with:

-ctl_bam1:1 /home/rommelhong/Chip-seq/picard/GSE45846_SOC1/marked_SOC1_input_rep1.bam -ctl_bam1:2 /home/rommelhong/Chip-seq/picard/GSE45846_SOC1/marked_SOC1_input_rep2.bam -ctl_bam1:3 /home/rommelhong/Chip-seq/picard/GSE45846_SOC1/marked_SOC1_input_rep3.bam

Thanks,

Jin

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[Jin]

Oops sorry,

Merging only works for FASTQs in the pipeline. You need to manually merge BAMs before pushing them into the pipeline.

Please forget about the example command line parameters I wrote previously. Just match number of chip replicates and controls or use a single control.

-bam1 BAM1 -bam2 BAM2 -ctl_bam1 CTL_BAM1 -ctl_bam2 CTL_BAM2

or

-bam1 BAM1 -bam2 BAM2 -ctl_bam1 CTL_BAM1

Thanks,

Jin

[洪彧丞]

Hello, thanks for your reply,

But I still have some questions:

According to your advice, it seems that AQUAS pipeline will limit the numbers of controls (inputs) and experiment (chips). What is the maximum of the input and chip number?

In addition, I have 3 replicated data, and all data have own chip data and input data.

Therefore, I should type the command like this?

-bam1 BAM1 -bam2 BAM2 -bam3 BAM3 -ctl_bam1 CTL_BAM1 -ctl_bam2 CTL_BAM2 -ctl_bam3 CTL_BAM3

 If this command is correct, why aquas pipeline can not read the parameter of rep3 input?

Thank you for your help.

Jin於 2017年9月14日星期四 UTC+8上午5時29分46秒寫道：

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[Jin]

You were right. I made a hotfix for this problem. It's in the branch named "can_use_more_than_two_ctrls".

$ git clone https://github.com/kundajelab/chipseq_pipeline

$ git checkout can_use_more_than_two_ctrls

Please let me know if it works so that I can merge that branch to master.

Thanks,

Jin

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[洪彧丞]

OK, I'll try it and tell you the result.

Thank you very much.

Yu-Cheng Hung

Jin於 2017年9月19日星期二 UTC+8上午4時46分29秒寫道：

[洪彧丞]

Hello, sorry for late reply.

I have tried the branch named "can_use_more_than_two_ctrls" and it could work normally.

But, I have two questions:

First, two warnings appeared like picture below when I install my genome data (ath10, Arabidopsis thaliana):

Second, the final report html (ex. GSE51537_JAG_report.html) doesn't have the information of ctl1, ctl2, ctl3 like picture below:

Thank you,

Yu-Cheng Hung

Jin於 2017年9月19日星期二 UTC+8上午4時46分29秒寫道：

You were right. I made a hotfix for this problem. It's in the branch named "can_use_more_than_two_ctrls".

[Jin]

1) You can use your own blacklist BED (with `-blacklist  [BED_GZ]`) if you want your final IDR peaks to be filtered through it. You can ignore the second warning. The latest pipeline no longer uses unique mappability tracks.

2) Please pull fix commit d23864655b16aaa3e0fc5385963f3c707380e135 from the "branch can_use_more_than_two_ctrls" and resume pipelines. Please let me know if it works.

Thanks,

Jin

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[洪彧丞]

It could work normally and the result like picture below:

 I'll try the pair-end data to test if it can process successfully.

Thank you.

Yu-Cheng Hung 

Jin於 2017年10月1日星期日 UTC+8上午9時15分46秒寫道：

[洪彧丞]

Hello, I have tried to process pair-end data (by format of BAM), but an error appeared and the pipeline discontinued immediately like picture below:

Thank you,

Yu-Cheng Hung

  

Jin於 2017年10月1日星期日 UTC+8上午9時15分46秒寫道：

[Jin]

It looks like cross-correlation analysis failed. Can you post a full log? Also, run the following command for debugging info.

$ tail [YOUR_OUT_DIR]/qc/rep*/*.cc.qc

Thanks,

Jin

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[洪彧丞]

Attach is the full log, I had changed the Filename Extension to .txt , please modify .txt to .js and .html when you want to check it.

I run the command and get the information like picture below:

Thank you,

Yu-Cheng Hung 

Jin於 2017年10月18日星期三 UTC+8下午4時35分54秒寫道：

[Jin]

The 3rd column in a cc.qc log file must be positive, but your is -5.

We need more information about the quality of your samples for debugging.

Please run pipelines with `-final_stage xcor` then pipeline will stop before peak calling so no error will occurs.

And then send us (lee...@gmail.com) HTML report (*_report.html) found in your [YOUR_OUT_DIR].

Thanks,

Jin

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[洪彧丞]

Hello, I had sent my HTML report to you, and my address is romme...@gmail.com.

Thank you,

Yu-Cheng Hung

Jin於 2017年10月21日星期六 UTC+8上午7時41分22秒寫道：

[洪彧丞]

Hi Jin,

Recently, I tried to analyze ChIP-seq data about histone modification, Do I just need add "-type histone" to do this?

Thank you,

Yu-Cheng Hung

Jin於 2017年10月21日星期六 UTC+8上午7時41分22秒寫道：

[Jin]

Yes `-type histone` or just `-histone` should work.

Jin

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