search

CCBR / Pipeliner

An open-source and scalable solution to NGS analysis powered by the NIH's Biowulf cluster.
4 stars 0 forks source link

Variant Calling for RNAseq (hg38) stopped at star_align_2 #464

Closed tangwei1129 closed 3 years ago

tangwei1129 commented 3 years ago

I tried to run RNA-seq analysis on hg38 for variant calling option, and it stopped at star_align_2, and gave errors see below. please advice me what to do. Thx. Wei NCI/NIH

[Fri Jun 11 19:59:52 2021] Error in rule star_align_2: [Fri Jun 11 19:59:52 2021] jobid: 0 [Fri Jun 11 19:59:52 2021] output: SL477789.p2Aligned.out.bam, SL477789.p2SJ.out.tab, SL477789.p2Log.final.out

[Fri Jun 11 19:59:52 2021] RuleException: [Fri Jun 11 19:59:52 2021] CalledProcessError in line 13 of /data/CCBR_Pipeliner/4.0.2/Pipeliner/Rules/star.align.2.rl:

skchronicles commented 3 years ago

Hello @tangwei1129,

Thank you for reaching out to us. Can you please attach the "HPC_usage_table.txt" in the output directory of the pipeline? I have a feeling the prior step may have failed due to the number of splice junctions it is trying to insert into the genome. Can you also look through one of the slurm output files for rule star_align_2 to see if it is reporting this error message:

EXITING because of FATAL ERROR: cannot insert junctions on the fly because of strand GstrandBit problem SOLUTION: please contact STAR author at https://groups.google.com/forum/#!forum/rna-star

Regards, @skchronicles

tangwei1129 commented 3 years ago

I have attached the HPC_usage_table.txt, and also found the error you mentioned. see below. How can I fix it? 'EXITING because of FATAL ERROR: cannot insert junctions on the fly because of strand GstrandBit problem Jun 12 00:18:19 ...... FATAL ERROR, exiting'

HPC_usage_table.txt

skchronicles commented 3 years ago

Hey @tangwei1129,

Yes, it is failing due to the number of splice junctions it is attempting to insert back into the reference genome. I have seen this issue occur once before and thought it was a one-off, sample specific issue . I will push a permanent fix to the activeDev branch of Pipeliner when I get some free time today or tomorrow.

I will keep you in the loop, and I will let you know when you can re-run the pipeline with the latest changes.

skchronicles commented 3 years ago

Hey @tangwei1129,

I have pulled in a patch 9febecd52bb594b6e60bd7563c9efb56f23f0029 into Biowulf.

Can you please re-try running Pipeliner's RNA variant calling pipeline with module 4.0.2 on Biowulf? 

module purge
module load ccbrpipeliner/4.0.2
ccbrpipe.sh

When the GUI pops up, please point to a new working directory. Do not point to the same output directory in the previous failed run. This will ensure that the new resources containing the patch get carried over.

image

Thank you for testing this out on your side, and please let me know if there are any issues.

tangwei1129 commented 3 years ago

it seems I can not initialize the directory. it says Permission denied. copying template into /data/tangw3/rnaseqSV1 cp: cannot open ‘/gpfs/gsfs5/users/CCBR_Pipeliner/4.0.2/Pipeliner/Results-template/Scripts/jaccard_score.py’ for reading: Permission denied cp: cannot open ‘/gpfs/gsfs5/users/CCBR_Pipeliner/4.0.2/Pipeliner/Results-template/Scripts/bam_filter_by_mapq.py’ for reading: Permission denied cp: cannot open ‘/gpfs/gsfs5/users/CCBR_Pipeliner/4.0.2/Pipeliner/Results-template/Scripts/scrnaQC.Rmd’ for reading: Permission denied cp: cannot open ‘/gpfs/gsfs5/users/CCBR_Pipeliner/4.0.2/Pipeliner/Results-template/Scripts/scrnaQC.R’ for reading: Permission denied

Skyler Kuhn @.***> 于2021年6月16日周三 下午5:12写道：

Hey @tangwei1129 https://github.com/tangwei1129,

I have pulled in a patch 9febecd https://github.com/CCBR/Pipeliner/commit/9febecd52bb594b6e60bd7563c9efb56f23f0029 into Biowulf.

Can you please re-try running Pipeliner's RNA variant calling pipeline with module 4.0.2 on Biowulf?

module purge module load ccbrpipeliner/4.0.2 ccbrpipe.sh

When the GUI pops up, please point to a new working directory. Do not point to the same output directory in the previous failed run. This will ensure that the new resources containing the patch get carried over.

[image: image] https://user-images.githubusercontent.com/18038345/122294134-be35ad00-cec5-11eb-84cb-e59120f1b1ea.png

Thank you for testing this out on your side, and please let me know if there are any issues.

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHub https://github.com/CCBR/Pipeliner/issues/464#issuecomment-862729980, or unsubscribe https://github.com/notifications/unsubscribe-auth/AB4EHUYIWBNZX3SPFENKLH3TTEHSVANCNFSM46RXAL4Q .

kopardev commented 3 years ago

@skchronicles can you please reset permissions like this: chmod -R a+rX /data/CCBR_Pipeliner/4.0.2/Pipeliner/Results-template/Scripts. Even I don't have permission to change these permissions.

kopardevn@cn0854 09:31 AM /data/CCBR_Pipeliner/4.0.2/Pipeliner/Results-template/Scripts
 % chmod a+rX *
chmod: changing permissions of ‘bam_filter_by_mapq.py’: Operation not permitted
chmod: changing permissions of ‘DiffBind_pipeliner.Rmd’: Operation not permitted
chmod: changing permissions of ‘filterMetrics’: Operation not permitted
chmod: changing permissions of ‘frip_plot.py’: Operation not permitted
chmod: changing permissions of ‘FRiP_plot.R’: Operation not permitted
chmod: changing permissions of ‘frip.py’: Operation not permitted
chmod: changing permissions of ‘integrateBatches.R’: Operation not permitted
chmod: changing permissions of ‘jaccard_score.py’: Operation not permitted
chmod: changing permissions of ‘scrnaQC_Matrix.R’: Operation not permitted
chmod: changing permissions of ‘scrnaQC.R’: Operation not permitted
chmod: changing permissions of ‘scrnaQC_RDS.R’: Operation not permitted
chmod: changing permissions of ‘scrnaQC_Reports.R’: Operation not permitted
chmod: changing permissions of ‘scrnaQC.Rmd’: Operation not permitted
chmod: changing permissions of ‘sjCollapseSamples.awk’: Operation not permitted
skchronicles commented 3 years ago

@tangwei1129 Sorry about that, when I pulled the patch onto the Biowulf, I forgot to update permissions for other.

I have corrected the issue. Please try re-run Pipeliner again. You shouldn't have any issues copying over the files into your working directory.

tangwei1129 commented 3 years ago

it still has a problem with the initial directory. see below. copying template into /data/tangw3/SV cp: cannot open ‘/gpfs/gsfs5/users/CCBR_Pipeliner/4.0.2/Pipeliner/Results-template/Scripts/.kopardevn_dir_bash_history’ for reading: Permission denied

Skyler Kuhn @.***> 于2021年6月17日周四 上午9:55写道：

@tangwei1129 https://github.com/tangwei1129 Sorry about that, when I pulled the patch onto the Biowulf, I forgot to update permissions for other.

I have corrected the issue. Please try re-run Pipeliner again. You shouldn't have any issues copying over the files into your working directory.

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHub https://github.com/CCBR/Pipeliner/issues/464#issuecomment-863260402, or unsubscribe https://github.com/notifications/unsubscribe-auth/AB4EHU23TJ5DVMCR7XSOWE3TTH5E7ANCNFSM46RXAL4Q .

kopardev commented 3 years ago

@tangwei1129 thats my fault...My history file gets created whenever I cd into any folder. I deleted it. Please try now.

tangwei1129 commented 3 years ago

I tried, and now it stopped after alignment. see below error.

859 of 6505 steps (13%) done [Thu Jun 17 15:00:14 2021] Removing temporary output file SL477774Aligned.out.bam. [Thu Jun 17 15:00:14 2021] Failed to remove marker file for job started ([Errno 2] No such file or directory: 'SL477774Aligned.out.bam'). Please ensure write permissions for the directory /gpfs/gsfs12/users/tangw3/hudsonalpha/haib20CY6295/RNA/SV/.snakemake [Thu Jun 17 15:00:14 2021] Finished job 6434. [Thu Jun 17 15:00:14 2021] 860 of 6505 steps (13%) done [Thu Jun 17 15:00:24 2021] Exiting because a job execution failed. Look above for error message [Thu Jun 17 15:00:24 2021] Complete log: /gpfs/gsfs12/users/tangw3/hudsonalpha/haib20CY6295/RNA/SV/.snakemake/log/2021-06-17T130927.374495.snakemake.log

Vishal Koparde @.***> 于2021年6月17日周四 上午11:55写道：

@tangwei1129 https://github.com/tangwei1129 thats my fault...My history file gets created whenever I cd into any folder. I deleted it. Please try now.

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHub https://github.com/CCBR/Pipeliner/issues/464#issuecomment-863360135, or unsubscribe https://github.com/notifications/unsubscribe-auth/AB4EHUYSTOPXIGIVKGQWSQ3TTILG7ANCNFSM46RXAL4Q .

skchronicles commented 3 years ago

@tangwei1129 Can you please send us the pipeline's new "HPC_usage_table.txt"?

tangwei1129 commented 3 years ago

yes sure. please see the attached. or the last couple lines of file pl:star1p 17293561 ccr FAILED 1 32 4-00:00:00 00:00:17 96.0GB/node 0.0GB cn2299 1 152 0.151111 ambss tangw3 pl:star1p 17293733 ccr FAILED 1 32 4-00:00:00 00:00:19 96.0GB/node 0.0GB cn2314 1 147 0.168889 ambss tangw3 pl:star1p 17293753 ccr FAILED 1 32 4-00:00:00 00:00:14 96.0GB/node 0.0GB cn2413 144 0.124444 ambss tangw3 pl:star1p 17293754 ccr FAILED 1 32 4-00:00:00 00:00:17 96.0GB/node 0.0GB cn4474 1 139 0.151111 ambss tangw3 pl:star1p 17293757 ccr COMPLETED 1 32 4-00:00:00 00:19:35 96.0GB/node 34.3GB cn2286 16 339 10.444445 ambss tangw3 pl:star1p 17293786 ccr FAILED 1 32 4-00:00:00 00:00:26 96.0GB/node 0.0GB cn2299 1 253 0.231111 ambss tangw3 pl:star1p 17293788 ccr FAILED 1 32 4-00:00:00 00:03:41 96.0GB/node 0.0GB cn2304 2 251 1.964444 ambss tangw3 pl:star1p 17293941 ccr FAILED 1 32 4-00:00:00 00:00:25 96.0GB/node 0.0GB cn2314 1 151 0.222222 ambss tangw3 pl:star1p 17293974 ccr FAILED 1 32 4-00:00:00 00:00:23 96.0GB/node 0.0GB cn2413 57 0.204444 ambss tangw3 pl:star1p 17293977 ccr FAILED 1 32 4-00:00:00 00:00:26 96.0GB/node 0.0GB cn4474 1 54 0.231111 ambss tangw3

HPC_usage_table.txt

skchronicles commented 3 years ago

@tangwei1129 Can we close this issue? I believe the changes we made to a local clone of Pipeliner on Biowulf addressed these issues (i.e. running STAR in a per sample 2-pass basic mode).

tangwei1129 commented 3 years ago

yes, please close it. Thank you so much for your help!

Skyler Kuhn @.***> 于2021年7月1日周四 上午10:41写道：

Reopened #464 https://github.com/CCBR/Pipeliner/issues/464.

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHub https://github.com/CCBR/Pipeliner/issues/464#event-4966450776, or unsubscribe https://github.com/notifications/unsubscribe-auth/AB4EHU5FBMIF5CSSIJVT7PDTVR5AZANCNFSM46RXAL4Q .

skchronicles commented 3 years ago

Sounds good, have a great 4th of the July weekend @tangwei1129!