Open caleblareau opened 5 years ago
Hi Caleb, Is it possible that RSEM is not in your default PATH (or you provided the wrong path with the -rsem parameter)? That could be the issue. If RSEM is indeed in your PATH, could you send me the commend line you used to run BRAPeS? That might help me understand what the problem is.
Best, Shaked
I aligned with STAR, so I supplied the same bam files twice. This was my command.
python2 BRAPeS/brapes.py -path aligned -g hg38 -o brapes_ss2 -sumF brapes_ss2 -bam aligned.bam -unmapped aligned.bam
I do have rsem
in my PATH
Do you also have bowtie2 in the PATH? If you don't that could cause bowtie2 to fail. Also, what is under your "aligned" directory? Does it have a subfolder for each cell, or is the folder only with data of a single cell? Are you running this command once or in parallel for several cells?
bowtie2
is in the PATH. The file structure looks like this:
aligned/GMLCL_108_S108:
-rw-rw----. 1 cl322 aryee 96650028 May 27 12:38 aligned.bam
-rw-rw----. 1 cl322 aryee 1873136 May 27 12:38 aligned.bam.bai
-rw-rw----. 1 cl322 aryee 1848 May 27 12:38 GMLCL_108_S108Log.final.out
-rw-rw----. 1 cl322 aryee 16491017 May 27 12:38 GMLCL_108_S108Log.out
-rw-rw----. 1 cl322 aryee 447 May 27 12:38 GMLCL_108_S108Log.progress.out
-rw-rw----. 1 cl322 aryee 434 May 27 12:38 GMLCL_108_S108Log.std.out
-rw-rw----. 1 cl322 aryee 374973 May 27 12:38 GMLCL_108_S108ReadsPerGene.out.tab
-rw-rw----. 1 cl322 aryee 1311551 May 27 12:38 GMLCL_108_S108SJ.out.tab
aligned/GMLCL_109_S109:
-rw-rw----. 1 cl322 aryee 175026 May 27 12:38 aligned.bam
-rw-rw----. 1 cl322 aryee 1449704 May 27 12:38 aligned.bam.bai
-rw-rw----. 1 cl322 aryee 1822 May 27 12:38 GMLCL_109_S109Log.final.out
-rw-rw----. 1 cl322 aryee 16490899 May 27 12:38 GMLCL_109_S109Log.out
-rw-rw----. 1 cl322 aryee 447 May 27 12:38 GMLCL_109_S109Log.progress.out
-rw-rw----. 1 cl322 aryee 434 May 27 12:38 GMLCL_109_S109Log.std.out
-rw-rw----. 1 cl322 aryee 353334 May 27 12:38 GMLCL_109_S109ReadsPerGene.out.tab
-rw-rw----. 1 cl322 aryee 1969 May 27 12:38 GMLCL_109_S109SJ.out.tab
** for about 300 cells**
So the idea was a subfolder for each cell. I had attempted to run the command once rather than multiple times.
Ok, good. You're supposed to only run it once, this error could happen if running BRAPeS multiple times in parallel while writing to the same output folder.
In the cell in which BRAPeS crashed on, is there a file with fasta sequences named brapes_ss2.lambda.full.BCRs.fa ? It's possible that this file (or lack of it) is what makes BRAPeS crash
I see. Thanks for the suggestion.
So BRAPeS crashed on the very first file. I don’t see any output, specifically not this fasta file that you are referencing.
On May 27, 2019, at 5:07 PM, shakea02 notifications@github.com wrote:
Ok, good. You're supposed to only run it once, this error could happen if running BRAPeS multiple times in parallel while writing to the same output folder.
In the cell in which BRAPeS crashed on, is there a file with fasta sequences named brapes_ss2.lambda.full.BCRs.fa ? It's possible that this file (or lack of it) is what makes BRAPeS crash
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Can you send me the full log of the run? If BRAPeS doesn't write any output I think there might be a problem with how BRAPeS interrupt the parameters
full log here a majority of it is warnings about read mate mappings, which I can figure out some other time
(thanks by the way for the active debugging help!)
Of course, happy to help! The read mate mappings warnings means that some of the reads are mapped to both strands in different mapping, so BRAPeS can't infer the correct orientation of their unmapped pair. That could be an issue if that happens to most of the reads, but I don't think it's the problem here.
Do you see any new files created under the folder aligned/GMLCL_02_S2 ? Specifically files with the prefix brapes_ss2 ? If you don't see any it means that BRAPeS is writing the files to the incorrect directory. Could you try running it again but for the -path parameter give it the full directory instead of the relative directory?
Makes sense regarding the umapped pairs
I do in fact see new files...
-rw-rw----. 1 cl322 aryee 713 May 27 17:14 brapes_ss2.heavy.junctions.txt
-rw-rw----. 1 cl322 aryee 1274020 May 27 17:14 brapes_ss2.heavy.R1.fa
-rw-rw----. 1 cl322 aryee 1274020 May 27 17:14 brapes_ss2.heavy.R2.fa
-rw-rw----. 1 cl322 aryee 676383 May 27 17:14 brapes_ss2.heavy.mapped.and.unmapped.fa
-rw-rw----. 1 cl322 aryee 0 May 27 17:14 brapes_ss2.kappa.junctions.txt
-rw-rw----. 1 cl322 aryee 0 May 27 17:14 brapes_ss2.kappa.mapped.and.unmapped.fa
-rw-rw----. 1 cl322 aryee 0 May 27 17:14 brapes_ss2.kappa.R1.fa
-rw-rw----. 1 cl322 aryee 0 May 27 17:14 brapes_ss2.kappa.R2.fa
-rw-rw----. 1 cl322 aryee 308 May 27 17:14 brapes_ss2.lambda.junctions.txt
-rw-rw----. 1 cl322 aryee 23600 May 27 17:14 brapes_ss2.lambda.R1.fa
-rw-rw----. 1 cl322 aryee 23600 May 27 17:14 brapes_ss2.lambda.R2.fa
-rw-rw----. 1 cl322 aryee 30408 May 27 17:14 brapes_ss2.lambda.mapped.and.unmapped.fa
-rw-rw----. 1 cl322 aryee 743 May 27 17:15 brapes_ss2.reconstructed.junctions.heavy.fa
-rw-rw----. 1 cl322 aryee 0 May 27 17:15 brapes_ss2.reconstructed.junctions.kappa.fa
-rw-rw----. 1 cl322 aryee 210 May 27 17:15 brapes_ss2.reconstructed.junctions.lambda.fa
-rw-rw----. 1 cl322 aryee 9803 May 27 17:15 brapes_ss2.lambda.full.BCRs.fa
drwxrws---. 2 cl322 aryee 4096 May 27 17:15 rsem_ind453604
Re-running with the absolute directory didn't change anything.
That's good. So for some reason there's a problem creating an RSEM index from the reconstructed sequences in the brapes_ss2.lambda.full.BCRs.fa file. Can you try running the following command from the commend line:
rsem-prepare-reference --bowtie2 full/Path/to/aligned/GMLCL_02_S2/brapes_ss2.lambda.full.BCRs.fa /full/Path/to/aligned/rsem_ind453604/VDJ.lambda.seq
and send me the log? It could tell if the problem is with RSEM itself or with how BRAPeS calls RSEM.
The version of RSEM that I'm using evidently doesn't have a --bowtie2
flag available based on this suggestion. Let me try updating and rerunning things. Thanks for the great suggestion.
Sure thing! Let me know if how it goes
Hi, I tried running BRAPeS for ~ 300 cells and execution stopped on my first cell with the following error message... any idea what to do here? maybe an RSEM issue?