RSEM issue - Githubissues

Dear BRAPeS developers,

I was trying to run BRAPeS on the example data but had no success. RSEM was installed with bioconda, and the version is 1.3.2.

The error message was "RSEM did not produce any transcript alignment files for heavy chain, please check the -rsem parameter." The output "BCR.out.BCRs.txt" only contains the header line, unlike the "example.output.BCRs.txt" file provided by you. The same error occurred when using the bioconda version 1.2.28-0 or a 1.3.2 version installed by our Linux administrator not using conda.

The command is: brapes.py -genome mm10_ncbi -rsem ~/data/bin/anaconda3/envs/py27/bin/ -path Example/proc_data/ -sumF Example/BRAPeS_out/BCR.out -output BRAPeS_out/BCR.out -score 15 -top 10 -byExp -unmapped unmapped.bam -bam sorted.bam

The full log is:

2019-12-04 16:47:23.910158 Working on: ERR1467921 2019-12-04 16:47:23.918773 Pre-processing heavy chain 2019-12-04 16:47:23.927829 Did not find any V-J reads, searching for V-C and J-C reads: Added 139 unmapped reads to the reads file 2019-12-04 16:47:27.259014 Pre-processing kappa chain Added 957 unmapped reads to the reads file 2019-12-04 16:47:30.819533 Pre-processing lambda chain 2019-12-04 16:47:30.826005 Did not find any V-J reads, searching for V-C and J-C reads: Added 222 unmapped reads to the reads file 2019-12-04 16:47:34.052156 Reconstructing heavy chains 2019-12-04 16:47:37.536691 Reconstructing kappa chains 2019-12-04 16:57:23.322620 Reconstructing lambda chains 2019-12-04 16:57:23.327186 Creating full BCR sequences 2019-12-04 16:57:23.418055 Running RSEM to quantify expression of all possible isotypes sorting bam file sorting bam file 2019-12-04 16:57:25.624145 Did not reconstruct any lambda chains, not running RSEM on this chain 2019-12-04 16:57:25.634136 Finding productive CDR3 2019-12-04 16:57:25.916152 Performing CDR1 and CDR2 reconstruction

Loading reads...

Working on IGHV9-2...

No IMGT reference for IGHJ4. Will skip FR4 reconstruction... No IMGT reference for IGHV9-2. Skipping...

Working on IGHV9-3...

No IMGT reference for IGHJ4. Will skip FR4 reconstruction... No IMGT reference for IGHV9-3. Skipping...

Loading reads...

Working on IGKV1-122...

No IMGT reference for IGKJ1. Will skip FR4 reconstruction... No IMGT reference for IGKV1-122. Skipping...

Working on IGKV1-110...

No IMGT reference for IGKJ1. Will skip FR4 reconstruction... No IMGT reference for IGKV1-110. Skipping...

Working on IGKV1-117...

No IMGT reference for IGKJ1. Will skip FR4 reconstruction... No IMGT reference for IGKV1-117. Skipping...

RSEM did not produce any transcript alignment files for heavy chain, please check the -rsem parameter RSEM did not produce any transcript alignment files for kappa chain, please check the -rsem parameter 2019-12-04 16:57:26.427233 Did not reconstruct any lambda chains, not running RSEM on this chain 2019-12-04 16:57:26.427649 Writing results to summary file

When I did not specify the "-rsem" argument (RSEM was already added to the PATH), another error occurred:

2019-12-04 17:05:44.695056 Working on: ERR1467921 2019-12-04 17:05:44.703218 Pre-processing heavy chain 2019-12-04 17:05:44.712471 Did not find any V-J reads, searching for V-C and J-C reads: Added 139 unmapped reads to the reads file 2019-12-04 17:05:47.875784 Pre-processing kappa chain Added 957 unmapped reads to the reads file 2019-12-04 17:05:51.229238 Pre-processing lambda chain 2019-12-04 17:05:51.235855 Did not find any V-J reads, searching for V-C and J-C reads: Added 222 unmapped reads to the reads file 2019-12-04 17:05:54.209072 Reconstructing heavy chains 2019-12-04 17:05:57.320026 Reconstructing kappa chains 2019-12-04 17:15:28.427234 Reconstructing lambda chains 2019-12-04 17:15:28.432419 Creating full BCR sequences 2019-12-04 17:15:28.523025 Running RSEM to quantify expression of all possible isotypes Traceback (most recent call last): File "/home/dsun3/bin/BRAPeS/brapes.py", line 2157, in args.Fr) File "/home/dsun3/bin/BRAPeS/brapes.py", line 52, in runBCRpipe hvr_path, genome) File "/home/dsun3/bin/BRAPeS/brapes.py", line 321, in runSingleCell runRsem(outDir, rsem, bowtie2, fullTcrFileHeavy, fullTcrFileKappa,fullTcrFileLambda, output, samtools, False) File "/home/dsun3/bin/BRAPeS/brapes.py", line 1185, in runRsem runRSEMonOneFile(fullTcrFileHeavy, rsem, bowtie2, rsemIndDir, output, samtools, "heavy", secondRound) File "/home/dsun3/bin/BRAPeS/brapes.py", line 1216, in runRSEMonOneFile '-q', resF, rsemIndDir + '/VDJ.' + chain + '.seq'], stdout=devnull, stderr=devnull) File "/home/dsun3/data/bin/anaconda3/envs/py27/lib/python2.7/subprocess.py", line 172, in call return Popen(*popenargs, **kwargs).wait() File "/home/dsun3/data/bin/anaconda3/envs/py27/lib/python2.7/subprocess.py", line 394, in init errread, errwrite) File "/home/dsun3/data/bin/anaconda3/envs/py27/lib/python2.7/subprocess.py", line 1047, in _execute_child raise child_exception OSError: [Errno 2] No such file or directory

Any ideas what might be going wrong? Thank you!

Hello,

Thank you for your interest in BRAPeS. Regarding the second issue (when RSEM is added to the PATH), there was a bug in the program and it's now fixed, thank you for noticing! As for the first issue, I downloaded the newest RSEM version and ran BRAPeS on the example data and it worked without any problem. Based on the output log you posted, I think you might be running an older version of BRAPeS, can you pull the new version and try again?

Thanks, Shaked

Thank you for the quick reply. I downloaded the latest github version. Without '-rsem', the second issue is now fixed. However, the first error persisted. I tried on a different computer but still got the same error message ('RSEM did not produce any transcript alignment files for heavy chain, please check the -rsem parameter').

Is there anything else that you think might be causing the issue? Thanks again!

python brapes.py -genome mm10_ncbi -path Example/proc_data/ -sumF Example/BRAPeS_out/BCR.out -output BRAPeS_out/BCR.out -score 15 -top 10 -byExp -unmapped unmapped.bam -bam sorted.bam

2019-12-04 22:02:53.932638 Working on: ERR1467921 2019-12-04 22:02:54.441392 Pre-processing heavy chain 2019-12-04 22:02:54.482511 Did not find any V-J reads, searching for V-C and J-C reads: Added 139 unmapped reads to the reads file 2019-12-04 22:03:00.373301 Pre-processing kappa chain Added 957 unmapped reads to the reads file 2019-12-04 22:03:06.693289 Pre-processing lambda chain 2019-12-04 22:03:06.705354 Did not find any V-J reads, searching for V-C and J-C reads: Added 222 unmapped reads to the reads file 2019-12-04 22:03:12.336326 Reconstructing heavy chains 2019-12-04 22:03:17.032362 Reconstructing kappa chains 2019-12-04 22:17:23.496863 Reconstructing lambda chains 2019-12-04 22:17:23.503861 Creating full BCR sequences 2019-12-04 22:17:23.656102 Running RSEM to quantify expression of all possible isotypes sorting bam file sorting bam file 2019-12-04 22:17:29.067154 Did not reconstruct any lambda chains, not running RSEM on this chain 2019-12-04 22:17:29.080890 Finding productive CDR3 2019-12-04 22:17:29.954443 Performing CDR1 and CDR2 reconstruction

Loading reads...

Working on IGHV9-2...

No IMGT reference for IGHJ4. Will skip FR4 reconstruction... No IMGT reference for IGHV9-2. Skipping...

Working on IGHV9-3...

No IMGT reference for IGHJ4. Will skip FR4 reconstruction... No IMGT reference for IGHV9-3. Skipping...

Loading reads...

Working on IGKV1-122...

No IMGT reference for IGKJ1. Will skip FR4 reconstruction... No IMGT reference for IGKV1-122. Skipping...

Working on IGKV1-110...

No IMGT reference for IGKJ1. Will skip FR4 reconstruction... No IMGT reference for IGKV1-110. Skipping...

Working on IGKV1-117...

No IMGT reference for IGKJ1. Will skip FR4 reconstruction... No IMGT reference for IGKV1-117. Skipping...

RSEM did not produce any transcript alignment files for heavy chain, please check the -rsem parameter RSEM did not produce any transcript alignment files for kappa chain, please check the -rsem parameter 2019-12-04 22:17:31.212076 Did not reconstruct any lambda chains, not running RSEM on this chain 2019-12-04 22:17:31.213223 Writing results to summary file

Hello,

The log output you sent is not of the latest version, since the somatic hypermutation correction was changed and the output it provides now is different. If you go to the github page and go to the HVR_rec folder, do you see the latest version (from 7/25)? I'm attaching below the log output of the newest version.

However, this might not be the issue. Can you try running these two command lines from the terminal and see if you get any error?

path-to-rsem/rsem-prepare-reference --bowtie2 path-to-BRAPeS/Example/proc_data/ERR1467921/BRAPeS_out/test.rsem.ver.heavy.full.BCRs.fa VDJ.test.seq

path-to-rsem/rsem-calculate-expression --no-qualities --bowtie2 --bowtie2-mismatch-rate 0.0 --paired-end path-to-BRAPeS/Example/proc_data/ERR1467921/BRAPeS_out/test.rsem.ver.heavy.R1.fa path-to-BRAPeS/Example/proc_data/ERR1467921/BRAPeS_out/test.rsem.ver.heavy.R2.fa VDJ.test.seq VDJ.test.rsem.out

Here's the log output that I'm getting from BRAPeS:

2019-12-05 11:26:02.702965 Working on: ERR1467921 2019-12-05 11:26:02.731251 Pre-processing heavy chain 2019-12-05 11:26:02.758475 Did not find any V-J reads, searching for V-C and J-C reads: Added 139 unmapped reads to the reads file 2019-12-05 11:26:08.922235 Pre-processing kappa chain Added 957 unmapped reads to the reads file 2019-12-05 11:26:15.217287 Pre-processing lambda chain 2019-12-05 11:26:15.240906 Did not find any V-J reads, searching for V-C and J-C reads: Added 222 unmapped reads to the reads file 2019-12-05 11:26:21.955844 Reconstructing heavy chains 2019-12-05 11:26:25.591583 Reconstructing kappa chains 2019-12-05 11:36:12.712462 Reconstructing lambda chains 2019-12-05 11:36:12.718622 Creating full BCR sequences 2019-12-05 11:36:12.973163 Running RSEM to quantify expression of all possible isotypes sorting bam file sorting bam file 2019-12-05 11:36:19.225158 Did not reconstruct any lambda chains, not running RSEM on this chain 2019-12-05 11:36:19.260914 Finding productive CDR3 2019-12-05 11:36:19.717973 Performing CDR1 and CDR2 reconstruction

Loading reads...

Working on IGHV9-2...

Reconstructing CDR1 Reconstruction Succeeded! Reconstructing CDR2 Reconstruction Succeeded! Reconstructing FR1 Reconstruction Succeeded! Reconstructing FR2 Reconstruction Succeeded! Reconstructing FR3 Reconstruction Succeeded! Reconstructing FR4 Reconstruction Succeeded!

Working on IGHV9-3...

Loading reads...

Working on IGKV1-122...

Working on IGKV1-117...

Working on IGKV1-110...

sorting bam file sorting bam file 2019-12-05 11:54:46.614080 Did not reconstruct any lambda chains, not running RSEM on this chain 2019-12-05 11:54:47.083402 Writing results to summary file

Thank you @shakea02 . I did the installation with 'git clone https://github.com/YosefLab/BRAPeS.git' last night, so I'm not sure why it's not the latest version. Should I do it another way?

Regarding the two commands, both finished without errors. However, when changing slightly your python code to output RSEM standard out and standard error, an error occurred in the RSEM reference preparation step, where the program complained "Number of transcripts in the reference is less than 1!". I checked the "BCR.out.heavy.full.BCRs.bestIso.CDR1.CDR2.reconstructions.fasta.BCRs.fasta" file and it is empty.

The full log is:

2019-12-05 14:45:04.515627 Working on: ERR1467921 2019-12-05 14:45:04.524096 Pre-processing heavy chain 2019-12-05 14:45:04.534343 Did not find any V-J reads, searching for V-C and J-C reads: Added 139 unmapped reads to the reads file 2019-12-05 14:45:07.971786 Pre-processing kappa chain Added 957 unmapped reads to the reads file 2019-12-05 14:45:11.679573 Pre-processing lambda chain 2019-12-05 14:45:11.686279 Did not find any V-J reads, searching for V-C and J-C reads: Added 222 unmapped reads to the reads file 2019-12-05 14:45:15.019389 Reconstructing heavy chains 2019-12-05 14:45:18.549340 Reconstructing kappa chains 2019-12-05 14:54:54.171313 Reconstructing lambda chains 2019-12-05 14:54:54.177234 Creating full BCR sequences 2019-12-05 14:54:54.268029 Running RSEM to quantify expression of all possible isotypes rsem-synthesis-reference-transcripts Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind5925621/VDJ.heavy.seq 1 0 Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.heavy.full.BCRs.fa

rsem-preref Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind5925621/VDJ.heavy.seq.transcripts.fa 1 Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind5925621/VDJ.heavy.seq -q

bowtie2-build -f -q Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind5925621/VDJ.heavy.seq.idx.fa Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind5925621/VDJ.heavy.seq Building a SMALL index

bowtie2 -f --sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.0 -I 1 -X 1000 --no-mixed --no-discordant -p 1 -k 200 --quiet -x Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind5925621/VDJ.heavy.seq -1 Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.heavy.R1.fa -2 Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.heavy.R2.fa | samtools view -S -b -o Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.heavy.rsem.out.temp/BCR.out.heavy.rsem.out.bam -

rsem-parse-alignments Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind5925621/VDJ.heavy.seq Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.heavy.rsem.out.temp/BCR.out.heavy.rsem.out Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.heavy.rsem.out.stat/BCR.out.heavy.rsem.out Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.heavy.rsem.out.temp/BCR.out.heavy.rsem.out.bam 2 -tag XM -q

rsem-build-read-index 32 0 1 Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.heavy.rsem.out.temp/BCR.out.heavy.rsem.out_alignable_1.fa Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.heavy.rsem.out.temp/BCR.out.heavy.rsem.out_alignable_2.fa

rsem-run-em Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind5925621/VDJ.heavy.seq 2 Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.heavy.rsem.out Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.heavy.rsem.out.temp/BCR.out.heavy.rsem.out Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.heavy.rsem.out.stat/BCR.out.heavy.rsem.out -p 1 -b Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.heavy.rsem.out.temp/BCR.out.heavy.rsem.out.bam 0 -q Warning: Read ERR1467921.638508 is ignored due to at least one of the mates' length < seed length (= 25)! Warning: Read ERR1467921.109703 is ignored due to at least one of the mates' length < seed length (= 25)! Warning: There are 2 reads ignored in total. Time Used for EM.cpp : 0 h 00 m 00 s

rm -rf Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.heavy.rsem.out.temp

rsem-synthesis-reference-transcripts Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind5925621/VDJ.kappa.seq 1 0 Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.kappa.full.BCRs.fa

rsem-preref Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind5925621/VDJ.kappa.seq.transcripts.fa 1 Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind5925621/VDJ.kappa.seq -q

bowtie2-build -f -q Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind5925621/VDJ.kappa.seq.idx.fa Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind5925621/VDJ.kappa.seq Building a SMALL index

bowtie2 -f --sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.0 -I 1 -X 1000 --no-mixed --no-discordant -p 1 -k 200 --quiet -x Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind5925621/VDJ.kappa.seq -1 Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.kappa.R1.fa -2 Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.kappa.R2.fa | samtools view -S -b -o Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.kappa.rsem.out.temp/BCR.out.kappa.rsem.out.bam -

rsem-parse-alignments Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind5925621/VDJ.kappa.seq Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.kappa.rsem.out.temp/BCR.out.kappa.rsem.out Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.kappa.rsem.out.stat/BCR.out.kappa.rsem.out Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.kappa.rsem.out.temp/BCR.out.kappa.rsem.out.bam 2 -tag XM -q

rsem-build-read-index 32 0 1 Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.kappa.rsem.out.temp/BCR.out.kappa.rsem.out_alignable_1.fa Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.kappa.rsem.out.temp/BCR.out.kappa.rsem.out_alignable_2.fa

rsem-run-em Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind5925621/VDJ.kappa.seq 2 Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.kappa.rsem.out Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.kappa.rsem.out.temp/BCR.out.kappa.rsem.out Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.kappa.rsem.out.stat/BCR.out.kappa.rsem.out -p 1 -b Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.kappa.rsem.out.temp/BCR.out.kappa.rsem.out.bam 0 -q Warning: Read ERR1467921.82207 is ignored due to at least one of the mates' length < seed length (= 25)! Warning: There are 1 reads ignored in total. Time Used for EM.cpp : 0 h 00 m 00 s

rm -rf Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.kappa.rsem.out.temp

2019-12-05 14:54:56.562326 Did not reconstruct any lambda chains, not running RSEM on this chain 2019-12-05 14:54:56.567939 Finding productive CDR3 2019-12-05 14:54:56.852498 Performing CDR1 and CDR2 reconstruction

Loading reads...

Working on IGHV9-2...

No IMGT reference for IGHJ4. Will skip FR4 reconstruction... No IMGT reference for IGHV9-2. Skipping...

Working on IGHV9-3...

No IMGT reference for IGHJ4. Will skip FR4 reconstruction... No IMGT reference for IGHV9-3. Skipping...

Loading reads...

Working on IGKV1-122...

No IMGT reference for IGKJ1. Will skip FR4 reconstruction... No IMGT reference for IGKV1-122. Skipping...

Working on IGKV1-110...

No IMGT reference for IGKJ1. Will skip FR4 reconstruction... No IMGT reference for IGKV1-110. Skipping...

Working on IGKV1-117...

No IMGT reference for IGKJ1. Will skip FR4 reconstruction... No IMGT reference for IGKV1-117. Skipping...

rsem-synthesis-reference-transcripts Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind54981819/VDJ.heavy.seq 1 0 Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.heavy.full.BCRs.bestIso.CDR1.CDR2.reconstructions.fasta.BCRs.fasta Number of transcripts in the reference is less than 1! "rsem-synthesis-reference-transcripts Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind54981819/VDJ.heavy.seq 1 0 Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.heavy.full.BCRs.bestIso.CDR1.CDR2.reconstructions.fasta.BCRs.fasta" failed! Plase check if you provide correct parameters/options for the pipeline! bowtie2 -f --sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.0 -I 1 -X 1000 --no-mixed --no-discordant -p 1 -k 200 --quiet -x Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind54981819/VDJ.heavy.seq -1 Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.heavy.R1.fa -2 Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.heavy.R2.fa | samtools view -S -b -o Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.afterCDRrec.heavy.rsem.out.temp/BCR.out.afterCDRrec.heavy.rsem.out.bam - Could not locate a Bowtie index corresponding to basename "Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind54981819/VDJ.heavy.seq" Error: Encountered internal Bowtie 2 exception (#1) Command: /home/dsun3/data/bin/anaconda3/envs/py27/bin/bowtie2-align-s --wrapper basic-0 -f --sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.0 -I 1 -X 1000 --no-mixed --no-discordant -p 1 -k 200 --quiet -x Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind54981819/VDJ.heavy.seq -1 Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.heavy.R1.fa -2 Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.heavy.R2.fa (ERR): bowtie2-align exited with value 1

rsem-parse-alignments Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind54981819/VDJ.heavy.seq Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.afterCDRrec.heavy.rsem.out.temp/BCR.out.afterCDRrec.heavy.rsem.out Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.afterCDRrec.heavy.rsem.out.stat/BCR.out.afterCDRrec.heavy.rsem.out Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.afterCDRrec.heavy.rsem.out.temp/BCR.out.afterCDRrec.heavy.rsem.out.bam 2 -tag XM -q Cannot open Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind54981819/VDJ.heavy.seq.grp! It may not exist. "rsem-parse-alignments Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind54981819/VDJ.heavy.seq Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.afterCDRrec.heavy.rsem.out.temp/BCR.out.afterCDRrec.heavy.rsem.out Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.afterCDRrec.heavy.rsem.out.stat/BCR.out.afterCDRrec.heavy.rsem.out Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.afterCDRrec.heavy.rsem.out.temp/BCR.out.afterCDRrec.heavy.rsem.out.bam 2 -tag XM -q" failed! Plase check if you provide correct parameters/options for the pipeline! rsem-synthesis-reference-transcripts Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind54981819/VDJ.kappa.seq 1 0 Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.kappa.full.BCRs.bestIso.CDR1.CDR2.reconstructions.fasta.BCRs.fasta Number of transcripts in the reference is less than 1! "rsem-synthesis-reference-transcripts Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind54981819/VDJ.kappa.seq 1 0 Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.kappa.full.BCRs.bestIso.CDR1.CDR2.reconstructions.fasta.BCRs.fasta" failed! Plase check if you provide correct parameters/options for the pipeline! bowtie2 -f --sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.0 -I 1 -X 1000 --no-mixed --no-discordant -p 1 -k 200 --quiet -x Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind54981819/VDJ.kappa.seq -1 Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.kappa.R1.fa -2 Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.kappa.R2.fa | samtools view -S -b -o Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.afterCDRrec.kappa.rsem.out.temp/BCR.out.afterCDRrec.kappa.rsem.out.bam - Could not locate a Bowtie index corresponding to basename "Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind54981819/VDJ.kappa.seq" Error: Encountered internal Bowtie 2 exception (#1) Command: /home/dsun3/data/bin/anaconda3/envs/py27/bin/bowtie2-align-s --wrapper basic-0 -f --sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.0 -I 1 -X 1000 --no-mixed --no-discordant -p 1 -k 200 --quiet -x Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind54981819/VDJ.kappa.seq -1 Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.kappa.R1.fa -2 Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.kappa.R2.fa (ERR): bowtie2-align exited with value 1

rsem-parse-alignments Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind54981819/VDJ.kappa.seq Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.afterCDRrec.kappa.rsem.out.temp/BCR.out.afterCDRrec.kappa.rsem.out Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.afterCDRrec.kappa.rsem.out.stat/BCR.out.afterCDRrec.kappa.rsem.out Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.afterCDRrec.kappa.rsem.out.temp/BCR.out.afterCDRrec.kappa.rsem.out.bam 2 -tag XM -q Cannot open Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind54981819/VDJ.kappa.seq.grp! It may not exist. "rsem-parse-alignments Example/proc_data/ERR1467921/BRAPeS_out/rsem_ind54981819/VDJ.kappa.seq Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.afterCDRrec.kappa.rsem.out.temp/BCR.out.afterCDRrec.kappa.rsem.out Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.afterCDRrec.kappa.rsem.out.stat/BCR.out.afterCDRrec.kappa.rsem.out Example/proc_data/ERR1467921/BRAPeS_out/BCR.out.afterCDRrec.kappa.rsem.out.temp/BCR.out.afterCDRrec.kappa.rsem.out.bam 2 -tag XM -q" failed! Plase check if you provide correct parameters/options for the pipeline! RSEM did not produce any transcript alignment files for heavy chain, please check the -rsem parameter RSEM did not produce any transcript alignment files for kappa chain, please check the -rsem parameter 2019-12-05 14:54:57.414421 Did not reconstruct any lambda chains, not running RSEM on this chain 2019-12-05 14:54:57.414748 Writing results to summary file

Ok, now I understand the problem better, thanks! The RSEM crash is because the somatic hypermutation correction isn't working properly and doesn't produce any reconstruction. I believe it's the same problem that causes you to get the different output log but I'm not sure what is causing it. @gabe-raulet do you have any suggestions as to why when cloning the git we get the older CDR1/2 reconstruction version?

The program can now run successfully with the updated version.

YosefLab / BRAPeS

RSEM issue #3