Trinity transcript_abundance.pl error using RSEM #305
Comments
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Hi Alex,
I've just encountered this myself as well. Try using 'bowtie2' instead of
'bowtie', as that solved it for me.
I'll aim to resolve the bowtie-1 issue in the next release.
best,
~b
…On Fri, Jul 7, 2017 at 12:08 PM, amartin44 ***@***.***> wrote:
Hello,
I am trying to estimate transcript abundance from a transcriptome
generated from paired-end RNA-seq data using Trinity. However, I am running
into a consistent error while using the transcript_abundance.pl script
that I cannot seem to debug. After submitting a PBS job using the following
options:
perl align_and_estimate_abundance.pl --transcripts Trinity.fasta
--seqType fq
--samples_file fundulus_bioreplicates.txt --SS_lib_type RF --est_method
RSEM
--aln_method bowtie --trinity_mode --prep_reference --output_dir
rsem_outdir
--thread_count 20
the reference files are successfully prepared. Subsequently, the program
begins to make .bam files for the different biological replicates, but
eventually the entire job fails in the rsem-calculate-expression phase. The
error is as follows:
Read HWI-ST1234:250:C96G9ACXX:2:1101:3338:2119: The adjacent two lines do
not represent the two mates of a paired-end read! (RSEM assumes the two
mates of a paired-end read should be adjacent)
Error, cmd: rsem-calculate-expression --paired-end -p 4 --forward-prob 0
--no-bam-output --bam fundulus_RSEM.bowtie.bam (...) died with ret: 65280
at align_and_estimate_abundance.pl line 766.
I have verified that both reads in the pair with the tag
"HWI-ST1234:250:C96G9ACXX:2:1101:3338:2119" exist in the .bam file. Has
anyone experienced a similar error and had success debugging it? I am
relatively new in the bioinformatics field and would greatly appreciate any
insight on what might be going wrong.
Thanks!
Alex.
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Thanks for getting back to me so quickly Brian; Bowtie2 seems to be working fine. |
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terrific! Great to hear.
~b
…On Mon, Jul 10, 2017 at 11:35 AM, amartin44 ***@***.***> wrote:
Thanks for getting back to me so quickly Brian; Bowtie2 seems to be
working fine.
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It only recently began happening BenLangmead/bowtie#52 Perhaps Bowtie will be updated soon and output ordered alignments, as it did before. You could also downgrade to version 1.1 in the meantime because Bowtie 1 is much faster (simpler algorithm) than Bowtie 2. |
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Hello,
However during processing I'm seeing a lot of warnings like bellow The commandline ends with an error... How to fix this? |
Could you send the script that you use to submit this task on PBS? |
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Hello, ~/software/trinityrnaseq-Trinity-v2.8.4/util/align_and_estimate_abundance.pl --transcripts Sspon.cds.fasta --seqType fq --left SES-MC-3_FRAS190026479-1a_1QC.fq.gz --right SES-MC-3_FRAS190026479-1a_2QC.fq.gz --est_method RSEM --output_dir MC --aln_method bowtie --thread_count 4 --prep_reference The commandline ends with an error... .Number of first and second mates in read A00808:49:HKYHNDSXX:2:1103:9462:31375's full alignments (both mates are aligned) are not matched! "rsem-scan-for-paired-end-reads 1 bowtie.bam.for_rsem.tmp.bam bowtie.bam.for_rsem.bam" failed! Plase check if you provide correct parameters/options for the pipeline! Thanks |
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hi - it looks like there could be some problem with your data:
.Number of first and second mates in read
A00808:49:HKYHNDSXX:2:1103:9462:31375's full alignments (both mates are
aligned) are not matched!
If it's not, then try using another tool such as salmon or kallisto and see
if that gives you any trouble. These others are both supported by the
Trinity script. If you need to use RSEM, you could follow up with the RSEM
developer for further support.
best,
~b
…On Mon, May 27, 2019 at 1:22 AM xiutinghua ***@***.***> wrote:
Hello,
*I am trying to estimate transcript abundance from a transcriptome
generated from paired-end RNA-seq data using Trinity. I'm running the
following commandline. In fact, I submitted three samples submitted in the
SGV, but only this sample(SES-MC-3_FRAS190026479-1a) failed.*
~/software/trinityrnaseq-Trinity-v2.8.4/util/
align_and_estimate_abundance.pl --transcripts Sspon.cds.fasta --seqType
fq --left SES-MC-3_FRAS190026479-1a_1QC.fq.gz --right
SES-MC-3_FRAS190026479-1a_2QC.fq.gz --est_method RSEM --output_dir MC
--aln_method bowtie --thread_count 4 --prep_reference
*The commandline ends with an error...*
.Number of first and second mates in read
A00808:49:HKYHNDSXX:2:1103:9462:31375's full alignments (both mates are
aligned) are not matched!
"rsem-scan-for-paired-end-reads 1 bowtie.bam.for_rsem.tmp.bam
bowtie.bam.for_rsem.bam" failed! Plase check if you provide correct
parameters/options for the pipeline!
Error, cmd: convert-sam-for-rsem bowtie.bam bowtie.bam.for_rsem died with
ret: 65280 at
/public1/home/stu_huaxiuting/software/trinityrnaseq-Trinity-v2.8.4/util/
align_and_estimate_abundance.pl line 790.
Thanks
xiuting
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hello, also got some problem running :
it ran correctly up until then it produced an error any help ? should i use bowtie(1)? or salmon? edit: i saw here https://groups.google.com/forum/#!topic/trinityrnaseq-users/LEBWTtzUKkI , that you said you have to --prep_reference first. I got that option in my command. Should i run first the command --prep_reference and then the rest? |
Hello,
I am trying to estimate transcript abundance from a transcriptome generated from paired-end RNA-seq data using Trinity. However, I am running into a consistent error while using the transcript_abundance.pl script that I cannot seem to debug. After submitting a PBS job using the following options:
perl align_and_estimate_abundance.pl --transcripts Trinity.fasta --seqType fq
--samples_file fundulus_bioreplicates.txt --SS_lib_type RF --est_method RSEM
--aln_method bowtie --trinity_mode --prep_reference --output_dir rsem_outdir
--thread_count 20
the reference files are successfully prepared. Subsequently, the program begins to make .bam files for the different biological replicates, but eventually the entire job fails in the rsem-calculate-expression phase. The error is as follows:
Read HWI-ST1234:250:C96G9ACXX:2:1101:3338:2119: The adjacent two lines do not represent the two mates of a paired-end read! (RSEM assumes the two mates of a paired-end read should be adjacent)
Error, cmd: rsem-calculate-expression --paired-end -p 4 --forward-prob 0 --no-bam-output --bam fundulus_RSEM.bowtie.bam (...) died with ret: 65280 at align_and_estimate_abundance.pl line 766.
I have verified that both reads in the pair with the tag "HWI-ST1234:250:C96G9ACXX:2:1101:3338:2119" exist in the .bam file. Has anyone experienced a similar error and had success debugging it? I am relatively new in the bioinformatics field and would greatly appreciate any insight on what might be going wrong.
Thanks!
Alex.
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