cannot open RSEM.grp file

[Andrew Thompson]

I am trying to run SEM with a bowtie BAM file that I have created.  However I am getting the following error:

Cannot open /home/andrewwt/Trinity_paired_end_assembly/Trinity.fasta.RSEM.grp! It may not exist.

Error, cmd: rsem-calculate-expression  --paired-end   -p 4   --forward-prob 0 --no-bam-output --bam /home/andrewwt/bowtieD1/bowtie_out.coordSorted.bam /home/andrewwt/Trinity_paired_end_assembly/Trinity.fasta.RSEM RSEM  died with ret: 65280 at /c1/apps/trinity/trinityrnaseq-2.0.3/util/align_and_estimate_abundance.pl line 632.

What is the RSEM.grp file and why might I not have it?  I can't find it anywhere. 

Any help would be great!

[Ken Field]

You need to run prep-reference first. That will create that file. 

Ken

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[Tiago Hori]

Andrew, that script has been deprecated for a few versions now. Is there a specific reason for not using align_and_estimate.pl?

T.

Ultimate stresses sportsmanship and fair play. Competitive play is encouraged, but NEVER AT THE EXPENSE OF RESPECT BETWEEN PLAYERS, adherence to the rules and the BASIC JOY OF PLAY.

[Tiago Hori]

That too. :) And if you move to align and estimate, you will still need the prep-reference flag!

T.

Ultimate stresses sportsmanship and fair play. Competitive play is encouraged, but NEVER AT THE EXPENSE OF RESPECT BETWEEN PLAYERS, adherence to the rules and the BASIC JOY OF PLAY.

On Mar 25, 2015, at 04:03 PM, Ken Field <kfi...@bucknell.edu> wrote:

You need to run prep-reference first. That will create that file. 

Ken

[Andrew Thompson]

Thanks guys, that got rid of that error.  I ran this first to capture both single and paired end reads that I had in my file:

$TRINITY_HOME/util/bowtie_PE_separate_then_join.pl --seqType fq --left left.fq --right right.fq --target Trinity.fasta --aligner bowtie -- -p 4 --all --best --strata -m 300

It output several bam files and I input the path to the bam file after the option  --aln_method when I ran rem

Now I am getting this error: 

 CMD: touch /home/andrewwt/Trinity_paired_end_assembly/Trinity.fasta.RSEM.rsem.prepped.started

CMD: rsem-prepare-reference  --transcript-to-gene-map /home/andrewwt/Trinity_paired_end_assembly/Trinity.fasta.gene_trans_map /home/andrewwt/Trinity_paired_end_assembly/Trinity.fasta /home/andrewwt/Trinity_paired_end_assembly/Trinity.fasta.RSEM

CMD: rsem-calculate-expression  --paired-end   -p 4   --forward-prob 0 --no-bam-output --bam /home/andrewwt/bowtieD1/bowtie_out.coordSorted.bam /home/andrewwt/Trinity_paired_end_assembly/Trinity.fasta.RSEM RSEM 

The two reads do not come from the same pair!Error, cmd: rsem-calculate-expression  --paired-end   -p 4   --forward-prob 0 --no-bam-output --bam /home/andrewwt/bowtieD1/bowtie_out.coordSorted.bam /home/andrewwt/Trinity_paired_end_assembly/Trinity.fasta.RSEM RSEM  died with ret: 65280 at /c1/apps/trinity/trinityrnaseq-2.0.6/util/align_and_estimate_abundance.pl line 653.

Could I be using the wrong bam file output from bowtie?  

On Wednesday, March 25, 2015 at 2:35:04 PM UTC-4, Tiago Hori wrote:

That too. :) And if you move to align and estimate, you will still need the prep-reference flag!

T.

Ultimate stresses sportsmanship and fair play. Competitive play is encouraged, but NEVER AT THE EXPENSE OF RESPECT BETWEEN PLAYERS, adherence to the rules and the BASIC JOY OF PLAY.

On Mar 25, 2015, at 04:03 PM, Ken Field <kfi...@bucknell.edu> wrote:

You need to run prep-reference first. That will create that file. 

Ken

On Mar 25, 2015, at 2:15 PM, Andrew Thompson <drew...@gmail.com> wrote:

I am trying to run SEM with a bowtie BAM file that I have created.  However I am getting the following error:

Cannot open /home/andrewwt/Trinity_paired_end_assembly/Trinity.fasta.RSEM.grp! It may not exist.

Error, cmd: rsem-calculate-expression  --paired-end   -p 4   --forward-prob 0 --no-bam-output --bam /home/andrewwt/bowtieD1/bowtie_out.coordSorted.bam /home/andrewwt/Trinity_paired_end_assembly/Trinity.fasta.RSEM RSEM  died with ret: 65280 at /c1/apps/trinity/trinityrnaseq-2.0.3/util/align_and_estimate_abundance.pl line 632.

What is the RSEM.grp file and why might I not have it?  I can't find it anywhere. 

Any help would be great!

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[Tiago Hori]

Yep. 

The script you used maps the reads individually and then find the pairs. RSEM uses bow tie in pair end-mode for paired data. 

I would advise you to use the align and estimate script, cause it will do both for you.

T.

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[Andrew Thompson]

So you suggest I don't run bowtie first?  Instead let the align_and_estimate_abundance.pl script do the mapping of reads? This will map reads that have mate pairs and those that do not?

[Tiago Hori]

Yep. Align and estimate will both the mapping and estimation using RSEM. In pair-end mode RSEM will ignore improper pairs for estimation even though the may be mapped.

T.

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[Andrew Thompson]

Good to know.  I have been confused on this.  Is there a way to take advantage of info in the improper pairs as far as abundance estimation?  For example, if the left or right is removed due to quality issues, the remaining read could still help with differential expression analyses if it is not thrown out by the software.  But RSEM is ignoring these improper pairs....

[Brian Haas]

You can always try running your reads as 'single' instead of 'paired' for the abundance estimation, and see how the results compare.  For those shorter transcripts/fragments that fail to recruit both pairs, you'll at least have expression values could be informative. Chances are, though, they're fragmented because the read depth is not sufficient, which means your power for expression and DE analysis with those transcripts is going to be lacking anyway.   

best,

~brian

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Brian J. Haas
The Broad Institute
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[Tiago Hori]

I would echo Brian's word of caution, this seams to me that is a question of the more or better realm. I think properly paired data is much more reliable data. Unless you know you are concerned with several lowly expressed transcripts, I am not sure the having more features "expressed" translates into having better data.

I work with fairly complex, duplicated genomes and I find that unpaired data is very ambiguous.

T.

Ultimate stresses sportsmanship and fair play. Competitive play is encouraged, but NEVER AT THE EXPENSE OF RESPECT BETWEEN PLAYERS, adherence to the rules and the BASIC JOY OF PLAY.