Re: rsem-calculate-expression: RSEM can not recognize reference sequence name 1! SAM/BAM file declares less reference sequences (194) than RSEM knows (196345)!

[Michalina Kosiorek]

hi guys!

sorry for bothering you! I forgot that I should use the files from  --QUANTMODE  from STAR. and it works very well!

:)

Michalina

W dniu sobota, 23 maja 2015 13:34:57 UTC+2 użytkownik Michalina Kosiorek napisał:

Dear All, 

I am new to RSEM, I have perfomed alignment by STAR and I have checked my bam files with RSEM and are valid! 

my command is:

./rsem-calculate-expression -p 24 --paired-end --bam /media/michalina/DC96D00796CFE05E/BAM_FINAL/TS.bam /media/michalina/DC96D00796CFE05E/REFSEQ_GENOMES/RSEM_refseq_38_GTF_bowtie/RSEM_ref38_gtf_bowtie /media/michalina/DC96D00796CFE05E/RSEM/TS_quals38_gtf_bowtie

and the exit from the above is that:

rsem-parse-alignments /media/michalina/DC96D00796CFE05E/REFSEQ_GENOMES/RSEM_refseq_38_GTF_bowtie/RSEM_ref38_gtf_bowtie /media/michalina/DC96D00796CFE05E/RSEM/TS_quals38_gtf_bowtie.temp/TS_quals38_gtf_bowtie /media/michalina/DC96D00796CFE05E/RSEM/TS_quals38_gtf_bowtie.stat/TS_quals38_gtf_bowtie b /media/michalina/DC96D00796CFE05E/BAM_FINAL/TS.bam -t 3 -tag XM

Warning: The SAM/BAM file declares less reference sequences (194) than RSEM knows (196345)!

RSEM can not recognize reference sequence name 1!

"rsem-parse-alignments /media/michalina/DC96D00796CFE05E/REFSEQ_GENOMES/RSEM_refseq_38_GTF_bowtie/RSEM_ref38_gtf_bowtie /media/michalina/DC96D00796CFE05E/RSEM/TS_quals38_gtf_bowtie.temp/TS_quals38_gtf_bowtie /media/michalina/DC96D00796CFE05E/RSEM/TS_quals38_gtf_bowtie.stat/TS_quals38_gtf_bowtie b /media/michalina/DC96D00796CFE05E/BAM_FINAL/TS.bam -t 3 -tag XM" failed! Plase check if you provide correct parameters/options for the pipeline!

The alignements with STAR I did to the same genome.fasta amd genes.gtf file that I used for RSEM reference preparation. The organism is human GRCh38.

the code for rsem reference preparation was:

./rsem-prepare-reference --gtf /media/michalina/DC96D00796CFE05E/REFSEQ_GENOMES/RSEM_refseq_38_GTF/genes.gtf --bowtie-path /usr/bin/bowtie /media/michalina/DC96D00796CFE05E/REFSEQ_GENOMES/RSEM_refseq_38_GTF/genome.fasta /media/michalina/DC96D00796CFE05E/REFSEQ_GENOMES/RSEM_refseq_38_GTF_bowtie/RSEM_ref38_gtf_bowtie

and it was generated correctlky I guess.. as below:

./rsem-prepare-reference --gtf /media/michalina/DC96D00796CFE05E/REFSEQ_GENOMES/RSEM_refseq_38_GTF/genes.gtf --bowtie-path /usr/bin/bowtie /media/michalina/DC96D00796CFE05E/REFSEQ_GENOMES/RSEM_refseq_38_GTF/genome.fasta /media/michalina/DC96D00796CFE05E/REFSEQ_GENOMES/RSEM_refseq_38_GTF_bowtie/RSEM_ref38_gtf_bowtie

Warning: If Bowtie is not used, no need to set --bowtie-path option!

rsem-extract-reference-transcripts /media/michalina/DC96D00796CFE05E/REFSEQ_GENOMES/RSEM_refseq_38_GTF_bowtie/RSEM_ref38_gtf_bowtie 0 /media/michalina/DC96D00796CFE05E/REFSEQ_GENOMES/RSEM_refseq_38_GTF/genes.gtf 0 /media/michalina/DC96D00796CFE05E/REFSEQ_GENOMES/RSEM_refseq_38_GTF/genome.fasta

Parsed 200000 lines

Parsed 400000 lines

Parsed 600000 lines

Parsed 800000 lines

Parsed 1000000 lines

Parsed 1200000 lines

Parsed 1400000 lines

Parsed 1600000 lines

Parsed 1800000 lines

Parsing gtf File is done!

/media/michalina/DC96D00796CFE05E/REFSEQ_GENOMES/RSEM_refseq_38_GTF/genome.fasta is processed!

196345 transcripts are extracted and 0 transcripts are omitted.

Extracting sequences is done!

Group File is generated!

Transcript Information File is generated!

Chromosome List File is generated!

Extracted Sequences File is generated!

rsem-preref /media/michalina/DC96D00796CFE05E/REFSEQ_GENOMES/RSEM_refseq_38_GTF_bowtie/RSEM_ref38_gtf_bowtie.transcripts.fa 1 /media/michalina/DC96D00796CFE05E/REFSEQ_GENOMES/RSEM_refseq_38_GTF_bowtie/RSEM_ref38_gtf_bowtie -l 125

Refs.makeRefs finished!

Refs.saveRefs finished!

/media/michalina/DC96D00796CFE05E/REFSEQ_GENOMES/RSEM_refseq_38_GTF_bowtie/RSEM_ref38_gtf_bowtie.idx.fa is generated!

/media/michalina/DC96D00796CFE05E/REFSEQ_GENOMES/RSEM_refseq_38_GTF_bowtie/RSEM_ref38_gtf_bowtie.n2g.idx.fa is generated!

I dont understand what is wrong with my rsem-calculate-expression. 

Could you help me with that?

Michalina

[Małgorzata Komór]

Hi Michalina!

What do you mean by STAR -quantmode ?

Is it possible to run RSEM on gene counts?

Thanks,
Gosia

[Bo Li]

Hi Gosia,

I think she refers to the STAR aligner's --quantMode option. See STAR
manual
(https://github.com/alexdobin/STAR/blob/master/doc/STARmanual.pdf) for
more details.

Best,
Bo

>>> WARNING: THE SAM/BAM FILE DECLARES LESS REFERENCE SEQUENCES (194)
>>> THAN RSEM KNOWS (196345)!
>>> RSEM CAN NOT RECOGNIZE REFERENCE SEQUENCE NAME 1!

> /media/michaldina/DC96D00796CFE05E/REFSEQ_GENOMES/RSEM_refseq_38_GTF_bowtie/RSEM_ref38_gtf_bowtie

>>> -l 125
>>> Refs.makeRefs finished!
>>> Refs.saveRefs finished!
>>>
>>
> /media/michalina/DC96D00796CFE05E/REFSEQ_GENOMES/RSEM_refseq_38_GTF_bowtie/RSEM_ref38_gtf_bowtie.idx.fa
>>> is generated!
>>>
>>
> /media/michalina/DC96D00796CFE05E/REFSEQ_GENOMES/RSEM_refseq_38_GTF_bowtie/RSEM_ref38_gtf_bowtie.n2g.idx.fa
>>> is generated!
>>>
>>> I dont understand what is wrong with my
>>> rsem-calculate-expression. 
>>> Could you help me with that?
>>> Michalina
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