rsem-calculate-expresion returns "The SAM/BAM file declares less than one reference sequence!"

[Fernando J.]

Hi! 

I am trying to replicate an experiment using RSEM to perform RNA analysis (https://www.nature.com/articles/nature16965). Basically, they use Ensembl rel.70 to do it. Si I downloaded both files:

• ftp://ftp.ensembl.org/pub/release-70/gtf/homo_sapiens/Homo_sapiens.GRCh37.70.gtf.gz
• ftp://ftp.ensembl.org/pub/release-70/fasta/homo_sapiens/dna/Homo_sapiens.GRCh37.70.dna.primary_assembly.fa.gz
• o unzip both files.

Unzip the gz:

• gzip -d *.gz  Then:

Prepare the reference:

• rsem-prepare-reference --gtf Homo_sapiens.GRCh37.70.gtf --bowtie Homo_sapiens.GRCh37.70.dna.primary_assembly.fa ref/human_ensembl

Then I tried to run RSEM to calculate de expression (In a loop across 100 paired samples):

• rsem-calculate-expression -p 60 --paired-end sample01_R1.fq.gz sample01_R2.fq.gz /RSEM_enseml70/ensembl_70/ref/human_ensembl ./rsem_calc_expr/sample01

I get the following error:

bowtie -q --phred33-quals -n 2 -e 99999999 -l 25 -I 1 -X 1000 -p 60 -a -m 200 -S /RSEM_enseml70/ensembl_70/ref/human_ensembl -1 sample01_R1.fq.gz -2 sample01_R2.fq.gz 2> ./rsem_calc_expr/sample01.log | samtools view -b -o ./rsem_calc_expr/sample01.temp/sample01.bam -

rsem-parse-alignments /RSEM_enseml70/ensembl_70/ref/human_ensembl ./rsem_calc_expr/sample01.temp/sample01 ./rsem_calc_expr/sample01.stat/sample01 ./rsem_calc_expr/sample01.temp/sample01.bam 3 -tag XM

The SAM/BAM file declares less than one reference sequence!

"rsem-parse-alignments /RSEM_enseml70/ensembl_70/ref/human_ensembl ./rsem_calc_expr/sample01.temp/sample01 ./rsem_calc_expr/sample01.stat/PDAC_001 ./rsem_calc_expr/sample01.temp/sample01.bam 3 -tag XM" failed! Plase check if you provide correct parameters/options for the pipeline!

In the sake of clarity.... I provide the output (nohup.out text file) from the rsem-prepare-reference.

I tried different thing but not sure what is going on... 

Any help will be very welcomed

Kind regards! And thank you!!

Fer