I have prepared reference using the command:

rsem-prepare-reference -p 5 --bowtie2 cd_hit_est.fasta ref_output_12

And the files i obtained are:

ref_12.1.bt2

ref_12.2.bt2

ref_12.3.bt2

ref_12.4.bt2

ref_12.grp

ref_12.idx.fa

ref_12.n2g.idx.fa

ref_12.rev.1.bt2

ref_12.rev.2.bt2

ref_12.seq

ref_12.ti

ref_12.transcripts.fa

then i tried running the command:

rsem-calculate-expression -p 5 --bowtie2 --ci-memory 5000 --paired-end SRR5183402_1.fastq SRR5183402_2.fastq ref_output_12 SSE_fm

and this is the error I am getting:

bowtie2 -q --phred33 --sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.1 -I 1 -X 1000 --no-mixed --no-discordant -p 5 -k 200 -x ref_output_12 -1 SRR5183402_1.fastq -2 SRR5183402_2.fastq 2> SSE_fm.log | samtools view -b -o SSE_fm.temp/SSE_fm.bam - [main_samview] fail to read the header from "-". "bowtie2 -q --phred33 --sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.1 -I 1 -X 1000 --no-mixed --no-discordant -p 5 -k 200 -x ref_output_12 -1 SRR5183402_1.fastq -2 SRR5183402_2.fastq 2> SSE_fm.log | samtools view -b -o SSE_fm.temp/SSE_fm.bam -" failed! Plase check if you provide correct parameters/options for the pipeline!

Previosly I had used trinity output, but i got same error. This time i am using clustered data, still the same error.

I have used bowtie2 option while creating both reference and rsem-calculate-expression.

Any help on this would be highly appreciated. Thank you very much.

Best regards,

Bhoomika