Hi there, I have trinity assembly for which i have to calculate isoform abundance, highest covered isoforms etc using RSEM. I used this following command perl $TRINITY_DIR/util/RSEM_util/run_RSEM_align_n_estimate.pl --transcripts <trinity output="" fasta="" file=""> --seqType fq --left <read1.cleaned.filtered> --right <read2.cleaned.filtered> --thread_count 4 --output_dir <outdir> --prefix <dataset id=""> by following this link https://bitbucket.org/yangya/optimize_assembler

but it gives error

bowtie -q --phred33-quals -n 2 -e 99999999 -l 25 -I 1 -X 1000 -p 4 -a -m 200 -S /media/madiha/800Gb/Madiha/softwares/RSEM-1.3.0/Trinity.31.fasta.TRANS -1 SMIX_1.fq -2 SMIX_2.fq | samtools view -S -b -o RSEM.temp/RSEM.bam - Could not locate a Bowtie index corresponding to basename "Trinity.31.fasta.TRANS" Command: bowtie --wrapper basic-0 -q --phred33-quals -n 2 -e 99999999 -l 25 -I 1 -X 1000 -p 4 -a -m 200 -S -1 SMIX_1.fq -2 SMIX_2.fq Trinity.31.fasta.TRANS

rsem-parse-alignments Trinity.31.fasta.TRANS RSEM.temp/RSEM RSEM.stat/RSEM RSEM.temp/RSEM.bam 3 -tag XM The SAM/BAM file declares less than one reference sequence!

although i have created all reference files snd index file successfully and have all output files and bam file according to this link http://deweylab.biostat.wisc.edu/rsem/rsem-prepare-reference.html

Any help or suggestion would be really appreciated Best Regards