Hello Community,

I am trying to to run rsem-calculate-expression using STAR to aligned reads.

the command that i use is:

for preparing the reference

rsem-prepare-reference --gtf GENOME_data/Homo_sapiens.GRCh38.90.gtf --star GENOME_data/Homo_sapiens.GRCh38.dna.genome.fa --star-path /users/data/applications/STAR-2.5.3a/bin/ -p 28 GENOME_data/rsem_ref

for Quantification with RSEM

rsem-calculate-expression --p 28 --star --star-gzipped-file --star-path /users/data/applications/STAR-2.5.3a/bin --paired-end --seed 5 --output-genome-bam --sort-bam-by-read-name --forward-prob=1.0 ../R1.read1.fastq.gz ../R1.read2.fastq.gz GENOME_data/rsem_ref/ RNASEQ_data/R1

Now i get this error:

Read A00182:360:HJM25DRXX:1:1102:8703:35728: the two mates do not align to the same transcript. RSEM does not support discordant alignments.

"rsem-parse-alignments GENOME_data/rsem-ref RNASEQ_data/R1.temp/R1 RNASEQ_data/R1.stat RNASEQ_data/R1.temp/R1.sorted.bam 3 -t ag XM" failed. Please check if you provide correct parameters/options for the pipeline

Any help on this is highly aprreciated. Thank you very much.

Abhilash Kannan