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4 months ago
Emily
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20
I've performed RNAseq alignment with STAR using --quantMode TranscriptomeSAM GeneCounts and obtained the Aligned.toTranscriptome.out.bam file.
I wanted to get the TPM of the transcripts using RSEM. I build the indice of RSEM using
rsem-prepare-reference --polyA-length 125 --gtf GCF_000002985.6_WBcel235_genomic.gtf GCF_000002985.6_WBcel235_genomic.fna rsem_ref
there is no error up to this step. However when I performed
rsem-calculate-expression --bam --no-bam-output -p 8 --paired-end --forward-prob 1 Aligned.toTranscriptome.out.bam rsem_ref outputRsem > OutputRSEM.log
it shows the error:
rsem-parse-alignments rsem_ref outputRsem.temp/outputRsem outputRsem.stat/outputRsem Aligned.toTranscriptome.out.bam 3 -tag XM
Warning: The SAM/BAM file declares less reference sequences (56716) than RSEM knows (56718)!
"rsem-parse-alignments rsem_ref outputRsem.temp/outputRsem outputRsem.stat/outputRsem Aligned.toTranscriptome.out.bam 3 -tag XM" failed! Plase check if you provide correct parameters/options for the pipeline!
Pls Help!
There is a mismatch between rsem reference and reference used for star, the difference is two transcripts. Check that references are the same.
thanks! not sure how this warning caused as I used the same gtf and fna for building STAR alginer indice, maybe I could just avoid this warning?
Sorry I have another error popped up: RSEM's indices might be corrupted, unassigned_transcript_572 appears more than once!
do you have any idea on this or if there's other method that could calculate TPM from STAR output bam file, pls kindly advice me.
I realised theres lines (approximately 3000 of them) showing unassigned transcript such as this one in my RSEM indice transcript.fa file