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Trinity transcript_abundance.pl error using RSEM (65280 at line 766)

Hi I am trying to estimate transcript abundance from a transcriptome generated from paired-end RNA-seq data using Trinity. However, I am running into a consistent error while using the-

perl align_and_estimate_abundance.pl --seqType fastq --left /home/nbri/nasreen/qf_leaf_1.fastq  --right /home/nbri/nasreen/qf_leaf_2.fastq  --transcripts /home/nbri/nasreen/Trinity_a.fasta --output_prefix RSEM_clr_mod --est_method RSEM --aln_method bowtie --trinity_mode --prep_reference --output_dir /home/nbri/nasreen/RSEM_leaf_1

The error is as follows:

CMD: touch /home/nbri/nasreen/Trinity_a.fasta.RSEM.rsem.prepped.started
CMD: rsem-prepare-reference  --transcript-to-gene-map /home/nbri/nasreen/Trinity_a.fasta.gene_trans_map /home/nbri/nasreen/Trinity_a.fasta /home/nbri/nasreen/Trinity_a.fasta.RSEM
rsem-synthesis-reference-transcripts /home/nbri/nasreen/Trinity_a.fasta.RSEM 0 1 /home/nbri/nasreen/Trinity_a.fasta.gene_trans_map /home/nbri/nasreen/Trinity_a.fasta
Sequence TRINITY_DN49245_c0_g1_i5 contains an unknown letter (ASCII code 13) at 0-based position 60!
"rsem-synthesis-reference-transcripts /home/nbri/nasreen/Trinity_a.fasta.RSEM 0 1 /home/nbri/nasreen/Trinity_a.fasta.gene_trans_map /home/nbri/nasreen/Trinity_a.fasta" failed! Plase check if you provide correct parameters/options for the pipeline!
Error, cmd: rsem-prepare-reference  --transcript-to-gene-map /home/nbri/nasreen/Trinity_a.fasta.gene_trans_map /home/nbri/nasreen/Trinity_a.fasta /home/nbri/nasreen/Trinity_a.fasta.RSEM died with ret: 65280 at align_and_estimate_abundance.pl line 766.

I am relatively new in the bioinformatics field and would greatly appreciate any insight on what might be going wrong.

Thanks!

Nasreen

rna-seq assembly sequencing next-gen rna-seq
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updated by h.mon h.mon • written by nasreenbano91 • nasreenbano91

1 answer

ASCII code 13 is the code for carriage return, which is not usually used in the unix world, and RSEM apparently abhors it. Did you edit your fasta file on windows? To fix it, use dos2unix, or use this perl one-liner:

perl -p -i -e 's/\r\n$/\n/' Trinity_a.fasta
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written by h.mon h.mon

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