Hi,
I have assembled RNA-Seq data do novo in Trinity, and am now trying to estimate the abundance of the transcripts that were identified. I used TopHat to align the RNA-Seq fastq reads against the Trinity-generated fasta file of transcripts.
All attempts to use the align_and_estimate_abundance.pl script that is packaged with Trinity have failed, however.
My input code is:
sampleName.Trinity.fasta was generated in Trinity, and accepted_hits.bam generated in TopHat
I always get the following error message:
I have tried using --alignment_method bowtie2 instead of accepted_hits.bam but got the same error message.
Replace the --seqType flag from fq to fa changed the result -t 3 to -t 2 in the output, but otherwise produced the same error.
Any suggestions as to why align_and_estimate_abundance.pl does not seem to be working correctly?
I have assembled RNA-Seq data do novo in Trinity, and am now trying to estimate the abundance of the transcripts that were identified. I used TopHat to align the RNA-Seq fastq reads against the Trinity-generated fasta file of transcripts.
All attempts to use the align_and_estimate_abundance.pl script that is packaged with Trinity have failed, however.
My input code is:
/usr/local/trinity/util/align_and_estimate_abundance.pl \
--transcripts "sampleName.Trinity.fasta" \
--seqType fq \
--left "/filepath/reads.1.fastq" \
--right "/filepath/reads.2.fastq" \
--est_method RSEM \
--aln_method accepted_hits.bam \
--thread_count 1 \
--trinity_mode \
--fragment_length 180 \
--output_dir "./" \
--output_prefix "S1"
--transcripts "sampleName.Trinity.fasta" \
--seqType fq \
--left "/filepath/reads.1.fastq" \
--right "/filepath/reads.2.fastq" \
--est_method RSEM \
--aln_method accepted_hits.bam \
--thread_count 1 \
--trinity_mode \
--fragment_length 180 \
--output_dir "./" \
--output_prefix "S1"
sampleName.Trinity.fasta was generated in Trinity, and accepted_hits.bam generated in TopHat
I always get the following error message:
CMD: rsem-calculate-expression --paired-end -p 4 --bam accepted_hits.bam /filepath/S1_RSEM/CB1_S1_L001.Trinity.fasta.RSEM S1
/usr/local/bin/rsem-parse-alignments /filepath/S1_RSEM/CB1_S1_L001.Trinity.fasta.RSEM S1.temp/S1 S1.stat/S1 b accepted_hits.bam -t 3 -tag XM
RSEM does not support gapped alignments, sorry!
"/usr/local/bin/rsem-parse-alignments /filepath/S1_RSEM/CB1_S1_L001.Trinity.fasta.RSEM S1.temp/S1 S1.stat/S1 b accepted_hits.bam -t 3 -tag XM" failed! Plase check if you provide correct parameters/options for the pipeline!
Error, cmd: rsem-calculate-expression --paired-end -p 4 --bam accepted_hits.bam /filepath/S1_RSEM/CB1_S1_L001.Trinity.fasta.RSEM S1 died with ret: 65280 at /usr/local/trinity/util/align_and_estimate_abundance.pl line 620
/usr/local/bin/rsem-parse-alignments /filepath/S1_RSEM/CB1_S1_L001.Trinity.fasta.RSEM S1.temp/S1 S1.stat/S1 b accepted_hits.bam -t 3 -tag XM
RSEM does not support gapped alignments, sorry!
"/usr/local/bin/rsem-parse-alignments /filepath/S1_RSEM/CB1_S1_L001.Trinity.fasta.RSEM S1.temp/S1 S1.stat/S1 b accepted_hits.bam -t 3 -tag XM" failed! Plase check if you provide correct parameters/options for the pipeline!
Error, cmd: rsem-calculate-expression --paired-end -p 4 --bam accepted_hits.bam /filepath/S1_RSEM/CB1_S1_L001.Trinity.fasta.RSEM S1 died with ret: 65280 at /usr/local/trinity/util/align_and_estimate_abundance.pl line 620
I have tried using --alignment_method bowtie2 instead of accepted_hits.bam but got the same error message.
Replace the --seqType flag from fq to fa changed the result -t 3 to -t 2 in the output, but otherwise produced the same error.
Any suggestions as to why align_and_estimate_abundance.pl does not seem to be working correctly?
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