Hey all, i've just run blastx against my transcriptome and am trying to generate the TPM's using Trinity. Keep getting stuck with this error, and am not quite sure why. Any help would be greatly appreciated...
[lcharala@titan17 util]$ ./align_and_estimate_abundance.pl --transcripts Pseudoneoponera-Trinity.fasta --seqType fq --left RFA006Pseudoneoponerafilteredreads_1P.fastq --right RFA006Pseudoneoponerafilteredreads_2P.fastq --est_method RSEM --aln_method bowtie --trinity_mode --prep_reference --output_dir PseudoOutput1 --thread_count 20
$VAR1 = [
{
'left' => '/home/lcharala/Desktop/trinityrnaseq-Trinity-v2.4.0/util/RFA006Pseudoneoponerafilteredreads_1P.fastq',
'right' => '/home/lcharala/Desktop/trinityrnaseq-Trinity-v2.4.0/util/RFA006Pseudoneoponerafilteredreads_2P.fastq',
'output_dir' => 'PseudoOutput1'
}
];
CMD: set -o pipefail && bowtie -q --all --best --strata -m 300 --chunkmbs 512 -X 800 -S -p 20 /home/lcharala/Desktop/trinityrnaseq-Trinity-v2.4.0/util/Pseudoneoponera-Trinity.fasta.bowtie -1 /home/lcharala/Desktop/trinityrnaseq-Trinity-v2.4.0/util/RFA006Pseudoneoponerafilteredreads_1P.fastq -2 /home/lcharala/Desktop/trinityrnaseq-Trinity-v2.4.0/util/RFA006Pseudoneoponerafilteredreads_2P.fastq | samtools view -F 4 -S -b -o bowtie.bam -
[samopen] SAM header is present: 230007 sequences.
# reads processed: 551750603
# reads with at least one reported alignment: 542669028 (98.35%)
# reads that failed to align: 9081575 (1.65%)
Reported 271334514 paired-end alignments to 1 output stream(s)
CMD: touch bowtie.bam.ok
CMD: rsem-calculate-expression --paired-end -p 20 --no-bam-output --bam bowtie.bam /home/lcharala/Desktop/trinityrnaseq-Trinity-v2.4.0/util/Pseudoneoponera-Trinity.fasta.RSEM RSEM
rsem-parse-alignments /home/lcharala/Desktop/trinityrnaseq-Trinity-v2.4.0/util/Pseudoneoponera-Trinity.fasta.RSEM RSEM.temp/RSEM RSEM.stat/RSEM bowtie.bam 3 -tag XM
Read K00115:209:H7G7JBBXX:3:1101:6938:1209: The adjacent two lines do not represent the two mates of a paired-end read! (RSEM assumes the two mates of a paired-end read should be adjacent)
"rsem-parse-alignments /home/lcharala/Desktop/trinityrnaseq-Trinity-v2.4.0/util/Pseudoneoponera-Trinity.fasta.RSEM RSEM.temp/RSEM RSEM.stat/RSEM bowtie.bam 3 -tag XM" failed! Plase check if you provide correct parameters/options for the pipeline!
Error, cmd: rsem-calculate-expression --paired-end -p 20 --no-bam-output --bam bowtie.bam /home/lcharala/Desktop/trinityrnaseq-Trinity-v2.4.0/util/Pseudoneoponera-Trinity.fasta.RSEM RSEM died with ret: 65280 at ./align_and_estimate_abundance.pl line 766.
[lcharala@titan17 util]$ ./align_and_estimate_abundance.pl --transcripts Pseudoneoponera-Trinity.fasta --seqType fq --left RFA006Pseudoneoponerafilteredreads_1P.fastq --right RFA006Pseudoneoponerafilteredreads_2P.fastq --est_method RSEM --aln_method bowtie --trinity_mode --prep_reference --output_dir PseudoOutput1 --thread_count 20
$VAR1 = [
{
'left' => '/home/lcharala/Desktop/trinityrnaseq-Trinity-v2.4.0/util/RFA006Pseudoneoponerafilteredreads_1P.fastq',
'right' => '/home/lcharala/Desktop/trinityrnaseq-Trinity-v2.4.0/util/RFA006Pseudoneoponerafilteredreads_2P.fastq',
'output_dir' => 'PseudoOutput1'
}
];
CMD: set -o pipefail && bowtie -q --all --best --strata -m 300 --chunkmbs 512 -X 800 -S -p 20 /home/lcharala/Desktop/trinityrnaseq-Trinity-v2.4.0/util/Pseudoneoponera-Trinity.fasta.bowtie -1 /home/lcharala/Desktop/trinityrnaseq-Trinity-v2.4.0/util/RFA006Pseudoneoponerafilteredreads_1P.fastq -2 /home/lcharala/Desktop/trinityrnaseq-Trinity-v2.4.0/util/RFA006Pseudoneoponerafilteredreads_2P.fastq | samtools view -F 4 -S -b -o bowtie.bam -
[samopen] SAM header is present: 230007 sequences.
# reads processed: 551750603
# reads with at least one reported alignment: 542669028 (98.35%)
# reads that failed to align: 9081575 (1.65%)
Reported 271334514 paired-end alignments to 1 output stream(s)
CMD: touch bowtie.bam.ok
CMD: rsem-calculate-expression --paired-end -p 20 --no-bam-output --bam bowtie.bam /home/lcharala/Desktop/trinityrnaseq-Trinity-v2.4.0/util/Pseudoneoponera-Trinity.fasta.RSEM RSEM
rsem-parse-alignments /home/lcharala/Desktop/trinityrnaseq-Trinity-v2.4.0/util/Pseudoneoponera-Trinity.fasta.RSEM RSEM.temp/RSEM RSEM.stat/RSEM bowtie.bam 3 -tag XM
Read K00115:209:H7G7JBBXX:3:1101:6938:1209: The adjacent two lines do not represent the two mates of a paired-end read! (RSEM assumes the two mates of a paired-end read should be adjacent)
"rsem-parse-alignments /home/lcharala/Desktop/trinityrnaseq-Trinity-v2.4.0/util/Pseudoneoponera-Trinity.fasta.RSEM RSEM.temp/RSEM RSEM.stat/RSEM bowtie.bam 3 -tag XM" failed! Plase check if you provide correct parameters/options for the pipeline!
Error, cmd: rsem-calculate-expression --paired-end -p 20 --no-bam-output --bam bowtie.bam /home/lcharala/Desktop/trinityrnaseq-Trinity-v2.4.0/util/Pseudoneoponera-Trinity.fasta.RSEM RSEM died with ret: 65280 at ./align_and_estimate_abundance.pl line 766.