rsem-prep_reference error

[Shenu Hudson]

Hello trinity users ,

I’m working on a plant pathogen (control and treated sample). I already completed my assembly using trinity then I found that my assembled data contains plant genes. Then I filtered out plant genes and tried to do the differential expression analysis using RSEM and EdgeR. Then I got the below error 

command line:

~/trinityrnaseq-Trinity-v2.8.3/util/align_and_estimate_abundance.pl --transcripts trinity_filtered.fasta --seqType fq --left PCCMS_1_paired.fastq --right PCCMS_2_paired.fastq --est_method RSEM --aln_method bowtie2 --trinity_mode  --prep_reference --output_dir rsem_outdir_control

Total time for backward call to driver() for mirror index: 00:02:49
CMD: touch /state/partition1/rani/trinity_filtered.fasta.RSEM.rsem.prepped.started
CMD: rsem-prepare-reference  --transcript-to-gene-map /state/partition1/rani/trinity_filtered.fasta.gene_trans_map /state/partition1/rani/trinity_filtered.fasta /state/partition1/rani/trinity_filtered.fasta.RSEM
rsem-synthesis-reference-transcripts /state/partition1/rani/trinity_filtered.fasta.RSEM 0 1 /state/partition1/rani/trinity_filtered.fasta.gene_trans_map /state/partition1/rani/trinity_filtered.fasta
 (ASCII code 13), at line 2, position 81!_filtered.fasta contains an unknown character,
"rsem-synthesis-reference-transcripts /state/partition1/rani/trinity_filtered.fasta.RSEM 0 1 /state/partition1/rani/trinity_filtered.fasta.gene_trans_map /state/partition1/rani/trinity_filtered.fasta" failed! Plase check if you provide correct parameters/options for the pipeline!
Error, cmd: rsem-prepare-reference  --transcript-to-gene-map /state/partition1/rani/trinity_filtered.fasta.gene_trans_map /state/partition1/rani/trinity_filtered.fasta /state/partition1/rani/trinity_filtered.fasta.RSEM died with ret: 65280 at /share/apps/trinityrnaseq-Trinity-v2.8.3/util/align_and_estimate_abundance.pl line 790.

i checked at line 2 and there is no unknown character in that line.

I would appreciate any help :)

[Mark Chapman]

Hi Shenu,

Could it be a hidden character? try sed -n 'l' myfile (or sed -n 'l' myfile | head -n 2 if the file is big)

you should see \t for a tab and $ for a linebreak

and try it on your pre-edited file too to see what the original format was)

cheers, Mark

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Dr. Mark A. Chapman

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[Shenu Hudson]

Hi Mark,

Thank you so much for your reply

i tried sed command as you said and this is what i got... could you please check and let me know what you think

sed -n 'l' trinity_filtered.fasta | head -n 2

>TRINITY_DN27143_c0_g1_i1 len=867 path=[0:0-866]\r$

cccttcttctgtccctttattccatccctctccctttactttgtcaccacccacaaatacaaaagagca\

sed -n 'l' Trinity.fasta | head -n 2
>TRINITY_DN27143_c0_g1_i1 len=867 path=[0:0-866]\r$
CCCTTCTTCTGTCCCTTTATTCCATCCCTCTCCCTTTACTTTGTCACCACCCACAAATACAAAAGAGCA\

[Mark Chapman]

Hi Shenu,

Nothing obvious, especially as both files have the same characters. The error was allegedly at position 81 but there are only 69 characters and a carriage return. Can you run it with head (ie without the '-n 2'). 

Also, What happens if you run your command with the unfiltered fasta (~/trinityrnaseq-Trinity-v2.8.3/util/align_and_estimate_abundance.pl --transcripts Trinity.fasta etc etc)? does it work?

Cheers, Mark

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[Shenu Hudson]

Hi Mark,

Sorry for late reply...

Thank you for pointing out about position 81. you were right i found a line break character .

I removed all the line break character and then ran the command for RSEM. still i'm getting same sort of error

Error:

Total time for backward call to driver() for mirror index: 00:00:27

CMD: touch /state/partition1/rani/trinity_filtered.fasta.RSEM.rsem.prepped.started
CMD: rsem-prepare-reference  --transcript-to-gene-map /state/partition1/rani/trinity_filtered.fasta.gene_trans_map /state/partition1/rani/trinity_filtered.fasta /state/partition1/rani/trinity_filtered.fasta.RSEM
rsem-synthesis-reference-transcripts /state/partition1/rani/trinity_filtered.fasta.RSEM 0 1 /state/partition1/rani/trinity_filtered.fasta.gene_trans_map /state/partition1/rani/trinity_filtered.fasta

 (ASCII code 13), at line 2, position 868!filtered.fasta contains an unknown character,

"rsem-synthesis-reference-transcripts /state/partition1/rani/trinity_filtered.fasta.RSEM 0 1 /state/partition1/rani/trinity_filtered.fasta.gene_trans_map /state/partition1/rani/trinity_filtered.fasta" failed! Plase check if you provide correct parameters/options for the pipeline!
Error, cmd: rsem-prepare-reference  --transcript-to-gene-map /state/partition1/rani/trinity_filtered.fasta.gene_trans_map /state/partition1/rani/trinity_filtered.fasta /state/partition1/rani/trinity_filtered.fasta.RSEM died with ret: 65280 at /share/apps/trinityrnaseq-Trinity-v2.8.3/util/align_and_estimate_abundance.pl line 790.

then i checked at line 2, position 868

sed -n 'l' trinity_filtered.fasta | head -n 14

>TRINITY_DN27143_c0_g1_i1 len=867 path=[0:0-866]\r$
cccttcttctgtccctttattccatccctctccctttactttgtcaccacccacaaatacaaaagagca\

tgcgaacccgatgatcggtagccttcagtttgggtaataacctgtccagcatttctaacttcccacata\
gcctaactattgtaggcagataatgtctgggtactaaggtttcaacctcatcggcatgaagctgactta\
gatatgggtgattgcaaatattccggagttccatcacagtgttatgaactgagcgacccttaacattac\
caatggatccaagattatcctccactcttttcatcaaaagcttctgataagcagacgcttcacatctga\
taagcctttcaattttttcaggcaattgatgttcgaccttgtgtttcagccttcttaggacaaatggcc\
gaagaacttgatgaagtcggtttataatcaacagattctcctcctctgatagtagagcttcttcaggtg\
aactatcaccattactctcaaacggtttattgaaccattgagaaaaatcctcagatgaattaaaaatgt\
taggcagcaagaagttaagcaaggcccagagctcctctaaattattctgaagcggagttccagtcaaga\
gcaagcgatgggaactttgataaagcttcaagtcagcgttcagcttacatgaggcattctttattcgat\
gcccctcgtctataataatgtaatgccagagaattttgctcagtttaggacgatcatgtttgttcatta\
aatattcataagtagtcaaaaggacattgaatttctgatgaacaatcctttccttgaataatttgcgcc\
tttcttctggaggacctgaatatgtaatcttattcacac\r$

>TRINITY_DN27196_c0_g1_i1 len=518 path=[0:0-517]\r$
cctggccacttttatgccagaggagcctggaagatttaaacaatttgcagagcaagtaatttccctttc\

 sed -n 'l' Trinity.fasta | head -n 14

>TRINITY_DN27143_c0_g1_i1 len=867 path=[0:0-866]\r$
CCCTTCTTCTGTCCCTTTATTCCATCCCTCTCCCTTTACTTTGTCACCACCCACAAATACAAAAGAGCA\

TGCGAACCCGATGATCGGTAGCCTTCAGTTTGGGTAATAACCTGTCCAGCATTTCTAACTTCCCACATA\
GCCTAACTATTGTAGGCAGATAATGTCTGGGTACTAAGGTTTCAACCTCATCGGCATGAAGCTGACTTA\
GATATGGGTGATTGCAAATATTCCGGAGTTCCATCACAGTGTTATGAACTGAGCGACCCTTAACATTAC\
CAATGGATCCAAGATTATCCTCCACTCTTTTCATCAAAAGCTTCTGATAAGCAGACGCTTCACATCTGA\
TAAGCCTTTCAATTTTTTCAGGCAATTGATGTTCGACCTTGTGTTTCAGCCTTCTTAGGACAAATGGCC\
GAAGAACTTGATGAAGTCGGTTTATAATCAACAGATTCTCCTCCTCTGATAGTAGAGCTTCTTCAGGTG\
AACTATCACCATTACTCTCAAACGGTTTATTGAACCATTGAGAAAAATCCTCAGATGAATTAAAAATGT\
TAGGCAGCAAGAAGTTAAGCAAGGCCCAGAGCTCCTCTAAATTATTCTGAAGCGGAGTTCCAGTCAAGA\
GCAAGCGATGGGAACTTTGATAAAGCTTCAAGTCAGCGTTCAGCTTACATGAGGCATTCTTTATTCGAT\
GCCCCTCGTCTATAATAATGTAATGCCAGAGAATTTTGCTCAGTTTAGGACGATCATGTTTGTTCATTA\
AATATTCATAAGTAGTCAAAAGGACATTGAATTTCTGATGAACAATCCTTTCCTTGAATAATTTGCGCC\
TTTCTTCTGGAGGACCTGAATATGTAATCTTATTCACAC\r$

>TRINITY_DN27196_c0_g1_i1 len=518 path=[0:0-517]\r$
CCTGGCCACTTTTATGCCAGAGGAGCCTGGAAGATTTAAACAATTTGCAGAGCAAGTAATTTCCCTTTC\

[Shenu Hudson]

It worked with unfiltered data

[Mark Chapman]

Hi Shenu,

Sorry for the late reply. Looks like there is a line break between contigs in the first fasta and not in your filtered fasta using sed. Not really sure why this is but I guess it means the line breaks have been modified in your file unfortunately. You might be able to fix this in notepad++, but it's hard to say exactly what has happened, sorry.

Cheers, Mark

On Thu, 8 Aug 2019, 08:00 Shenu Hudson, <shenuhu...@gmail.com> wrote:

It worked with unfiltered data

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