align_and_estimate_abundance error Trinty

Question

[Princy]

Hello, I am trying to prepare a reference for alignment and abundance estimation. I have taken the transcriptome fasta file, do I need to use genomic fasta file or gtf file? I don't understand this point. Please guide me

I am using this code

perl /cabinfs/opt/applications/trinity/trinityrnaseq-Trinity-v2.5.1/util/align_and_estimate_abundance.pl \
 --transcripts /cabinfs/home/princy889/cucumber/trim/trinity_out_dir/Trinity.fasta \
 --est_method RSEM --aln_method bowtie2 --trinity_mode \
 --prep_reference /cabinfs/home/princy889/cucumber/trim/GCF_000004075.3_Cucumber_9930_V3_rna.fa

The error is

Error, don't understand arguments: /cabinfs/home/princy889/cucumber/trim/GCF_000004075.3_Cucumber_9930_V3_rna.fa  at /cabinfs/opt/applications/trinity/trinityrnaseq-Trinity-v2.5.1/util/align_and_estimate_abundance.pl line 293.

[andres.firrincieli]

Can you print the usage of align_and_estimate_abundance.pl

perl /cabinfs/opt/applications/trinity/trinityrnaseq-Trinity-v2.5.1/util/align_and_estimate_abundance.pl --help

[Princy]

Thanks! I correct my command according to manual

perl /cabinfs/opt/applications/trinity/trinityrnaseq-Trinity-v2.5.1/util/align_and_estimate_abundance.pl \ --transcripts /cabinfs/home/princy889/cucumber/trim/trinity_out_dir/Trinity.fasta \ --est_method RSEM --aln_method bowtie2 --trinity_mode --prep_reference

now this error is showing Only prepping reference. Stopping now.

where I am wrong pl let me know

[andres.firrincieli]

Does not look like an error. With that command line you just prepared the reference. Here the different usage of align_and_estimate_abundance.pl

## Just prepare the reference for alignment and abundance estimation
#
#    $0 --transcripts Trinity.fasta --est_method RSEM --aln_method bowtie --trinity_mode --prep_reference
#
#   ## Run the alignment and abundance estimation (assumes reference has already been prepped, errors-out if prepped reference not located.)
#
#    $0 --transcripts Trinity.fasta --seqType fq --left reads_1.fq --right reads_2.fq --est_method RSEM --aln_method bowtie --trinity_mode --output_dir rsem_outdir
#
##  ## prep the reference and run the alignment/estimation
#
#    $0 --transcripts Trinity.fasta --seqType fq --left reads_1.fq --right reads_2.fq --est_method RSEM --aln_method bowtie --trinity_mode --prep_reference --output_dir rsem_outdir
#
#   ## Use a samples.txt file:
#
#    $0 --transcripts Trinity.fasta --est_method RSEM --aln_method bowtie2 --prep_reference --trinity_mode --samples_file samples.txt --seqType fq  
#

[Princy]

yes, it's an error because the output file is empty.

[andres.firrincieli]

ok, right now I can't check this "error" on my computer but I will keep you updated. In the meanwhile:

output file is empty.

Are you refereeing to the index files?

Also, can you try the third command line?

--transcripts Trinity.fasta --seqType fq --left reads_1.fq --right reads_2.fq --est_method RSEM --aln_method bowtie --trinity_mode --prep_reference --output_dir rsem_outdir

[andres.firrincieli]

I double-checked and it is not an error! That command line is used only to prepare the index files:

Trinity.fasta.bowtie2.1.bt2
Trinity.fasta.bowtie2.2.bt2
Trinity.fasta.bowtie2.3.bt2
Trinity.fasta.bowtie2.4.bt2
Trinity.fasta.bowtie2.ok
Trinity.fasta.bowtie2.rev.1.bt2
Trinity.fasta.bowtie2.rev.2.bt2
Trinity.fasta.RSEM.grp
Trinity.fasta.RSEM.idx.fa
Trinity.fasta.RSEM.n2g.idx.fa
Trinity.fasta.RSEM.rsem.prepped.ok
Trinity.fasta.RSEM.seq
Trinity.fasta.RSEM.ti
Trinity.fasta.RSEM.transcripts.fa

[Princy]

ok, Thaks!