align_and_estimate_abundance.pl ERROR/QUESTION

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Nikelle Petrillo

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Jan 27, 2016, 1:58:12 PM1/27/16
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Hello. I am new to RNA seq. I have a trinity.fast file I would like to align reads to. I believe I am supposed to be using the align_and_estimate_abundance.pl command. However, when i input my arguments. I get warnings and error back. I am not sure what to make of them. Does anyone have any advice? Thanks for your help. - Nikelle 


[npetrill@trogdor util]$ perl align_and_estimate_abundance.pl --transcripts /home/richardsonlab/AMMA_transcripts/trinity_out_dir/Trinity.fasta --seqType fq --left R1V22_B9F8St_ACTTGA_L002_R1_001.qualitytrimmer30.fastq --right R1V22_B9F8St_ACTTGA_L002_R2_001.qualitytrimmer30.fastq --est_method RSEM --aln_method bowtie --trinity_mode

CMD: set -o pipefail && bowtie -q --all --best --strata -m 300 --chunkmbs 512 -X 800 -S -p 4 /home/richardsonlab/AMMA_transcripts/trinity_out_dir/Trinity.fasta.bowtie -1 R1V22_B9F8St_ACTTGA_L002_R1_001.qualitytrimmer30.fastq -2 R1V22_B9F8St_ACTTGA_L002_R2_001.qualitytrimmer30.fastq | samtools view -F 4 -S -b -o bowtie.bam -

Warning: Could not open read file "R1V22_B9F8St_ACTTGA_L002_R1_001.qualitytrimmer30.fastq" for reading; skipping...

Command: bowtie --wrapper basic-0 -q --all --best --strata -m 300 --chunkmbs 512 -X 800 -S -p 4 -1 R1V22_B9F8St_ACTTGA_L002_R1_001.qualitytrimmer30.fastq -2 R1V22_B9F8St_ACTTGA_L002_R2_001.qualitytrimmer30.fastq /home/richardsonlab/AMMA_transcripts/trinity_out_dir/Trinity.fasta.bowtie 

[E::hts_open_format] fail to open file 'bowtie.bam'

samtools view: failed to open "bowtie.bam" for writing: Permission denied

Error, cmd: set -o pipefail && bowtie -q --all --best --strata -m 300 --chunkmbs 512 -X 800 -S -p 4 /home/richardsonlab/AMMA_transcripts/trinity_out_dir/Trinity.fasta.bowtie -1 R1V22_B9F8St_ACTTGA_L002_R1_001.qualitytrimmer30.fastq -2 R1V22_B9F8St_ACTTGA_L002_R2_001.qualitytrimmer30.fastq | samtools view -F 4 -S -b -o bowtie.bam - died with ret: 256 at align_and_estimate_abundance.pl line 653.

Mark Chapman

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Jan 27, 2016, 3:09:32 PM1/27/16
to Nikelle Petrillo, trinityrnaseq-users
​Hi Nikelle,

Some possibilities:

Have you prepped reference? I think you need --prep_reference the first time you run the pl script:

In the example on the github:
./util/align_and_estimate_abundance.pl --transcripts Trinity.fasta --seqType fq --left reads_1.fq --right reads_2.fq --est_method RSEM --aln_method bowtie --trinity_mode --prep_reference

or:

It seems to not recognise your reads - have you tried with the full path specified?

Are your reads definitely in a format that doesnt have a bunch of hidden characters? eg ctrl-M or newline characters?

Was the trinity.fasta assembled in trinity? If not you'll have to modify the transcript names.

Hopefully one of these will work :)

Best wishes, Mark


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Nikelle Petrillo

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Jan 27, 2016, 3:57:18 PM1/27/16
to trinityrnaseq-users, nikelle....@gmail.com
Thanks Mark! 

I added in -prep_reference and got back these errors. 

[npetrill@trogdor util]$ perl align_and_estimate_abundance.pl --transcripts /home/richardsonlab/AMMA_transcripts/trinity_out_dir/Trinity.fasta --seqType fq --left /home/richardsonlab/AMMA_transcripts/R1V22_B9F8St_ACTTGA_L002_R1_001.qualitytrimmer30.fastq --right /home/richardsonlab/AMMA_transcripts/R1V22_B9F8St_ACTTGA_L002_R2_001.qualitytrimmer30.fastq --est_method RSEM --aln_method bowtie --trinity_mode --output_dir /home/npetrill/aligning --prep_reference

CMD: set -o pipefail && bowtie -q --all --best --strata -m 300 --chunkmbs 512 -X 800 -S -p 4 /home/richardsonlab/AMMA_transcripts/trinity_out_dir/Trinity.fasta.bowtie -1 /home/richardsonlab/AMMA_transcripts/R1V22_B9F8St_ACTTGA_L002_R1_001.qualitytrimmer30.fastq -2 /home/richardsonlab/AMMA_transcripts/R1V22_B9F8St_ACTTGA_L002_R2_001.qualitytrimmer30.fastq | samtools view -F 4 -S -b -o bowtie.bam -

Error: reads file does not look like a FASTQ file

terminate called after throwing an instance of 'int'

bash: line 1:  1911 Aborted                 (core dumped) bowtie -q --all --best --strata -m 300 --chunkmbs 512 -X 800 -S -p 4 /home/richardsonlab/AMMA_transcripts/trinity_out_dir/Trinity.fasta.bowtie -1 /home/richardsonlab/AMMA_transcripts/R1V22_B9F8St_ACTTGA_L002_R1_001.qualitytrimmer30.fastq -2 /home/richardsonlab/AMMA_transcripts/R1V22_B9F8St_ACTTGA_L002_R2_001.qualitytrimmer30.fastq

      1912 Done                    | samtools view -F 4 -S -b -o bowtie.bam -

Error, cmd: set -o pipe fail && bowtie -q --all --best --strata -m 300 --chunkmbs 512 -X 800 -S -p 4 /home/richardsonlab/AMMA_transcripts/trinity_out_dir/Trinity.fasta.bowtie -1 /home/richardsonlab/AMMA_transcripts/R1V22_B9F8St_ACTTGA_L002_R1_001.qualitytrimmer30.fastq -2 /home/richardsonlab/AMMA_transcripts/R1V22_B9F8St_ACTTGA_L002_R2_001.qualitytrimmer30.fastq | samtools view -F 4 -S -b -o bowtie.bam - died with ret: 34304 at align_and_estimate_abundance.pl line 653.



It seems its not recognizing my reads and i am not sure why... I had fastq reads and then ran them through the FastX toolkit (i quality trimmed them) so I'm assuming they are still fastq? What do you mean by specifying the full path? And yes, the trinity.fasta file was assembled in trinity! 


-Nikelle 



On Wednesday, January 27, 2016 at 3:09:32 PM UTC-5, Mark Chapman wrote:
​Hi Nikelle,

Some possibilities:

Have you prepped reference? I think you need --prep_reference the first time you run the pl script:

In the example on the github:
./util/align_and_estimate_abundance.pl --transcripts Trinity.fasta --seqType fq --left reads_1.fq --right reads_2.fq --est_method RSEM --aln_method bowtie --trinity_mode --prep_reference

or:

It seems to not recognise your reads - have you tried with the full path specified?

Are your reads definitely in a format that doesnt have a bunch of hidden characters? eg ctrl-M or newline characters?

Was the trinity.fasta assembled in trinity? If not you'll have to modify the transcript names.

Hopefully one of these will work :)

Best wishes, Mark

On 27 January 2016 at 18:58, Nikelle Petrillo <nikelle....@gmail.com> wrote:

Hello. I am new to RNA seq. I have a trinity.fast file I would like to align reads to. I believe I am supposed to be using the align_and_estimate_abundance.pl command. However, when i input my arguments. I get warnings and error back. I am not sure what to make of them. Does anyone have any advice? Thanks for your help. - Nikelle 


[npetrill@trogdor util]$ perl align_and_estimate_abundance.pl --transcripts /home/richardsonlab/AMMA_transcripts/trinity_out_dir/Trinity.fasta --seqType fq --left R1V22_B9F8St_ACTTGA_L002_R1_001.qualitytrimmer30.fastq --right R1V22_B9F8St_ACTTGA_L002_R2_001.qualitytrimmer30.fastq --est_method RSEM --aln_method bowtie --trinity_mode

CMD: set -o pipefail && bowtie -q --all --best --strata -m 300 --chunkmbs 512 -X 800 -S -p 4 /home/richardsonlab/AMMA_transcripts/trinity_out_dir/Trinity.fasta.bowtie -1 R1V22_B9F8St_ACTTGA_L002_R1_001.qualitytrimmer30.fastq -2 R1V22_B9F8St_ACTTGA_L002_R2_001.qualitytrimmer30.fastq | samtools view -F 4 -S -b -o bowtie.bam -

Warning: Could not open read file "R1V22_B9F8St_ACTTGA_L002_R1_001.qualitytrimmer30.fastq" for reading; skipping...

Command: bowtie --wrapper basic-0 -q --all --best --strata -m 300 --chunkmbs 512 -X 800 -S -p 4 -1 R1V22_B9F8St_ACTTGA_L002_R1_001.qualitytrimmer30.fastq -2 R1V22_B9F8St_ACTTGA_L002_R2_001.qualitytrimmer30.fastq /home/richardsonlab/AMMA_transcripts/trinity_out_dir/Trinity.fasta.bowtie 

[E::hts_open_format] fail to open file 'bowtie.bam'

samtools view: failed to open "bowtie.bam" for writing: Permission denied

Error, cmd: set -o pipefail && bowtie -q --all --best --strata -m 300 --chunkmbs 512 -X 800 -S -p 4 /home/richardsonlab/AMMA_transcripts/trinity_out_dir/Trinity.fasta.bowtie -1 R1V22_B9F8St_ACTTGA_L002_R1_001.qualitytrimmer30.fastq -2 R1V22_B9F8St_ACTTGA_L002_R2_001.qualitytrimmer30.fastq | samtools view -F 4 -S -b -o bowtie.bam - died with ret: 256 at align_and_estimate_abundance.pl line 653.

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Mark Chapman

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Jan 27, 2016, 5:17:01 PM1/27/16
to Nikelle Petrillo, trinityrnaseq-users

Hi Nikelle,
By full path I mean specify /home/foldername/otherfolder/fwd_reads.fastq not just fwd_reads.fastq
But I think it's found them just fine.
What do you get if you run 'head' on your fastq files?

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University of Southampton
Life Sciences Building 85
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Nikelle Petrillo

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Jan 28, 2016, 2:15:59 PM1/28/16
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Hi Mark, 

This is what I get when I run head. This does not look like a fastq file to me.... any reason why once I trimmed my original fastq reads via fast tool kit they may have changed?

Thanks, 
Nikelle 

[npetrill@trogdor AMMA_transcripts]$ head R1V22_B9F8St_ACTTGA_L002_R1_001.qualitytrimmer30.fastq

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Mark Chapman

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Jan 28, 2016, 3:41:38 PM1/28/16
to Nikelle Petrillo, trinityrnaseq-users

Hi Nikelle,
Definitely something up there. It looks like they've been opened in a text editor or something else then saved as some other format. I don't use fastool so I couldn't say what's wrong. But trimmomatic is easy and is now bundled into trinity so is easy to run. Just add the relevant command to your assembly.
Cheers, Mark

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