align_and_estimate_abundance.pl ERROR/ QUESTION

Question

[nikelle.petrillo]

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.

Answer 1

[dally]

I see two errors. One is that the fastq file could not be opened, this means that it does not exist or something is wrong with the installation.

The other is giving a permission denied. Usually this can be fixed with the sudo command (though there may be a more elegant solution).

[nikelle.petrillo]

Thank you. I fixed my command a little bit and I am getting a new error that I am still not sure what to make of:

[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
which: no rsem-calculate-expression in (/home/npetrill/trinityrnaseq-2.1.1/util/../trinity-plugins/rsem:/usr/lib64/qt-3.3/bin:/usr/local/bin:/bin:/usr/bin:/usr/local/sbin:/usr/sbin:/sbin:/home/npetrill/bin:/usr/local/bin/trinityrnaseq-2.0.6:/usr/local/bin/bowtie-1.1.2:/home/npetrill/samtools-0.1.19:home/npetrill/cmake-3.4.2:/home/npetrill:/home/npetrill/scripts)
ERROR, cannot find rsem-calculate-expression in PATH setting: /home/npetrill/trinityrnaseq-2.1.1/util/../trinity-plugins/rsem:/usr/lib64/qt-3.3/bin:/usr/local/bin:/bin:/usr/bin:/usr/local/sbin:/usr/sbin:/sbin:/home/npetrill/bin:/usr/local/bin/trinityrnaseq-2.0.6:/usr/local/bin/bowtie-1.1.2:/home/npetrill/samtools-0.1.19:home/npetrill/cmake-3.4.2:/home/npetrill:/home/npetrill/scripts
Please be sure bowtie and express are installed and the utilities samtools bowtie-build bowtie rsem-calculate-expression are available via your PATH setting.

[dally]

You need to have samtools, bowtie, and rsem-calculate-expression available in your PATH.

This usually means that you can call up samtools by simply typing in samtools in your command line (as an example). You have to figure out which tool uses rsem-calculate-expression and then add that tool to your path. (it's probably a trinity tool).

You can add a tool to your path by going to your home directory and typing in vim .bash_profile and then adding a path by typing export PATH=$PATH:/path/goes/here and then exiting and typing source .bash_profile in the command line.

[nikelle.petrillo]

Thanks for your quick reply. Here is my path:

PATH=$PATH:$HOME/bin:/usr/local/bin/trinityrnaseq-2.0.6:/usr/local/bin/bowtie-1.1.2:/home/npetrill/samtools-0.1.19:/home/npetrill/cmake-3.4.2:/home/npetrill:/usr/loca$
export PATH="$PATH:$HOME/scripts:"

If I already have an "export" line, am I supposed to add the path to rsem tool onto this line?

[dally]

It's a very messy way of doing it but I believe if you add the line to the tool itself then it should work.