Entering edit mode
7.4 years ago
nikelle.petrillo ▴ 100
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.
[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.
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:
You need to have
rsem-calculate-expressionavailable 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_profileand then adding a path by typing
export PATH=$PATH:/path/goes/hereand then exiting and typing
source .bash_profilein the command line.
Thanks for your quick reply. Here is my path:
If I already have an "export" line, am I supposed to add the path to rsem tool onto this line?
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.