tophat error with output directory

[colaneri]

I'm running tophat in a remote server and I having this error message (see below the command and the error)
Can some one tell to me what could be going on?

# LSBATCH: User input
tophat -p 4 --min-intron-length 40 --max-intron-length 2000 --library-type fr-firststrand --no-novel-juncs -G /proj/seq/data/TAIR10_Ensembl/Annotation/Archives/archive-2013-03-06-09-54-25/Genes/genes.gtf -o/--output-dir tophat/Cnaa_rep1 /proj/seq/data/TAIR10_Ensembl/Sequence/Bowtie2Index/genome AGB_SPK_02-2-Cnaa-rep1_SpikeMix1_TAGCTT_L005_R1_001.fastq.gz, AGB_SPK_02-2-Cnaa-rep1_SpikeMix1_TAGCTT_L006_R1_001.fastq.gz
------------------------------------------------------------

Exited with exit code 1.

Resource usage summary:

CPU time : 0.07 sec.
Max Processes : 1
Max Threads : 1

The output (if any) follows:

Traceback (most recent call last):
File "/nas02/apps/tophat-2.0.13/bin/tophat", line 4088, in ?
sys.exit(main())
File "/nas02/apps/tophat-2.0.13/bin/tophat", line 3866, in main
prepare_output_dir()
File "/nas02/apps/tophat-2.0.13/bin/tophat", line 1242, in prepare_output_dir
os.mkdir(output_dir)
OSError: [Errno 13] Permission denied: '/--output-dir/'

[Brian Bushnell]

Rather than "-o/--output-dir", you need to just use "-o".

[colaneri]

Things get weird

Tx Brian, after change to -o
now I getting this error message
here I have less clue of what is going on

# LSBATCH: User input
tophat -p 4 --min-intron-length 40 --max-intron-length 2000 --library-type fr-firststrand --no-novel-juncs -G /proj/seq/data/TAIR10_Ensembl/Annotation/Archives/archive-2013-03-06-09-54-25/Genes/genes.gtf -o Cnaa_rep1 /proj/seq/data/TAIR10_Ensembl/Sequence/Bowtie2Index/genome AGB_SPK_02-2-Cnaa-rep1_SpikeMix1_TAGCTT_L005_R1_001.fastq, AGB_SPK_02-2-Cnaa-rep1_SpikeMix1_TAGCTT_L006_R1_001.fastq
------------------------------------------------------------

Exited with exit code 1.

Resource usage summary:

CPU time : 0.25 sec.
Max Processes : 1
Max Threads : 1

The output (if any) follows:


[2014-11-08 12:47:39] Beginning TopHat run (v2.0.13)
-----------------------------------------------
[2014-11-08 12:47:39] Checking for Bowtie
Bowtie version: 2.2.1.0
[2014-11-08 12:47:41] Checking for Bowtie index files (genome)..
[2014-11-08 12:47:41] Checking for reference FASTA file
[2014-11-08 12:47:41] Generating SAM header for /proj/seq/data/TAIR10_Ensembl/Sequence/Bowtie2Index/genome
Traceback (most recent call last):
File "/nas02/apps/tophat-2.0.13/bin/tophat", line 4088, in ?
sys.exit(main())
File "/nas02/apps/tophat-2.0.13/bin/tophat", line 3942, in main
params.read_params = check_reads_format(params, reads_list)
File "/nas02/apps/tophat-2.0.13/bin/tophat", line 1837, in check_reads_format
zf = ZReader(f_name, params)
File "/nas02/apps/tophat-2.0.13/bin/tophat", line 1790, in __init__
self.file=open(filename)
IOError: [Errno 2] No such file or directory: ''

[Brian Bushnell]

AGB_SPK_02-2-Cnaa-rep1_SpikeMix1_TAGCTT_L005_R1_001.fastq, AGB_SPK_02-2-Cnaa-rep1_SpikeMix1_TAGCTT_L006_R1_001.fastq

That looks wrong. Are your reads paired? If so, you should be using R1 and R2, and there should NOT be a comma in between, just a space. You are trying to use reads from two different lanes. E.G. the correct input files might look like this:

AGB_SPK_02-2-Cnaa-rep1_SpikeMix1_TAGCTT_L005_R1_001.fastq AGB_SPK_02-2-Cnaa-rep1_SpikeMix1_TAGCTT_L005_R2_001.fastq

[colaneri]

technical replicates

Yes they are files from the same library that was sequenced in two different lines. I use the command because the tophat manual.

Using TopHat

Usage: tophat [options]* <genome_index_base> <reads1_1[,...,readsN_1]> [reads1_2,...readsN_2]

I probably get it wrong, but it look to me from there that
reads1_1 and reads N_1 are technical replicates or different fastq files originated from the same library.

[Brian Bushnell]

You have both a comma and a space. Presumably, you need only one or the other.

[colaneri]

thanks!

You have both a comma and a space. Presumably, you need only one or the other.

However your suggestion looks corrects since the job has not returned any error so far.
How can I be sure that both files were used in the alignment?

[Brian Bushnell]

The output file will have some reads marked as "read 1" and others marked as "read 2" in the sam bitflag; you can get statistics about pairing rates with samtools flagstat. I think Tophat will also display the pairing rate once it finishes.

[colaneri]

not paired ends

But this is not paired end..
The two fastq are the same library sequenced in two different lines of the flow cell. It is possible to align together two files from the same library in this way?
sorry for all my confusion and thanks a lot for your help

[Brian Bushnell]

If the reads are single-ended, you should separate them with commas. If the reads are paired, you should separate them with spaces. You can always make things simpler by combining them first:

cat reads_A.fq reads_B.fq > combined.fq

...then run things on the combined file.

[colaneri]

back to the beggining

OK, you are right, the run ended and said that there were problems with pair end. But now I can back to do not understand the previously reported error:

# LSBATCH: User input
tophat -p 4 --min-intron-length 40 --max-intron-length 2000 --library-type fr-firststrand --no-novel-juncs -G /proj/seq/data/TAIR10_Ensembl/Annotation/Archives/archive-2013-03-06-09-54-25/Genes/genes.gtf -o Cnaa_rep1 /proj/seq/data/TAIR10_Ensembl/Sequence/Bowtie2Index/genome AGB_SPK_02-2-Cnaa-rep1_SpikeMix1_TAGCTT_L005_R1_001.fastq, AGB_SPK_02-2-Cnaa-rep1_SpikeMix1_TAGCTT_L006_R1_001.fastq
------------------------------------------------------------

Exited with exit code 1.

Resource usage summary:

CPU time : 0.25 sec.
Max Processes : 1
Max Threads : 1

The output (if any) follows:


[2014-11-08 12:47:39] Beginning TopHat run (v2.0.13)
-----------------------------------------------
[2014-11-08 12:47:39] Checking for Bowtie
Bowtie version: 2.2.1.0
[2014-11-08 12:47:41] Checking for Bowtie index files (genome)..
[2014-11-08 12:47:41] Checking for reference FASTA file
[2014-11-08 12:47:41] Generating SAM header for /proj/seq/data/TAIR10_Ensembl/Sequence/Bowtie2Index/genome
Traceback (most recent call last):
File "/nas02/apps/tophat-2.0.13/bin/tophat", line 4088, in ?
sys.exit(main())
File "/nas02/apps/tophat-2.0.13/bin/tophat", line 3942, in main
params.read_params = check_reads_format(params, reads_list)
File "/nas02/apps/tophat-2.0.13/bin/tophat", line 1837, in check_reads_format
zf = ZReader(f_name, params)
File "/nas02/apps/tophat-2.0.13/bin/tophat", line 1790, in __init__
self.file=open(filename)
IOError: [Errno 2] No such file or directory: ''

[Brian Bushnell]

Like I said, the problem is here:

001.fastq, AGB_SPK

Remove the space.

[colaneri]

Like I said, the problem is here:

001.fastq, AGB_SPK

Remove the space.

Brian, I really appreciate your help. I have all the aligments done! thanks a lot.