Tophat/Bowtie

[TPH]

Hi, I am self learning tuxedo suite for RNA-seq analysis. I keep getting error messages and some how I can not go forward beyond tophat. I downloaded .gtf file using UCSC browser. Here is the command i used.

tophat --num-threads 8 \
--solexa-quals --max-multihits 10 \
--coverage-search --microexon-search \
--mate-inner-dist 150 \
-o tophat-tumor-out \
--keep-tmp \
-G /path_to_folder_having_.gtf_file/hg19.gtf \
/bowtie-2-2.1.0/indexes/hg19 \
/path_to_folder_having_fastq_files/SRR057634_1.fastq \
/path_to_folder_having_fastq_files/SRR057634_2.fastq

But I get this error message again and again.
/path_to_folder_having_fastq_files/SRR057634_1.fastq: Permission denied

Could anyone can think of any thing why this happens?

Thank you very much.

TPH

[GenoMax]

It looks like you don't have read permission to the folder where the sequence files are stored. Is that true?

Are you able to run the following command and see an output?

$ head -6 /path_to_folder_having_fastq_files/SRR057634_1.fastq

[GenoMax]

You may be better off downloading a coherent bundle of sequence/annotation/indexes from iGenomes. If your GTF file does not match the sequence/indexes your results could be off.

[TPH]

Hi,
Thank you so much for your kind help.
Anyway this is the output I get

@SRR057634.1 HWUSI-EAS230-R:1:2:28:1908 length=36
CCCCCCCCCCCCCCCCCCCCCNCCCCCCCCCCCCCC
+SRR057634.1 HWUSI-EAS230-R:1:2:28:1908 length=36
@AA>?;55555565555555.!-1444444441144
@SRR057634.2 HWUSI-EAS230-R:1:2:29:1475 length=36
CCCCCCCCCCCCCCCCCCCCCNCCCCCCCCCCCCCC

[TPH]

Thank you very much. I'll try doing that.

[GenoMax]

Hi,
Thank you so much for your kind help.
Anyway this is the output I get

@SRR057634.1 HWUSI-EAS230-R:1:2:28:1908 length=36
CCCCCCCCCCCCCCCCCCCCCNCCCCCCCCCCCCCC
+SRR057634.1 HWUSI-EAS230-R:1:2:28:1908 length=36
@AA>?;55555565555555.!-1444444441144
@SRR057634.2 HWUSI-EAS230-R:1:2:29:1475 length=36
CCCCCCCCCCCCCCCCCCCCCNCCCCCCCCCCCCCC

That means you do have read permissions on the sequence file so that is not an issue. The error you are getting is misleading.

Can you tell me where you are trying to run this? On a stand alone server/cluster? Can you look at the directory to see if tophat wrote a log file and if it has any additional clues?

This may sound arcane but can you try to change "-o tophat-tumor-out" to "-o tophat_tumor_out" (underscores instead of hyphens) and see what happens?

[TPH]

Hi,
Thank you very much for your help. I am working on a cluster. They have moved index files to another folder location. I Guess that's the reason I was getting those error messages. Now the script works fine.
Thanks again for your kindness.