Tophat/Bowtie

TPH · 01-12-2016, 03:32 PM

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 · 01-12-2016, 03:41 PM

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?

Code:

$ head -6 /path_to_folder_having_fastq_files/SRR057634_1.fastq

GenoMax · 01-12-2016, 03:42 PM

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 · 01-12-2016, 03:46 PM

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 · 01-12-2016, 03:55 PM

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

GenoMax · 01-12-2016, 05:34 PM

Quote:

Originally Posted by 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

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 · 01-15-2016, 02:46 PM

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.

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01-12-2016, 03:32 PM	#1
TPH Member Location: USA Join Date: Jan 2016 Posts: 19	Tophat/Bowtie 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

01-12-2016, 03:41 PM	#2
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,080	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? Code: $ head -6 /path_to_folder_having_fastq_files/SRR057634_1.fastq

01-12-2016, 03:42 PM	#3
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,080	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.

01-12-2016, 03:46 PM	#4
TPH Member Location: USA Join Date: Jan 2016 Posts: 19	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

01-12-2016, 03:55 PM	#5
TPH Member Location: USA Join Date: Jan 2016 Posts: 19	Thank you very much. I'll try doing that.

01-15-2016, 02:46 PM	#7
TPH Member Location: USA Join Date: Jan 2016 Posts: 19	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.