Error running bowtie
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3.4 years ago
[2018-03-17 18:25:31] Beginning TopHat run (v2.1.1)
-----------------------------------------------
[2018-03-17 18:25:31] Checking for Bowtie
          Bowtie version:    2.3.2.0
[2018-03-17 18:25:31] Checking for Bowtie index files (genome)..
[2018-03-17 18:25:31] Checking for reference FASTA file
[2018-03-17 18:25:31] Generating SAM header for /media/amit/New Volume/genome/Homo_sapiens/UCSC/hg19/Sequence/Bowtie2Index/genome
[2018-03-17 18:25:31] Reading known junctions from GTF file
[2018-03-17 18:25:36] Preparing reads
     left reads: min. length=50, max. length=2469, 230262 kept reads (8215 discarded)
[2018-03-17 18:26:08] Building transcriptome data files ./tophat_out/tmp/genes
[2018-03-17 18:27:51] Building Bowtie index from genes.fa
[2018-03-17 18:40:12] Mapping left_kept_reads to transcriptome genes with Bowtie2 
[2018-03-17 19:31:47] Resuming TopHat pipeline with unmapped reads
[2018-03-17 19:31:47] Mapping left_kept_reads.m2g_um to genome genome with Bowtie2 
    [FAILED]
Error running bowtie:
Warning: Output file '-' was specified without -S.  This will not work in future Bowtie 2 versions.  Please use -S instead.
stat: No such file or directory
Warning: Could not open read file "Volume/genome/Homo_sapiens/UCSC/hg19/Sequence/Bowtie2Index/genome" for reading; skipping...
Error: No input read files were valid
(ERR): bowtie2-align exited with value 1
RNA-Seq alignment rna-seq next-gen genome • 1.8k views
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Here we go again:

You should know that the old 'Tuxedo' pipeline of Tophat(2) and Cufflinks is no longer the "advisable" tool for RNA-seq analysis. The software is deprecated/ in low maintenance and should be replaced by HISAT2, StringTie and ballgown. See this paper: Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. (If you can't get access to that publication, let me know and I'll -cough- help you.) There are also other alternatives, including alignment with STAR and bbmap, or pseudo-alignment using salmon.

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That was already conveyed half-way up this thread: C: Error running bowtie

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Oh right, I see. While I understand you and OP like to solve this issue, there is no much use in spending time on it, is there?

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After running HiSAT2 its showing error like: (ERR): hisat2-align exited with value 1

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Then wouldn't you rather try to solve the HISAT error? When my car breaks down I'm not getting a horse, I fix the car.

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Please provide the full command you are using for this run.

There seem to be multiple issues. You may be missing a leading / before the Volume in your genome index path specification. You are not providing correct path for the fastq files and/or they may not be in right format.

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$tophat -G '/media/amit/New Volume/genome/Homo_sapiens/UCSC/hg19/Annotation/Archives/archive-2010-09-27-22-25-17/genes.gtf' '/media/amit/New Volume/genome/Homo_sapiens/UCSC/hg19/Sequence/Bowtie2Index/genome' '/home/amit/Desktop/AMIT/SRR012172.fastq'

I have all file in external disk except fastq file, Is it rit for run this command?

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Before we go forward consider using some other program that is more current compared to TopHat. STAR/bbmap etc. TopHat is no longer recommended for use even by its authors.

You have a space in the file paths that is causing this problem. You could try following command (or eliminate the space in the path name and try again).

$tophat -G '/media/amit/New\ Volume/genome/Homo_sapiens/UCSC/hg19/Annotation/Archives/archive-2010-09-27-22-25-17/genes.gtf' '/media/amit/New\ Volume/genome/Homo_sapiens/UCSC/hg19/Sequence/Bowtie2Index/genome' '/home/amit/Desktop/AMIT/SRR012172.fastq'
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[2018-03-17 21:27:10] Beginning TopHat run (v2.1.1)
-----------------------------------------------
[2018-03-17 21:27:10] Checking for Bowtie
          Bowtie version:    2.3.2.0
Error: cannot find transcript file /media/amit/New\ Volume/genome/Homo_sapiens/UCSC/hg19/Annotation/Archives/archive-2010-09-27-22-25-17/genes.gtf

It iS not working.

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Rename New Volume to New_Volume and try with the new name minus the space in name. You know for sure that the file is present in that directory?

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Again it showing same error.

$ tophat -G '/media/amit/New_Volume/genome/Homo_sapiens/UCSC/hg19/Annotation/Archives/archive-2010-09-27-22-25-17/genes.gtf' '/media/amit/New_Volume/genome/Homo_sapiens/UCSC/hg19/Sequence/Bowtie2Index/genome' '/home/amit/Desktop/AMIT/SRR012172.fastq'  

[2018-03-17 21:49:42] Beginning TopHat run (v2.1.1)
-----------------------------------------------
[2018-03-17 21:49:42] Checking for Bowtie
          Bowtie version:    2.3.2.0
Error: cannot find transcript file /media/amit/New_Volume/genome/Homo_sapiens/UCSC/hg19/Annotation/Archives/archive-2010-09-27-22-25-17/genes.gtf
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what does this command show?

ls -lh /media/amit/New_Volume/genome/Homo_sapiens/UCSC/hg19/Annotation/Archives/archive-2010-09-27-22-25-17/genes.gtf

and try this next

$tophat -G /media/amit/New_Volume/genome/Homo_sapiens/UCSC/hg19/Annotation/Archives/archive-2010-09-27-22-25-17/genes.gtf -o /home/amit/Desktop/AMIT/out /media/amit/New_Volume/genome/Homo_sapiens/UCSC/hg19/Sequence/Bowtie2Index/genome /home/amit/Desktop/AMIT/SRR012172.fastq
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Witthout "_" showing path of directory but not with "_"

With New_Volume

$ ls -lh '/media/amit/New_Volume/genome/Homo_sapiens/UCSC/hg19/Annotation/Archives/archive-2010-09-27-22-25-17/genes.gtf'ls: cannot access '/media/amit/New_Volume/genome/Homo_sapiens/UCSC/hg19/Annotation/Archives/archive-2010-09-27-22-25-17/genes.gtf': No such file or directory

Without New Volume

amit@amit-Inspiron-1564:~/Desktop/AMIT$ ls -lh '/media/amit/New Volume/genome/Homo_sapiens/UCSC/hg19/Annotation/Archives/archive-2010-09-27-22-25-17/genes.gtf'
-rwxrwxrwx 1 amit amit 64M Mar 16  2012 /media/amit/New Volume/genome/Homo_sapiens/UCSC/hg19/Annotation/Archives/archive-2010-09-27-22-25-17/genes.gtf
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You did not rename the directory like I suggested you to. Do

$ mv /media/amit/New\ Volume /media/amit/New_Volume

they try tophat command I gave above with the new name without the space in New Volume..

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