Question: Error running bowtie
0
gravatar for amitunited0532
11 months ago by
amitunited053210 wrote:
[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
ADD COMMENTlink modified 11 months ago by genomax62k • written 11 months ago by amitunited053210
1

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.

ADD REPLYlink written 11 months ago by WouterDeCoster36k

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

ADD REPLYlink modified 11 months ago • written 11 months ago by genomax62k

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?

ADD REPLYlink written 11 months ago by WouterDeCoster36k

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

ADD REPLYlink written 11 months ago by amitunited053210

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.

ADD REPLYlink written 11 months ago by WouterDeCoster36k

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.

ADD REPLYlink written 11 months ago by genomax62k
$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?

ADD REPLYlink modified 11 months ago by genomax62k • written 11 months ago by amitunited053210
1

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'
ADD REPLYlink written 11 months ago by genomax62k
[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.

ADD REPLYlink modified 11 months ago by genomax62k • written 11 months ago by amitunited053210

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?

ADD REPLYlink modified 11 months ago • written 11 months ago by genomax62k

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
ADD REPLYlink modified 11 months ago by genomax62k • written 11 months ago by amitunited053210

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
ADD REPLYlink modified 11 months ago • written 11 months ago by genomax62k

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
ADD REPLYlink modified 11 months ago by genomax62k • written 11 months ago by amitunited053210

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..

ADD REPLYlink modified 11 months ago • written 11 months ago by genomax62k
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