Couldn't Build Bowtie index with err = 1

Question

[nathan.yeung]

I'm trying to run tophat2 on RNA-seq data but it can't be accomplished as it continues to give the error message of Couldn't Build Bowtie Index with err = 1. The genome.fa and genes.gtf are both from the same source of UCSC hg19. Any advice and assistance would be greatly appreciated! Thank you!

Here is the code:

tophat2 -p 1 -G ~/Desktop/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf -o ~/Desktop/Junior\ Project/tophatout/ ~/Desktop/Homo_sapiens/UCSC/hg19/Sequence/Bowtie2Index/genome SRR085471.fastq 

[2018-02-25 23:56:38] Beginning TopHat run (v2.1.1)
-----------------------------------------------
[2018-02-25 23:56:38] Checking for Bowtie
          Bowtie version:    2.3.4.1
[2018-02-25 23:56:38] Checking for Bowtie index files (genome)..
[2018-02-25 23:56:38] Checking for reference FASTA file
[2018-02-25 23:56:38] Generating SAM header for /home/nathan/Desktop/Homo_sapiens/UCSC/hg19/Sequence/Bowtie2Index/genome
[2018-02-25 23:57:52] Reading known junctions from GTF file
[2018-02-25 23:58:08] Preparing reads
     left reads: min. length=36, max. length=36, 15253905 kept reads (2847 discarded)
[2018-02-26 00:05:51] Building transcriptome data files /home/nathan/Desktop/Junior Project/tophatout//tmp/genes
[2018-02-26 00:07:36] Building Bowtie index from genes.fa
    [FAILED]
Error: Couldn't build bowtie index with err = 1

[WouterDeCoster]

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.

Please stop using Tophat https://t.co/Es4ohxOEyx Cole and I developed the method in *2008*. It was greatly improved in TopHat2 then HISAT & HISAT2. There is no reason to use it anymore. I have been saying this for years yet it has more citations this year than last #methodsmatter

— Lior Pachter (@lpachter) December 2, 2017

[Ram]

Please use the formatting bar (especially the code option) to present your post better. I've done it for you this time.

[Kritika]

Error seems like you have not index fasta file

Please index your fasta file with bowtie-build command in bowtie tool

Then align tophat with providing options for input fastq file ,reference file, bowtie--very sensitive , number of core and output file

[nathan.yeung]

do you mean to use the bowtie-build command to index the fasta file provided by the UCSC hg19 genome.fa?

[Kritika]

Yes also as said by WouterDeCoster Tuxedo protocol is no longer needed you can switch to hisat2 Hisat2 work like bowtie you need to index the reference sequence which you got it from ucsc next you use stringtie which works like tophat next you calculate Differential Gene Exprs by using ballgown which is R package