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link error : hard link not allowed for directory

Hi,

i performed RNA-seq with tophat protocol. but, I am unable to run tophat as I get an error . Here is the command I run:

$ tophat -p 8 -G ./Drosophila_melanogaster/Ensembl/BDGP5.25/Annotation/Genes/genes.gtf -o ./C1_R1_thout ./genome ~/biostar/RNA-seq/my_rnaseq_exp/GSM794483_C1_R1_1.fq ~/biostar/RNA-seq/my_rnaseq_exp/GSM794483_C1_R1_2.fq

[Thu Aug 13 20:56:29 2020] Beginning TopHat run (v1.3.2)
-----------------------------------------------
[Thu Aug 13 20:56:29 2020] Preparing output location ./C1_R1_thout/
[Thu Aug 13 20:56:29 2020] Checking for Bowtie index files
Error: Could not find Bowtie index files ./genome.*

current directory is ~/biostar/RNA-seq/tophat-1.3.2.Linux_x86_64

Could anyone please comment or advise on this. Thanks for your time.

rna-seq
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written by seabed0705 • seabed0705

and i also couldn't this :

$ ln –s ./Drosophila_melanogaster/Ensembl/BDGP5.25/Annotation/Genes/genes.gtf .
ln: `\342\200\223s': hard link not allowed for directory
ln: creating hard link `./genes.gtf': File exists
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written by seabed0705 • seabed0705

can you post the result of ls -l ./Drosophila_melanogaster/Ensembl/BDGP5.25/Annotation/Genes/ ? it seems that you already have a (regular) file there called genes.gtf ... you can't make a link name if that file name already exists

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written by lieven.sterck lieven.sterck

Thank you all! A link already existed.

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written by seabed0705 • seabed0705

can you confirm that you have built the bowtie indices and that they are in the location ( this ./genome folder )you expect them to be?

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written by lieven.sterck lieven.sterck

slipped my attention but tophat is kinda deprecated and has been replaced by newer versions/tools.

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. There are also other alternatives, including alignment with STAR and bbmap, or pseudo-alignment using salmon.

https://twitter.com/lpachter/status/937055346987712512

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written by lieven.sterck lieven.sterck

Thank you so much for the offer! I'll try. But using the tophat tool is the request I received.

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written by seabed0705 • seabed0705

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