Question: How do I run Tophat?
2
gravatar for from the mountains
4.2 years ago by
United States
from the mountains50 wrote:

I am using the following command:

./tophat2 -r 210 -p 8 -G ~/Data/*.gtf ~/Data/hg19f/hg19 ~/Data/thyroid1,~/Data/testes1 ~/Data/thyroid2,~/Data/testes2

but I'm getting the error:

[2015-04-11 21:58:47] Checking for reference FASTA file

    Warning: Could not find FASTA file /home/anoronh4/HW3/hg19f/hg19.fa

[2015-04-11 21:58:47] Reconstituting reference FASTA file from Bowtie index

Error locating program: bowtie-inspect

The files in hg19f are:

hg19.1.ebwt 

hg19.3.ebwt 

hg19.rev.1.ebwt 

make_hg19.sh

hg19.2.ebwt 

hg19.4.ebwt 

hg19.rev.2.ebwt

Since when do I need a .fa file?

 

bowtie tophat • 11k views
ADD COMMENTlink modified 4.2 years ago • written 4.2 years ago by from the mountains50
2

one correction:

./tophat2 -r 210 -p 8 -G ~/Data/*.gtf ~/Data/hg19f/hg19 ~/Data/thyroid1,~/Data/testes1 ~/Data/thyroid2,~/Data/testes2

Give full names of fasta/q files. Why its *.gtf ?

ADD REPLYlink written 4.2 years ago by geek_y9.7k
1

Is bowtie2 installed?  Is bowtie-inspect in the path?  

ADD REPLYlink written 4.2 years ago by Sean Davis25k
2
gravatar for Mehmet
4.2 years ago by
Mehmet460
Japan
Mehmet460 wrote:

Hi,

I think you want to align RNAseq reads to your reference genomes. If so, and If you have paired-end reads of RNAseq (R1 and R2), you should follow these steps:

1. Build index files using bowtie2 (bowtie2 is for tophat2) using "bowtie2-build your fasta file"  After generating bowtie index files, do not comprise them. just leave in your directory. in your case "The files in hg19f are:" you put all bowtie index files in a file and used the file in your tophat command. you should not do this. first, you will need to use your fast file to make bowtie index file, you will use your the same genome file in tophat command, not index files.

2. Run tophat command. in tophat command, you need to use your fasta (genome file) and RNAseq files (R1 and R2). The fast file is in .fa format. Ransom files are generally in .gz (gunzip) format.

here is a sample tophat command:

tophat -o OUT.dir2 --library-type fr-secondstrand --mate-std-dev 50 -a 6 --microexon-search --min-segment-intron 10 --max-segment-intron 20000 -i 30 -I 20000 -r 0 genome.fa R1.fastq.gz R2.fastq.gz

 

ADD COMMENTlink written 4.2 years ago by Mehmet460

hello sir i follow this instructions what u told about RNAseq but im gettin an error like this Error: Could not find Bowtie 2 index files (hg38.fa.*.bt2l).

ADD REPLYlink written 2.3 years ago by mrmkanni930

Hi , Sorry for the delay. Before running Tophat, you have to make an index file that will be used by Tophat.

For this; bowtie2-build your fasta file indexname

just give any index name (for ex; myindexname).

then, in Tophat analysis,

for the argument index in Tophat command, just type "myindexname" and run.

ADD REPLYlink written 2.2 years ago by Mehmet460

Hello I run the Tophat command as you mention in the last post. The program run but throw me a message at the end:

[2017-04-05 09:53:57] Checking for reference FASTA file
Warning: Could not find FASTA file TAIR10_chr_all.fa

[2017-04-05 09:53:57] Reconstituting reference FASTA file from Bowtie index Executing: /data/software/bowtie2-2.2.9/bowtie2-inspect TAIR10_chr_all > tophat_mapping/tmp/TAIR10_chr_all.fa

Is that message normal?

ADD REPLYlink written 2.2 years ago by irving.jgalo230
2
gravatar for Parham
4.2 years ago by
Parham1.4k
Sweden
Parham1.4k wrote:

Very good protocol for learning tuxedo! However RNA-STAR is way faster than tophat and bowtie. 

ADD COMMENTlink modified 4.2 years ago • written 4.2 years ago by Parham1.4k
0
gravatar for from the mountains
4.2 years ago by
United States
from the mountains50 wrote:

it was because i had added some bowtie functionalities to my path, but not all. silly oversight.

ADD COMMENTlink written 4.2 years ago by from the mountains50
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