Problem with Tophat / Bowtie pipeline

Question

[tebb]

Hi everyone,

Im very new into Tophat and Bowtie RNA analysis. I had made a script for RNA raw data analysis but i got an error. It is very probably that the script have a lot of errors, but here is:

#TOPHAT & BOWTIE2 PROCEDURES

#STEP 1 - Set up

iDirectory="/home/lgts/Desktop/TopBow/Input"

oDirectory="/home/lgts/Desktop/TopBow/Output"

Temp="/home/lgts/Desktop/TopBow/Temp"

#Resources

Reference="/home/lgts/Desktop/TopBow/Reference/hg19"

GtfRefecence="/home/lgts/Desktop/TopBow/Reference/hg19_genes.gtf"

#Parameters

NumCores="8"

#Samples, one by line

mySamples="KG-1_Ch48h-23_9

KG-1_Ch48h-23_9"

#STEP 2- Run alignments

for i in $mySamples 
do

tophat2 -p $NumCores -G $GtfReference --output-/home/lgts/Desktop/TopBow/Output $Temp/$i.out $Reference $iDirectory/${i}_1.fastq.gz $iDirectory/${i}_2.fastq.gz

done

I'd checked all the directories and they are ok.

The error message is at follows:

[2018-04-19 19:33:36] Beginning TopHat run (v2.1.0)
-----------------------------------------------
[2018-04-19 19:33:36] Checking for Bowtie
          Bowtie version:    2.2.6.0
**Error: cannot find transcript file --output-/home/lgts/Desktop/TopBow/Output**

[2018-04-19 19:33:36] Beginning TopHat run (v2.1.0)
-----------------------------------------------
[2018-04-19 19:33:36] Checking for Bowtie
          Bowtie version:    2.2.6.0
**Error: cannot find transcript file --output-/home/lgts/Desktop/TopBow/Output**

I really dont know how to solve this, so any help or insight into this will be very very appreciated.

NOTE: I have both tophat 1 & tophat2, and bowtie1 & bowtie2 installed. I dont know if this could be the origin of this error..

[GenoMax]

For new projects you should seriously consider using something other than TopHat suite. STAR/BBMap/HISAT2 are all excellent current alternatives.

[tebb]

Hi, i tried with --output-$oDirectory, but i got the same error..

I dont know what do you mean with --output-dir. dir= output directory?

Thank you.

[GenoMax]

That option needs to be --output-dir $oDirectory.

Answer 1

[GenoMax]

You have the wrong option for output directory. It should be --output-dir.

You can do a special run of TopHat with -G option to create a transcriptome index separately. Check the manual.

Please note that starting with version 2.0.10 TopHat can be invoked with just the -G/--GTF and --transcriptome-index options but without providing any input reads (the <genome_index_base> argument is still required). This is a special usage directing TopHat to only build the transcriptome index data files for the given annotation and then exit. Note: Only after the transcriptome files are built with one of the methods above, by a single TopHat process, it is safe to run multiple TopHat processes simultaneously making use of the same pre-built transcriptome index data. For example, in order to just prepare the transcriptome index files for a specific annotation, an initial, single TopHat run could be invoked like this:

tophat -G known_genes.gtf \
    --transcriptome-index=transcriptome_data/known \
    hg19

Answer 2

[Drew.Judell]

You seem to be missing two key variables in your tophat command. Your command lacks a pointer for the single end or paired end sequences that you would like to map as well as the bowtie index associated with your reference annotation file (I'm assuming 'Reference="/home/lgts/Desktop/TopBow/Reference/hg19"' is your index).