Tophat2 RNA seq mapping

Question

[tikshyadav19]

Hi, I am trying to run tophat2 using following tophat command:

tophat -p 8 -G /scratch/sbag/Tikshana/Ath/index/Bowtie_Ath/Bowtie_Ath -o /scratch/sbag/Tikshana/Ath/map/Athctrl_tophat/ genome  /scratch/sbag/Tikshana/Ath/Athctrl_1P.fastq /scratch/sbag/Tikshana/Ath/Athctrl_2P.fastq

I am getting the following error:

Error: cannot find transcript file /scratch/sbag/Tikshana/Ath/index/Bowtie_Ath/Bowtie_Ath*

But the file contains files for indexed reference.

please help me to troubleshoot the problem

[Mehmet]

If you want to do RNA reads mapping to your reference genome, please use hisat2 tool.

[tikshyadav19]

may I know difference between Tophat2 and Hisat2

[ATpoint]

But the file contains files for indexed reference. What does that mean? Please show the content of the folder where the index files are in using ls.

[Carlo Yague]

Can you post the output of

ls /scratch/sbag/Tikshana/Ath/index/Bowtie_Ath/
ls /scratch/sbag/Tikshana/Ath/index/Bowtie_Ath/Bowtie_Ath/

[tikshyadav19]

These are the index output files: Bowtie_Ath.1.bt2 Bowtie_Ath.2.bt2 Bowtie_Ath.3.bt2 Bowtie_Ath.4.bt2 Bowtie_Ath.rev.1.bt2 Bowtie_Ath.rev.2.bt2

[ATpoint]

That is fine but in which folder are they?

[tikshyadav19]

in the above mentioned folder : /scratch/sbag/Tikshana/Ath/index/Bowtie_Ath/

Answer 1

[ATpoint]

I think this is simply wrong syntax.

Usage: tophat [options]* <genome_index_base> <reads1_1[,...,readsN_1]> [reads1_2,...readsN_2]

So -G must be a GTF file not a folder. Also why is there a whitespace in Athctrl_tophat/ genome.

It probably must be

tophat -p 8 -G AnyGTFfile.gtf -o /scratch/sbag/Tikshana/Ath/map/Athctrl_tophat/genome /scratch/sbag/Tikshana/Ath/index/Bowtie_Ath/ /scratch/sbag/Tikshana/Ath/Athctrl_1P.fastq /scratch/sbag/Tikshana/Ath/Athctrl_2P.fastq

[Carlo Yague]

nice catch ! alternatively:

tophat -p 8 /scratch/sbag/Tikshana/Ath/index/Bowtie_Ath/Bowtie_Ath -o /scratch/sbag/Tikshana/Ath/map/Athctrl_tophat/genome  /scratch/sbag/Tikshana/Ath/Athctrl_1P.fastq /scratch/sbag/Tikshana/Ath/Athctrl_2P.fastq

[GenoMax]

Ideally one would do a special transcriptome index creation run one-time as described in TopHat2 manual.

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

Then use the index produced for actual read alignments

tophat -o out_sample1 -p4 \
    --transcriptome-index=transcriptome_data/known \
    hg19 sample1_1.fq.z sample1_2.fq.z &

tophat -o out_sample2 -p4 \
    --transcriptome-index=transcriptome_data/known \
    hg19 sample2_1.fq.z sample2_2.fq.z &