Tophat: Genome indexing error

Question

[Adyasha]

Hi everyone,

I have 2 samples, paired end RNA sequence from illumina. I have done the QC and trimming.

Now after trimming with trimgalore I have

A_R1.val_fq
A_R2.val_fq
B_R1.val_fq
B_R2.val_fq

Now I performed tophat for genome mapping. I ran this command

 tophat -p4 -G GRCh38_latest_genomic.fna -o tophat_result bowtie_index A_R1_val.fq

but its throwing this error.

[FAILED]
Error: Couldn't build bowtie index with err = 1

I have a genome annotation file: gencode.v43.chr_patch_hapl_scaff.annotation.gtf and a reference genome: GRCh38_latest_genomic.fna. I tried making a bowtie index by using this command :

bowtie2-build GRCh38_latest_genomic.fna bowtie_index

This gave me files like this:

bowtie_index.1.bt2
bowtie_index.2.bt2
bowtie_index.3.bt2
bowtie_index.4.bt2
bowtie_index.rev.1.bt2
bowtie_index.rev.2.bt2

Please help me on this step. I could not figure out where I am doing mistake.

Thank you

Answer 1

[colindaven]

Tophat is many years out of date, use STAR or Hisat instead. The whole community have been warning users about this since about 2015.

[Adyasha]

Hi sir,

I have tried using hisat2 also,

hisat2 -x BOWTIE_INDEX/ -1 A_R1.val.fq -2 A_R2.val.fq -S out.sam

but its saying :

(ERR): "BOWTIE_INDEX/" does not exist

I have

bowtie_index.1.bt2
bowtie_index.2.bt2
bowtie_index.3.bt2
bowtie_index.4.bt2
bowtie_index.rev.1.bt2
bowtie_index.rev.2.bt

These files are in my BOWTIE_INDEX folder.

[Nicolas Rosewick]

hisat2 indexes are not the same as bowtie indexes.

Please read hisat2 manual : http://daehwankimlab.github.io/hisat2/manual/