Error running 'prep_reads' on TopHat (v2.0.9)

Question

[Veli Kaan Aydin]

Hello everyone,

Using this command tophat -p 8 -G genes.gtf -o c1_R1_thout genome SRR121576.fq SRR121577.fq I got this error:

[2014-08-14 14:49:06] Beginning TopHat run (v2.0.9)
-----------------------------------------------
[2014-08-14 14:49:06] Checking for Bowtie
          Bowtie version:     2.1.0.0
[2014-08-14 14:49:06] Checking for Samtools
        Samtools version:     0.1.19.0
[2014-08-14 14:49:06] Checking for Bowtie index files (genome)..
[2014-08-14 14:49:06] Checking for reference FASTA file
[2014-08-14 14:49:06] Generating SAM header for genome
    format:         fastq
    quality scale:     phred33 (default)
[2014-08-14 14:50:19] Reading known junctions from GTF file
[2014-08-14 14:50:22] Preparing reads
    [FAILED]
Error running 'prep_reads'
terminate called after throwing an instance of 'int'

I cannot figure out what it's going wrong here. Any thoughts?

Thank you.

[Devon Ryan]

Aside from the error, aligning two separate single-end libraries as though they were paired-end can also cause problems. You wanted to execute tophat -p 8 -G genes.gtf -o c1_R1_thout genome SRR121576.fq,SRR121577.fq or something like that.

[Veli Kaan Aydin]

Normally I wanted to execute CD4 and CD8 cells rna-seq data cd4 has one fastq file but CD8 has 3 so I think to execute codes like this order;

tophat -p 8 -G genes.gtf -o C1_R1_thout genome SRR121576.fq SRR121577.fq
tophat -p 8 -G genes.gtf -o C1_R2_thout genome SRR121576.fq SRR121578.fq
tophat -p 8 -G genes.gtf -o C1_R3_thout genome SRR121576.fq SRR121579.fq

Then cuffmerge it. Is it wrong?

[Devon Ryan]

That's wrong. What you want is:

tophat -p 8 -G genes.gtf -o SRR1211576 genome SRR1211576.fq
tophat -p 8 -G genes.gtf -o SRR1211577 genome SRR1211577.fq
tophat -p 8 -G genes.gtf -o SRR1211578 genome SRR1211578.fq
tophat -p 8 -G genes.gtf -o SRR1211579 genome SRR1211579.fq

You don't have paired-end reads, so you'll just get wrong results doing what you wrote.

[Veli Kaan Aydin]

When I tried that command it just gave me TopHat index.

[Devon Ryan]

Oops! I forgot "genome". I updated that comment.

[Veli Kaan Aydin]

I still get the "terminate called after throwing an instance of 'int'" error

[Devon Ryan]

Yeah, that's its own cryptic problem. Often this means a Phred score is out of range.

Answer 1

[Devon Ryan]

The "throwing an instance of 'int'" error is because these are colorspace files rather than regular fastq files. You'll need the --bowtie1 option as well as -C. You'll also need to use a colorspace index.

[Veli Kaan Aydin]

I am newbie to this (I started a week ago) Can you give me an example how to use this?