Tophat error message

Question

[saj98]

I am running tophat2 on rat genome using fastq file. I am using the following command r2d2@r2d2:~/RAID/Shame$ tophat -p5 -o WS001_R1_thout --library-type=fr-firststrand -G Rn6/Rn6.gtf Rn6/Rn6_index WS001_TTAGGC_L002_R1_001.fastq,WS001_TTAGGC_L003_R1_001.fastq,WS001_TTAGGC_L004_R1_001.fastq WS001_TTAGGC_L002_R2_001.fastq,WS001_TTAGGC_L003_R2_001.fastq,WS001_TTAGGC_L004_R1_001.fastq

So I got this error which I do not know how to solve it. I appreciate any suggestion and help.

[2017-03-11 16:02:04] Beginning TopHat run (v2.0.9)
-----------------------------------------------
[2017-03-11 16:02:04] Checking for Bowtie
          Bowtie version:    2.1.0.0
[2017-03-11 16:02:04] Checking for Samtools
        Samtools version:    0.1.19.0
[2017-03-11 16:02:04] Checking for Bowtie index files (genome)..
[2017-03-11 16:02:04] Checking for reference FASTA file
[2017-03-11 16:02:04] Generating SAM header for Rn6/Rn6_index
    format:      fastq
    quality scale:   phred33 (default)
[2017-03-11 16:02:06] Reading known junctions from GTF file
[2017-03-11 16:02:12] Preparing reads
    [FAILED]
Error running 'prep_reads'
terminate called after throwing an instance of 'int'

Thanks Shaima

[WouterDeCoster]

What type of data (technology) do you have? Is the phred33 scale correct for your data?

You should also know that Tophat is no longer the "advisable" tool for RNA-seq analysis. The software is deprecated/ in low maintenance and should be replaced by HISAT2, StringTie and ballgown. See this paper: Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. (If you can't get access to that publication, let me know and I'll -cough- help you.) There are also alternatives such as STAR followed by htseqcounts or featureCounts and DESeq2 or edgeR...

[saj98]

Hello Thank you so much for answering, I am not sure how to find the phred33 scale? I did the samples in Columbia genomic center, they are using Illumina pipeline. I am going to try switching to use HISAT2, StringTie and ballgown.

[Ram]

Sorry about my laziness (and the minor hijack), but could you explain why TopHat has been deprecated and what HISAT2 has as an advantage over TopHat please?

If this information is part of the paper, I can read it in its entirety when I get to work tomorrow (where I have institutional access to the paper)

[WouterDeCoster]

For example on the tophat webpage

TopHat 2.1.1 release 2/23/2016 Please note that TopHat has entered a low maintenance, low support stage as it is now largely superseded by HISAT2 which provides the same core functionality (i.e. spliced alignment of RNA-Seq reads), in a more accurate and much more efficient way.

[Ram]

Thank you, Wouter! #FILLERFILLERFILLER #WHYDOIHAVETODOTHIS

[saj98]

Actually, I am going to read the paper too. I got the error message today. They recommended me to update tophat or to switch to other tool to solve the error. I am going to spend tonight reading the article and let u know what I got.

[GenoMax]

You are using old versions of a program that itself is now deprecated. If you must use TopHat consider upgrading to the latest versions for it and bowtie etc. Ideally you should switch to using HISAT2/STAR/BBmap.

[saj98]

Thank you for answering, I am going to try to update the system or to switch to use HISAT2/STAR/BBmap.