[Tophat2] - Warning: junction database is empty!

[Puhekupla]

Hello All,

I'm doing Tophat2 on 10 samples, from which 3 samples should contain RNA-seq spikes sequences. I've build my own index files from +- 8 spike sequences via bowtie2-build. Now i'm trying to use Tophat2 whether to check if the spike sequences are found in the samples or not. Everything works fine untill I get one error I can't fix:

[2014-02-19 09:06:01] Beginning TopHat run (v2.0.9)
-----------------------------------------------
[2014-02-19 09:06:01] Checking for Bowtie
		  Bowtie version:	 2.1.0.0
[2014-02-19 09:06:01] Checking for Samtools
		Samtools version:	 0.1.19.0
[2014-02-19 09:06:01] Checking for Bowtie index files (genome)..
[2014-02-19 09:06:01] Checking for reference FASTA file
[2014-02-19 09:06:01] Generating SAM header for /indexes_spikes/spikes
	format:		 fastq
	quality scale:	 phred33 (default)
[2014-02-19 09:06:02] Preparing reads
	 left reads: min. length=139, max. length=151, 424139 kept reads (30263 discarded)
[2014-02-19 09:06:19] Mapping left_kept_reads to genome spikes with Bowtie2 
[2014-02-19 09:09:37] Mapping left_kept_reads_seg1 to genome spikes with Bowtie2 (1/6)
[2014-02-19 09:09:54] Mapping left_kept_reads_seg2 to genome spikes with Bowtie2 (2/6)
[2014-02-19 09:10:12] Mapping left_kept_reads_seg3 to genome spikes with Bowtie2 (3/6)
[2014-02-19 09:10:28] Mapping left_kept_reads_seg4 to genome spikes with Bowtie2 (4/6)
[2014-02-19 09:10:44] Mapping left_kept_reads_seg5 to genome spikes with Bowtie2 (5/6)
[2014-02-19 09:10:58] Mapping left_kept_reads_seg6 to genome spikes with Bowtie2 (6/6)
[2014-02-19 09:11:11] Searching for junctions via segment mapping
Warning: junction database is empty!
[2014-02-19 09:12:10] Reporting output tracks
	[FAILED]

Anyone knows whats going wrong?

[gfmgfm]

Hello,

We get the same error with aligning RNA seq spikes with tophat2.
Did you find the problem?

Thanks

[wokai001]

Probably, TopHat does not find any junctions.
Try an alignment with Bowtie and look for unmapped reads.

You may try to extract these reads from BAM files, convert them into fastq and then re-align using TopHat.

[gfmgfm]

Thanks a lot for the answer.

This is indeed because of empty junction.
The way to overcome this error is by adding --no-novel-juncs