tophat failed: Warning: junction database is empty!

Question

[biolab]

Hi everyone

I embedded tophat command in a perl script. When running the script, I found a tophat failure error. I searched similar problem posts, but none of them were resolved

• http://seqanswers.com/forums/showthread.php?t=41021
• http://seqanswers.com/forums/showthread.php?t=27454
• Tophat Warning: Junction Database Is Empty!

Could you please provide me some advice? I appreciate any of your answers. THANKS

Error message:

[2015-03-01 18:02:45] Searching for junctions via segment mapping
Warning: junction database is empty!
[2015-03-01 18:02:45] Reporting output tracks
    [FAILED]
Error running /usr/local/tophat/bin/tophat_reports --min-anchor 8 --splice-mismatches 0 --min-
... ...

FASTQ file:

@read:C:Chr1:m:29063581:29064263:267 length=100
CACTGGTGATCAGATTGAAGTTCTTAGCAACGGAAGCTGGTGAACCATGGTATTCCATTGGAGCTTCTAAATAAGGTGAAGAAGCTGAGCTCAGATTGCT
+
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
@read1
AGGAACTTGATTAGCAAAATCTCCAAGGATTATGTGAAGCTaCTCAAGTATGGTGATTCTTTGTACCGCTGCAATTGTTCCCAATGGGAAGGAGTTCGTT
+
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
@read2
ACTCTCAGGTACCAAGTCATTGATACCCCCGGGATTTTGGATAGGCCCTTCCATGCGAAGCTCTCTGCAATCATTGACGACTTGCTGCTCTTGGTCGTAC
+
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

Reference File:

>genome
aagaaaaaggcacacacatttatattcaaaaactaagaacataggtcacacaagaaagattcatacatcacatggcaaaacaaaaaaaaacaaaaaaatataaagcctggagagcagatgccaaatttttaactatataatttggcatttatagcttaagagaaaatacataatgggtgtagataaaacagtgatagtactgtaaaaataaataaaatagtacatTTAGAGAGACTTTACAGCTAGAAAACGAGGCTCTTTAGGCATGAACTTCTGGTTTGCATAAACATCCATGTAATCTCCAAACACAAACTTTGGATACTTCTTCTCACTTCCTTCCTCTTCCGCCACCGCGGCTGGCCCTATCGCCGCCTTGTACGACGGATTGTAGAAGGAAGCTATAGACCTTCTGTTTCCTTCCTCCCTCGCCAACACCCTGTGCCACGCACTCTTGTACctgccaaccacattttggttaaaactttatactcaaaattgtatattttgtactagtgttgtcgtcgtgtcaccactgtttccggatcaagtcgagattagtgttttggttcgtaaaagatgagtgaaaaatacCTTCCGTTGCTAAGAACTTCAATCTGATCACCAGTGTTGATAACAATGGCATTAGGTAGAGGCTGAACATCGATCCACTCGCCGTCTTTCAAGACCTGAAGGCCATCATATTCATCGTCTTGGAAAAGCAAAACGACACCTCCTGCATCAGTATGAGCTCGAAGGCCATTGACTAGCTCAGGATGAGGACAAGGAGGGTAATGGCTGACTTTAGTCCCAAAGAAAGCTGTCTCTTCTCCATCTTCCATTCCTTCATTGAAAGCTTTCTTTATGTAACCTTTAGGCAAACCCAAATTCTCATCCATCACTTCCATCATCTTGCTCGCTAGCTTCCTCACTTCTTCTCTGTATTCTCCCATAGTCTCTctgtttttacgtacaaatagaaaaacacaaaggtgagatttttgaaaatgggtttgaaagattttgactagttttaaaagagaaaatgtttacTTAATGTTGGATGGCCATTCGTTTTGGTTATGGTCCAAGAGAGTGAAGACATCTTCCCAGTCCACGTTTTCTAGCTTCTCGCCAGAGTTCTTTTGAACCAATTCGTTGAGCAACTTCACGGGATTAGAAGTCTTGAATGCTTCTTCTCTCTCTGTTTTGTAGCAATCTGAGCTCAGCTTCTTCACCTTATTTAGAAGCTCCAATGGAATACCATGGTTCACCAGctacatacatacacagataagcacatgaaaacagagagacagagagagtgtgtgttctgttttttgttagttagatccgaacCTGAAAAAATCCCCACTCTTCGCAAGCTCTAGCGATTTCAGACAGTGTCTTCTCTCTTTCTTCACCATTGAGTTTGGAGAAATCGATAACAGGAATCGCCATttcagatccgcaaagagagagagagagacagatgtgtttttggcaagtcaaaaaagactctgtttagtagtaatcttctgatttagttgt

[GouthamAtla]

Can you post the exact commands?

[biolab]

My command is

tophat2 -p 8 --b2-very-sensitive INDEX in.fq > tophat.log

[GouthamAtla]

Can you run just bowtie2 and see what happens?

[biolab]

Running bowtie2 (bowtie2 -x INDEX -U in.fq -S out.sam) is OK. THANKS!

[GouthamAtla]

I am not suggesting you to run bowtie2. I just wanted to know if bowtie2 fails. If its RNA-Seq data and you are aligning to reference genome instead of transcriptome, you should use splice aware aligner like tophat2.

Which genome it is ?

[biolab]

Hi Geeky, it is my test data, which contains only one sequence. I have run true data, and tophat works now. However, I am still confused of the error in testing.