Tophat error! Warning: junction database is empty! Could anybody help?

Question

[svaturose]

Hi! I'm trying to run tophat for rna-seq analysis. but encountered some errors. First of all,

>[2016-09-03 11:13:45] Beginning TopHat run (v2.1.1)
>-----------------------------------------------
>[2016-09-03 11:13:45] Checking for Bowtie
>         Bowtie version:    2.2.9.0
>[2016-09-03 11:13:45] Checking for Bowtie index files (genome)..>
>[2016-09-03 11:13:45] Checking for reference FASTA file
>   Warning: Could not find FASTA file ./index_files/BDGP75.fa
>[2016-09-03 11:13:45] Reconstituting reference FASTA file from Bowtie index
>  Executing: /Users/chungmyung/Documents/bowtie2-2.2.9/bowtie2-inspect ./index_files/BDGP75 > SRR1281994_1_mapped/tmp/BDGP75.fa
>[2016-09-03 11:13:45] Generating SAM header for ./index_files/BDGP75
>[2016-09-03 11:13:45] Preparing reads
>    left reads: min. length=36, max. length=70, 41923399 kept reads (1896 discarded)
>   right reads: min. length=36, max. length=70, 41923770 kept reads (1525 discarded)
>[2016-09-03 13:42:37] Mapping left_kept_reads to genome BDGP75 with Bowtie2 
>[2016-09-03 17:14:22] Mapping left_kept_reads_seg1 to genome BDGP75 with Bowtie2 (1/3)
>[2016-09-03 17:18:54] Mapping left_kept_reads_seg2 to genome BDGP75 with Bowtie2 (2/3)
>[2016-09-03 17:23:46] Mapping left_kept_reads_seg3 to genome BDGP75 with Bowtie2 (3/3)
>[2016-09-03 17:27:49] Mapping right_kept_reads to genome BDGP75 with Bowtie2 
>[2016-09-03 18:14:22] Mapping right_kept_reads_seg1 to genome BDGP75 with Bowtie2 (1/3)
>[2016-09-03 18:19:10] Mapping right_kept_reads_seg2 to genome BDGP75 with Bowtie2 (2/3)
>[2016-09-03 18:24:41] Mapping right_kept_reads_seg3 to genome BDGP75 with Bowtie2 (3/3)
>[2016-09-03 18:27:29] Searching for junctions via segment mapping
>   [FAILED]
>Error: segment-based junction search failed with err =-5
>  Reason: image not found

---

happened. So I had searched for the troubleshooting and ppl said it could happen on the latest version of tophat. (I was using tophat-2.1.1.OSX_x86_64) So I re-download it(ver.2.0.14) and ran it again.

Chungs-MacBook-Air:RNA_seq_analysis chungmyung$ tophat -o SRR1281994_1_mapped ./index_files/BDGP75 /Users/chungmyung/Desktop/Users/chungmyung/Desktop/RNA_seq_analysis/After_trimmomatic/SRR1281994_1_paired_output_1.fq /Users/chungmyung/Desktop/RNA_seq_analysis/After_trimmomatic/SRR1281994_1_paired_output_2.fq 

[2016-09-03 22:02:00] Beginning TopHat run (v2.0.14)
-----------------------------------------------
[2016-09-03 22:02:00] Checking for Bowtie
          Bowtie version:    2.2.9.0
[2016-09-03 22:02:00] Checking for Bowtie index files (genome)..
[2016-09-03 22:02:00] Checking for reference FASTA file
    Warning: Could not find FASTA file ./index_files/BDGP75.fa
[2016-09-03 22:02:00] Reconstituting reference FASTA file from Bowtie index
  Executing: /Users/chungmyung/Documents/bowtie2-2.2.9/bowtie2-inspect ./index_files/BDGP75 > SRR1281994_1_mapped/tmp/BDGP75.fa
[2016-09-03 22:02:00] Generating SAM header for ./index_files/BDGP75
Traceback (most recent call last):
  File "/Users/chungmyung/Documents/tophat-2.0.14.OSX_x86_64/tophat", line 4095, in <module>
    sys.exit(main())
  File "/Users/chungmyung/Documents/tophat-2.0.14.OSX_x86_64/tophat", line 3949, in main
    params.read_params = check_reads_format(params, reads_list)
  File "/Users/chungmyung/Documents/tophat-2.0.14.OSX_x86_64/tophat", line 1844, in check_reads_format
    zf = ZReader(f_name, params)
  File "/Users/chungmyung/Documents/tophat-2.0.14.OSX_x86_64/tophat", line 1797, in __init__
    self.file=open(filename)
IOError: [Errno 2] No such file or directory: '/Users/chungmyung/Desktop/Users/chungmyung/Desktop/RNA_seq_analysis/After_trimmomatic/SRR1281994_1_paired_output_1.fq'
Chungs-MacBook-Air:RNA_seq_analysis chungmyung$ tophat -o SRR1281994_1_mapped ./index_files/BDGP75 /Users/chungmyung/Desktop/RNA_seq_analysis/After_trimmomatic/SRR1281994_1_paired_output_1.fq /Users/chungmyung/Desktop/RNA_seq_analysis/After_trimmomatic/SRR1281994_1_paired_output_2.fq 

[2016-09-03 22:02:36] Beginning TopHat run (v2.0.14)
-----------------------------------------------
[2016-09-03 22:02:36] Checking for Bowtie
          Bowtie version:    2.2.9.0
[2016-09-03 22:02:36] Checking for Bowtie index files (genome)..
[2016-09-03 22:02:36] Checking for reference FASTA file
    Warning: Could not find FASTA file ./index_files/BDGP75.fa
[2016-09-03 22:02:36] Reconstituting reference FASTA file from Bowtie index
  Executing: /Users/chungmyung/Documents/bowtie2-2.2.9/bowtie2-inspect ./index_files/BDGP75 > SRR1281994_1_mapped/tmp/BDGP75.fa
[2016-09-03 22:02:36] Generating SAM header for ./index_files/BDGP75
[2016-09-03 22:02:36] Preparing reads
     left reads: min. length=36, max. length=70, 41923399 kept reads (1896 discarded)
    right reads: min. length=36, max. length=70, 41923770 kept reads (1525 discarded)
[2016-09-03 22:37:58] Mapping left_kept_reads to genome BDGP75 with Bowtie2 
[2016-09-03 23:28:47] Mapping left_kept_reads_seg1 to genome BDGP75 with Bowtie2 (1/3)
[2016-09-03 23:32:49] Mapping left_kept_reads_seg2 to genome BDGP75 with Bowtie2 (2/3)
[2016-09-03 23:37:07] Mapping left_kept_reads_seg3 to genome BDGP75 with Bowtie2 (3/3)
[2016-09-03 23:40:40] Mapping right_kept_reads to genome BDGP75 with Bowtie2 
[2016-09-04 00:27:35] Mapping right_kept_reads_seg1 to genome BDGP75 with Bowtie2 (1/3)
[2016-09-04 00:31:53] Mapping right_kept_reads_seg2 to genome BDGP75 with Bowtie2 (2/3)
[2016-09-04 00:36:56] Mapping right_kept_reads_seg3 to genome BDGP75 with Bowtie2 (3/3)
[2016-09-04 00:39:22] Searching for junctions via segment mapping
Warning: junction database is empty!
[2016-09-04 00:51:40] Retrieving sequences for splices
[2016-09-04 00:51:40] Indexing splices
Warning: Empty fasta file: 'SRR1281994_1_mapped/tmp/segment_juncs.fa'
Warning: All fasta inputs were empty
Error: Encountered internal Bowtie 2 exception (#1)
Command: bowtie2-build --wrapper basic-0 SRR1281994_1_mapped/tmp/segment_juncs.fa SRR1281994_1_mapped/tmp/segment_juncs 
    [FAILED]
Error: Splice sequence indexing failed with err =1

And now I have no idea. (+I am a very beginner. I have just studied rna-seq analysis for around a week and even don't know how to upload the code properly on this site.) Could anybody help me with this situation? Can I still use the mapped sequences? Should I download something else? It would be very helpful. Thanks.

[GenoMax]

The errors are likely not due to the version of tophat but they are due to the way you are issuing the tophat command. For example.

Warning: Could not find FASTA file ./index_files/BDGP75.fa

No such file or directory: '/Users/chungmyung/Desktop/Users/chungmyung/Desktop/RNA_seq_analysis/After_trimmomatic/SRR1281994_1_paired_output_1.fq'

file not found errors. That means that you are not providing correct full file paths or relative paths.

If you are new to command line then learning a bit of unix would be the best couple of hours you will spend (more would be needed to become proficient). You can take a look at the unix tutorial here.

Can you provide the command you are using for starting this search?

[svaturose]

Thank you for the comment. However, it seems there is the file named SRR1281994_1_paired_1.fq at the correct place. I am still not sure what the problem is.

I deleted the command line I used unfortunately :(

+Thank you so much for the link!

[Satyajeet Khare]

Try tophat with option --no-coverage-search. It might be a temporary fix though. It looks like Mac specific error on most of the forums.

[svaturose]

Thank you for the comment. could you give me more details or related links? Hmm... if it is Mac specific error, is there any solution for it? Should I move it to Window?(I only use Mac and Window... No Linux)