Question: Tophat error! Warning: junction database is empty! Could anybody help?
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gravatar for svaturose
3.2 years ago by
svaturose0
svaturose0 wrote:

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.

transcriptome rna-seq tophat • 2.3k views
ADD COMMENTlink modified 3.2 years ago by Satyajeet Khare1.5k • written 3.2 years ago by svaturose0
2

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?

ADD REPLYlink modified 3.2 years ago • written 3.2 years ago by genomax74k

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!

ADD REPLYlink written 3.2 years ago by svaturose0

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.

ADD REPLYlink modified 3.2 years ago • written 3.2 years ago by Satyajeet Khare1.5k

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)

ADD REPLYlink written 3.2 years ago by svaturose0
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