Write Failure In Tophat 1.3.1 X86_64
7
0
Entering edit mode
14.0 years ago
Gabrielw ▴ 60

using tophat 1.3.1 x86_64...

this is my entire command: 

tophat -o /storage/gwajnberg/outputtophat/ \
          /storage/gwajnberg/bowtie_splitted/ \
          /storage/gwajnberg/SRP000614/SRX002932/SRR013983.fastq

where .../bowtie_splitted/ is the directory with the reference .fa file that originated the bowtie indexes files ( they are in the same directory). My reads have between 250 and 300 bp.

thats what happened:

[Fri Jul  1 12:49:59 2011] Beginning TopHat run (v1.3.1)
-----------------------------------------------
[Fri Jul  1 12:49:59 2011] Preparing output location ./tophat_out/
[Fri Jul  1 12:49:59 2011] Checking for Bowtie index files
[Fri Jul  1 12:49:59 2011] Checking for reference FASTA file
    Warning: Could not find FASTA file /storage/gwajnberg/bowtie_splitted/.fa
[Fri Jul  1 12:49:59 2011] Reconstituting reference FASTA file from Bowtie index
Executing: /storage/app/bowtie-0.12.5/bowtie-0.12.5/bowtie-inspect /storage/gwajnberg/bowtie_splitted/ > ./tophat_out/tmp/.fa
[Fri Jul  1 13:07:57 2011] Checking for Bowtie
    Bowtie version:             0.12.5.0
[Fri Jul  1 13:07:57 2011] Checking for Samtools
    Samtools Version: 0.1.16
[Fri Jul  1 13:07:58 2011] Generating SAM header for /storage/gwajnberg/bowtie_splitted/
[Fri Jul  1 13:08:00 2011] Preparing reads
    format:         fastq
    quality scale:     phred33 (default)
    Left  reads: min. length=25, count=121868
[Fri Jul  1 13:08:08 2011] Mapping left_kept_reads against  with Bowtie 
[Fri Jul  1 13:08:40 2011] Processing bowtie hits
[Fri Jul  1 13:10:32 2011] Mapping left_kept_reads_seg1 against  with Bowtie (1/33)
.......
[Fri Jul  1 13:34:37 2011] Mapping left_kept_reads_seg33 against segment_juncs with Bowtie (33/33)
[Fri Jul  1 13:34:38 2011] Joining segment hits
sort: write failed: standard output: Broken pipe
sort: write error
[Fri Jul  1 13:37:11 2011] Reporting output tracks
    [FAILED]
Error: [Errno 2] No such file or directory
Found 0 junctions from happy spliced reads

Does anyone know how to help me? thanks!
tophat sequencing splicing • 8.6k views
ADD COMMENT •
0
Entering edit mode

A quick guess: you forgot to provide the path to your reference fasta file.

ADD REPLY •
0
Entering edit mode

Haven't run tophat (yet), but it looks like the "joining segment hits" step failed. Broken Pipe means one command was expecting to eat the output of another command via a Unix pipe, and one of the two commands failed. Then tophat tried to write some output at "Reporting output tracks", but that probably failed because of the earlier problem.

ADD REPLY •
0
Entering edit mode

Well, there isn´t any option to specify where is the fasta file....the command is :

tophat [options] [?] < read file> there is only an option to choose the output directory, but its not necessary, because there is a default directory!

somewhere I read that the reference file ( fa or fasta) must be in the same directory of bowtie indexes... and that´s what I did!

I built the indexes from this specified index! Somwhere I also read that if there isn´t a ref file, the programa builds a reference file from the recognized indexes files. I´ll try some othere things later, s

ADD REPLY •
2
Entering edit mode
14.0 years ago
seidel 11k

From the tophat documentation: When you run tophat, the first argument should be the full path to the directory containing the index plus the prefix of the index files.

Your command has a path, but no index listed. For instance, if you're studying Nematostella, and your index files all start with nematostella, then presumably you would also have a fasta file with that same prefix. So your index argument would look like this: /storage/gwajnberg/bowtie_splitted/nematostella
ADD COMMENT •
0
Entering edit mode

I´m moved my ref to /bowtie_splitted/hg19 no my reference name is hg19.fa and I´m building a new index from it...with prefix hg19 .... so I´ll try to run tophat again tomorrow ...so pray :)

ADD REPLY •
0
Entering edit mode

I voted for you because your answer helped me to understand a little bit more, but I realized that my installation isn 't working with the test file from tophat

ADD REPLY •
1
Entering edit mode
14.0 years ago
Docroberson ▴ 30

I think Michael is probably right.

Warning: Could not find FASTA file /storage/gwajnberg/bowtie_splitted/.fa

The fasta file was not specified correctly. The error is actually rather cryptic. If specifying the FASTA file correct doesn't work try posting the command you used to dissect any problems there.
ADD COMMENT •
0
Entering edit mode

Well, there isn´t any option to specify where is the fasta file....the command is :

tophat [options] [?] < read file> there is only an option to choose the output directory, but its not necessary, because there is a default directory!

somewhere I read that the reference file ( fa or fasta) must be in the same directory of bowtie indexes... and that´s what I did!

I built the indexes from this specified index! Somwhere I also read that if there isn´t a ref file, the programa builds a reference file from the recognized indexes files. I´ll try some othere things later, s

ADD REPLY •
1
Entering edit mode
14.0 years ago
Darked89 4.7k

You have to post your entire command used to run tophat. The message:

Could not find FASTA file /storage/gwajnberg/bowtie_splitted/.fa

indicates that you may have not specified your bowtie index correctly. If your index has the name prefix: your_genome.bowtie then your reference fasta should be your_genome.bowtie.fa. BTW, if the your_genome.bowtie.fa is absent it should be not a big deal except for some wasting some CPU time for recreating it from indexes.

The more troubling thing is the length of your sequences. If you got 25bp reads these can not be split with tophat, for sure not running with default options, and possibly not even with "--segment-length 12" if you do not have genome annotation and large genome. Either use untruncated, full length reads (preferably longer than 50bp) or split reads into 50bp+ vs shorter ones and map these in different modes (spliced with tophat, unspliced with bowtie).

Last but not least: update to bowtie 0.12.7
ADD COMMENT •
0
Entering edit mode

size of reads : between 250 and 300 bp .fa file in the same directory of bowtie indexes .... using this command : >tophat [options] [?] < read file>

ADD REPLY •
0
Entering edit mode
14.0 years ago
Gabrielw ▴ 60

this is my entire command: tophat -o /storage/gwajnberg/outputtophat/ /storage/gwajnberg/bowtie_splitted/ /storage/gwajnberg/SRP000614/SRX002932/SRR013983.fastq

where .../bowtie_splitted/ is the directory with the reference .fa file that originated the bowtie indexes files ( they are in the same directory). My reads have between 250 and 300 bp...

I'm trying using 0.12.7 version of bowtie, I was using 0.12.5 ....
ADD COMMENT •
0
Entering edit mode

the same thing happened :(

ADD REPLY •
0
Entering edit mode

/storage/gwajnberg/bowtie_splitted/ does not correspond to the bowtie index prefix. You are missing the indexed genome for mapping.

ADD REPLY •
0
Entering edit mode
14.0 years ago
Gabrielw ▴ 60

I Tried what everyone said and continued happening this.... my splitted index bowtie is with prefix hg19 and my fasta file is hg19.fa in the same directory.... :(

gwajnberg@lbbc02:~/tophat-1.3.1.Linux_x86_64$ ./tophat -o /storage/gwajnberg/outputtophat/ /storage/gwajnberg/bowtie_splitted/hg19/hg19 /storage/gwajnberg/SRP000614/SRX002932/SRR013983.fastq
[Tue Jul 5 12:59:34 2011] Beginning TopHat run (v1.3.1)
[Tue Jul 5 12:59:34 2011] Preparing output location /storage/gwajnberg/outputtophat//
[Tue Jul 5 12:59:34 2011] Checking for Bowtie index files
[Tue Jul 5 12:59:34 2011] Checking for reference FASTA file
[Tue Jul 5 12:59:34 2011] Checking for Bowtie
Bowtie version: 0.12.7.0
[Tue Jul 5 12:59:34 2011] Checking for Samtools
Samtools Version: 0.1.16
[Tue Jul 5 12:59:34 2011] Generating SAM header for /storage/gwajnberg/bowtie_splitted/hg19/hg19
[Tue Jul 5 12:59:58 2011] Preparing reads
format: fastq
quality scale: phred33 (default)
Left reads: min. length=25, count=121868
[Tue Jul 5 13:00:06 2011] Mapping left_kept_reads against hg19 with Bowtie
[Tue Jul 5 13:00:38 2011] Processing bowtie hits
[Tue Jul 5 13:02:24 2011] Mapping left_kept_reads_seg1 against hg19 with Bowtie (1/33)
[Tue Jul 5 13:04:09 2011] Mapping left_kept_reads_seg2 against hg19 with Bowtie (2/33)
[Tue Jul 5 13:06:00 2011] Mapping left_kept_reads_seg3 against hg19 with Bowtie (3/33)
[Tue Jul 5 13:07:48 2011] Mapping left_kept_reads_seg4 against hg19 with Bowtie (4/33)
[Tue Jul 5 13:09:35 2011] Mapping left_kept_reads_seg5 against hg19 with Bowtie (5/33)
[Tue Jul 5 13:11:20 2011] Mapping left_kept_reads_seg6 against hg19 with Bowtie (6/33)
[Tue Jul 5 13:13:00 2011] Mapping left_kept_reads_seg7 against hg19 with Bowtie (7/33)
[Tue Jul 5 13:14:36 2011] Mapping left_kept_reads_seg8 against hg19 with Bowtie (8/33)
[Tue Jul 5 13:16:04 2011] Mapping left_kept_reads_seg9 against hg19 with Bowtie (9/33)
[Tue Jul 5 13:17:21 2011] Mapping left_kept_reads_seg10 against hg19 with Bowtie (10/33)
[Tue Jul 5 13:18:16 2011] Mapping left_kept_reads_seg11 against hg19 with Bowtie (11/33)
[Tue Jul 5 13:18:47 2011] Mapping left_kept_reads_seg12 against hg19 with Bowtie (12/33)
[Tue Jul 5 13:18:56 2011] Mapping left_kept_reads_seg13 against hg19 with Bowtie (13/33)
[Tue Jul 5 13:19:00 2011] Mapping left_kept_reads_seg14 against hg19 with Bowtie (14/33)
[Tue Jul 5 13:19:03 2011] Mapping left_kept_reads_seg15 against hg19 with Bowtie (15/33)
[Tue Jul 5 13:19:06 2011] Mapping left_kept_reads_seg16 against hg19 with Bowtie (16/33)
[Tue Jul 5 13:19:08 2011] Mapping left_kept_reads_seg17 against hg19 with Bowtie (17/33)
[Tue Jul 5 13:19:11 2011] Mapping left_kept_reads_seg18 against hg19 with Bowtie (18/33)
[Tue Jul 5 13:19:14 2011] Mapping left_kept_reads_seg19 against hg19 with Bowtie (19/33)
[Tue Jul 5 13:19:16 2011] Mapping left_kept_reads_seg20 against hg19 with Bowtie (20/33)
[Tue Jul 5 13:19:19 2011] Mapping left_kept_reads_seg21 against hg19 with Bowtie (21/33)
[Tue Jul 5 13:19:21 2011] Mapping left_kept_reads_seg22 against hg19 with Bowtie (22/33)
[Tue Jul 5 13:19:24 2011] Mapping left_kept_reads_seg23 against hg19 with Bowtie (23/33)
[Tue Jul 5 13:19:27 2011] Mapping left_kept_reads_seg24 against hg19 with Bowtie (24/33)
[Tue Jul 5 13:19:29 2011] Mapping left_kept_reads_seg25 against hg19 with Bowtie (25/33)
[Tue Jul 5 13:19:32 2011] Mapping left_kept_reads_seg26 against hg19 with Bowtie (26/33)
[Tue Jul 5 13:19:34 2011] Mapping left_kept_reads_seg27 against hg19 with Bowtie (27/33)
[Tue Jul 5 13:19:37 2011] Mapping left_kept_reads_seg28 against hg19 with Bowtie (28/33)
[Tue Jul 5 13:19:39 2011] Mapping left_kept_reads_seg29 against hg19 with Bowtie (29/33)
[Tue Jul 5 13:19:42 2011] Mapping left_kept_reads_seg30 against hg19 with Bowtie (30/33)
[Tue Jul 5 13:19:45 2011] Mapping left_kept_reads_seg31 against hg19 with Bowtie (31/33)
[Tue Jul 5 13:19:47 2011] Mapping left_kept_reads_seg32 against hg19 with Bowtie (32/33)
[Tue Jul 5 13:19:50 2011] Mapping left_kept_reads_seg33 against hg19 with Bowtie (33/33)
[Tue Jul 5 13:19:52 2011] Searching for junctions via segment mapping
[Tue Jul 5 13:21:31 2011] Retrieving sequences for splices
[Tue Jul 5 13:25:29 2011] Indexing splices
[Tue Jul 5 13:25:30 2011] Mapping left_kept_reads_seg1 against segment_juncs with Bowtie (1/33)
[Tue Jul 5 13:25:34 2011] Mapping left_kept_reads_seg2 against segment_juncs with Bowtie (2/33)
[Tue Jul 5 13:25:39 2011] Mapping left_kept_reads_seg3 against segment_juncs with Bowtie (3/33)
[Tue Jul 5 13:25:43 2011] Mapping left_kept_reads_seg4 against segment_juncs with Bowtie (4/33)
[Tue Jul 5 13:25:48 2011] Mapping left_kept_reads_seg5 against segment_juncs with Bowtie (5/33)
[Tue Jul 5 13:25:52 2011] Mapping left_kept_reads_seg6 against segment_juncs with Bowtie (6/33)
[Tue Jul 5 13:25:57 2011] Mapping left_kept_reads_seg7 against segment_juncs with Bowtie (7/33)
[Tue Jul 5 13:26:01 2011] Mapping left_kept_reads_seg8 against segment_juncs with Bowtie (8/33)
[Tue Jul 5 13:26:05 2011] Mapping left_kept_reads_seg9 against segment_juncs with Bowtie (9/33)
[Tue Jul 5 13:26:08 2011] Mapping left_kept_reads_seg10 against segment_juncs with Bowtie (10/33)
[Tue Jul 5 13:26:12 2011] Mapping left_kept_reads_seg11 against segment_juncs with Bowtie (11/33)
[Tue Jul 5 13:26:13 2011] Mapping left_kept_reads_seg12 against segment_juncs with Bowtie (12/33)
[Tue Jul 5 13:26:14 2011] Mapping left_kept_reads_seg13 against segment_juncs with Bowtie (13/33)
[Tue Jul 5 13:26:15 2011] Mapping left_kept_reads_seg14 against segment_juncs with Bowtie (14/33)
[Tue Jul 5 13:26:16 2011] Mapping left_kept_reads_seg15 against segment_juncs with Bowtie (15/33)
[Tue Jul 5 13:26:16 2011] Mapping left_kept_reads_seg16 against segment_juncs with Bowtie (16/33)
[Tue Jul 5 13:26:17 2011] Mapping left_kept_reads_seg17 against segment_juncs with Bowtie (17/33)
[Tue Jul 5 13:26:17 2011] Mapping left_kept_reads_seg18 against segment_juncs with Bowtie (18/33)
[Tue Jul 5 13:26:18 2011] Mapping left_kept_reads_seg19 against segment_juncs with Bowtie (19/33)
[Tue Jul 5 13:26:19 2011] Mapping left_kept_reads_seg20 against segment_juncs with Bowtie (20/33)
[Tue Jul 5 13:26:19 2011] Mapping left_kept_reads_seg21 against segment_juncs with Bowtie (21/33)
[Tue Jul 5 13:26:20 2011] Mapping left_kept_reads_seg22 against segment_juncs with Bowtie (22/33)
[Tue Jul 5 13:26:21 2011] Mapping left_kept_reads_seg23 against segment_juncs with Bowtie (23/33)
[Tue Jul 5 13:26:21 2011] Mapping left_kept_reads_seg24 against segment_juncs with Bowtie (24/33)
[Tue Jul 5 13:26:22 2011] Mapping left_kept_reads_seg25 against segment_juncs with Bowtie (25/33)
[Tue Jul 5 13:26:22 2011] Mapping left_kept_reads_seg26 against segment_juncs with Bowtie (26/33)
[Tue Jul 5 13:26:23 2011] Mapping left_kept_reads_seg27 against segment_juncs with Bowtie (27/33)
[Tue Jul 5 13:26:24 2011] Mapping left_kept_reads_seg28 against segment_juncs with Bowtie (28/33)
[Tue Jul 5 13:26:24 2011] Mapping left_kept_reads_seg29 against segment_juncs with Bowtie (29/33)
[Tue Jul 5 13:26:25 2011] Mapping left_kept_reads_seg30 against segment_juncs with Bowtie (30/33)
[Tue Jul 5 13:26:25 2011] Mapping left_kept_reads_seg31 against segment_juncs with Bowtie (31/33)
[Tue Jul 5 13:26:26 2011] Mapping left_kept_reads_seg32 against segment_juncs with Bowtie (32/33)
[Tue Jul 5 13:26:27 2011] Mapping left_kept_reads_seg33 against segment_juncs with Bowtie (33/33)
[Tue Jul 5 13:26:27 2011] Joining segment hits
sort: write failed: standard output: Broken pipe
sort: write error
[Tue Jul 5 13:29:01 2011] Reporting output tracks
[FAILED]
Error: [Errno 2] No such file or directory
Found 0 junctions from happy spliced reads
view raw biostars-9903 hosted with ❤ by GitHub

ADD COMMENT •
0
Entering edit mode

Can you verify if your installation of tophat works? At http://tophat.cbcb.umd.edu/tutorial.html there is a section called: "Testing the Installation". It's a simple test. If that works, you can add complication until it breaks. If nothing else, at least you made it one step further.

ADD REPLY •
0
Entering edit mode

you were right !!! I used "testing the installation" and happened the same thing!!! thanksss now I'll try to install again :(

ADD REPLY •
0
Entering edit mode
14.0 years ago
Gabrielw ▴ 60

Well thanks all for helping me....that's what happened... tophat 1.3.1 x86_64 and tophat 1.3.0 x86_64 are bugged and when I used them with the test data from tophat homepage , it happened the same error that happened with my files... so I downloaded the version 1.2.0 x86_64 and worked very well... thanks all for the support :)
ADD COMMENT •
0
Entering edit mode

Sorry, should have comment here: Hey gabrielw. Where did you found that tophat 1.3.1 x86_64 and tophat 1.3.0 x86_64 are bugged?

Thanks!

ADD REPLY •
0
Entering edit mode
13.7 years ago
Rodrigo • 0

Hey gabrielw. Where did you found that tophat 1.3.1 x86_64 and tophat 1.3.0 x86_64 are bugged?

Thanks!
ADD COMMENT •

Login before adding your answer.

Similar Posts
Error! readyState=0 status=error text=
Loading Similar Posts
Traffic: 2935 users visited in the last hour
Content Search
Users
Tags
Badges
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6