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Old 05-22-2012, 07:39 AM   #1
wangli
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Default Tophat error "read #1 does not have a matching mate in the same order"

Hi, all

When using tophat commands, I am confronted with the following errors.

[root@** bin]# /home/bin/tophat -p 8 -G Ptrichocarpa_156_gene.gff3 -o CAR01 --max-intron-length 5000 -r 150 -m 2 --solexa1.3-quals /home/bin/bowtie-0.12.8/indexes/Pop_trichocarpa /home/****/Fastq/CAR01_1.fastq /home/****/Fastq/CAR01_2.fastq

[2012-05-22 10:24:03] Beginning TopHat run (v2.0.1)
-----------------------------------------------
[2012-05-22 10:24:03] Checking for Bowtie
Bowtie 2 not found, checking for older version..
Bowtie version: 0.12.8.0
[2012-05-22 10:24:03] Checking for Samtools
Samtools version: 0.1.18.0
[2012-05-22 10:24:03] Checking for Bowtie index files
[2012-05-22 10:24:03] Checking for reference FASTA file
[2012-05-22 10:24:03] Generating SAM header for /home/bin/bowtie-0.12.8/indexes/Pop_trichocarpa
format: fastq
quality scale: phred64 (reads generated with GA pipeline version >= 1.3)
[2012-05-22 10:24:03] Reading known junctions from GTF file
[2012-05-22 10:24:05] Preparing reads
[FAILED]
Error running 'prep_reads'
terminate called after throwing an instance of 'int'

I cannot figure out the problem. I am wondering about the flag " --solexa1.3-quals". Our reads are generated by Illumia Hi-seq. So, I assume it is >= 1.3. Is that right?

Any comments and suggestions are very appreciated.
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Old 05-22-2012, 01:24 PM   #2
harryzs
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Just remove --solexa1.3-quals and try.

My data are also from Illumia Hi-seq, and without --solexa1.3-quals, everything is fine with tophat v2.0.1
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Old 05-22-2012, 01:57 PM   #3
wangli
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Thank you, harryzs! I tried without --solexa1.3-quals, it worked.
But finally, I got the following error:

[2012-05-22 16:37:26] Reporting output tracks
[FAILED]
Error running /home/bin/tophat_reports --min-anchor 8 --splice-mismatches 2 --min-report-intron 50 --max-report-intron 5000 --min-isoform-fraction 0.15 --output-dir CAR01/ --max-multihits 20 --max-seg-multihits 40 --segment-length 25 --segment-mismatches 2 --min-closure-exon 100 --min-closure-intron 50 --max-closure-intron 5000 --min-coverage-intron 50 --max-coverage-intron 5000 --min-segment-intron 50 --max-segment-intron 5000 --max-mismatches 2 --max-insertion-length 3 --max-deletion-length 3 --bowtie1 -z gzip -p8 --inner-dist-mean 150 --inner-dist-std-dev 20 --gtf-annotations Ptrichocarpa_156_gene.gff3 --gtf-juncs CAR01/tmp/Ptrichocarpa_156_gene.juncs --no-closure-search --no-coverage-search --no-microexon-search --sam-header CAR01/tmp/Pop_trichocarpa_genome.bwt.samheader.sam --samtools=/home/bin/samtools --bowtie2-max-penalty 6 --bowtie2-min-penalty 2 --bowtie2-penalty-for-N 1 --bowtie2-read-gap-open 5 --bowtie2-read-gap-cont 3 --bowtie2-ref-gap-open 5 --bowtie2-ref-gap-cont 3 /home/bin/bowtie-0.12.8/indexes/Pop_trichocarpa.fa CAR01/junctions.bed CAR01/insertions.bed CAR01/deletions.bed CAR01/fusions.out CAR01/tmp/accepted_hits CAR01/tmp/left_kept_reads.m2g_um.candidates_and_unspl.bam CAR01/tmp/left_kept_reads.bam CAR01/tmp/right_kept_reads.m2g_um.candidates_and_unspl.bam CAR01/tmp/right_kept_reads.bam

./SeqAn-1.3/seqan/sequence/segment_infix.h:81 Assertion failed : data_begin_position <= data_end_position was: 18446744073709551569 > 51

In the output files, the accepted_hits.bam file doesnot exist.

Any idea?
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Old 05-22-2012, 11:15 PM   #4
harryzs
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Sorry, I have no idea about this.
You may post your question here :
http://seqanswers.com/forums/forumdisplay.php?f=18
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Old 05-23-2012, 01:13 PM   #5
wangli
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Thanks. I got answers from email to tophat.cufflinks@gmail.com
I am told that it is a bug of software. With application of the newly released version 2.0.2, it should be okay.
I will try again.
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Old 05-23-2012, 03:12 PM   #6
npochet
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I ran into the same error. Do you have any idea when we may expect the new release 2.0.2? Thanks much!
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Old 05-24-2012, 10:31 AM   #7
wangli
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The v.2.0.2 has been released recently. But the author said it is not totally ready to go. He still suggested to use v.2.0.0.
You can contact the above-mentioned email address. tophat.cufflinks@gmail.com
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Old 05-29-2012, 09:40 AM   #8
bmejake
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Hey all, even with the v2.0.3 release I'm getting this error:

[2012-05-29 13:16:00] Beginning TopHat run (v2.0.3)
-----------------------------------------------
[2012-05-29 13:16:00] Checking for Bowtie
Bowtie version: 0.12.7.0
[2012-05-29 13:16:00] Checking for Samtools
Samtools version: 0.1.18.0
[2012-05-29 13:16:00] Checking for Bowtie index files
[2012-05-29 13:16:00] Checking for reference FASTA file
Warning: Could not find FASTA file /lab/solexa_ploegh/jake/IGHlocus/MmusIGH.fa
[2012-05-29 13:16:00] Reconstituting reference FASTA file from Bowtie index
Executing: /usr/local/bin/bowtie-inspect /lab/solexa_ploegh/jake/IGHlocus/MmusIGH > /lab/solexa_ploegh/jake/IGHlocus/Bcell_counts/tmp/MmusIGH.fa
[2012-05-29 13:16:00] Generating SAM header for /lab/solexa_ploegh/jake/IGHlocus/MmusIGH
format: fastq
quality scale: phred64 (reads generated with GA pipeline version >= 1.3)
[2012-05-29 13:16:01] Preparing reads
[FAILED]
Error running 'prep_reads'
Error: read #1 (WIGTC-HISEQ:4:1101:1155:2046#ACAGTG/1;0) does not have a matching mate in the same order (WIGTC-HISEQ:4:1101:1155:2046#ACAGTG/2;0 found instead)


With this input:
tophat --solexa1.3-quals -o /lab/solexa_ploegh/jake/IGHlocus/Bcell_counts -p 8 --segment-length=20 --bowtie1 /lab/solexa_ploegh/jake/IGHlocus/MmusIGH /ACAGTG-s_4_1_sequence.txt /ACAGTG-s_4_2_sequence.txt

Any ideas why? I never had this error before the new releases, using these same datas.
Thanks for anyones thoughts!

Best,
Jake
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Old 05-29-2012, 11:41 AM   #9
wangli
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Currently, I am working with v 2.0.0, which works well.
From your error, it seems that your sequences are paired-end. In this case, -r flag is required.
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Old 05-29-2012, 12:06 PM   #10
bmejake
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Quote:
Originally Posted by wangli View Post
Currently, I am working with v 2.0.0, which works well.
From your error, it seems that your sequences are paired-end. In this case, -r flag is required.
Thanks for the advice, but after running with the -r option, I receive the same error.
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Old 06-03-2012, 10:14 AM   #11
planseq
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Hi Jake,

I had exactly the same issue (and on exactly the same computing cluster if I am not mistaken ) and just found that if you remove the ";0" or ";1" at the ends of the identifiers all runs fine.

Best,
Josien

Quote:
Originally Posted by bmejake View Post
Hey all, even with the v2.0.3 release I'm getting this error:

[2012-05-29 13:16:00] Beginning TopHat run (v2.0.3)
-----------------------------------------------
[2012-05-29 13:16:00] Checking for Bowtie
Bowtie version: 0.12.7.0
[2012-05-29 13:16:00] Checking for Samtools
Samtools version: 0.1.18.0
[2012-05-29 13:16:00] Checking for Bowtie index files
[2012-05-29 13:16:00] Checking for reference FASTA file
Warning: Could not find FASTA file /lab/solexa_ploegh/jake/IGHlocus/MmusIGH.fa
[2012-05-29 13:16:00] Reconstituting reference FASTA file from Bowtie index
Executing: /usr/local/bin/bowtie-inspect /lab/solexa_ploegh/jake/IGHlocus/MmusIGH > /lab/solexa_ploegh/jake/IGHlocus/Bcell_counts/tmp/MmusIGH.fa
[2012-05-29 13:16:00] Generating SAM header for /lab/solexa_ploegh/jake/IGHlocus/MmusIGH
format: fastq
quality scale: phred64 (reads generated with GA pipeline version >= 1.3)
[2012-05-29 13:16:01] Preparing reads
[FAILED]
Error running 'prep_reads'
Error: read #1 (WIGTC-HISEQ:4:1101:1155:2046#ACAGTG/1;0) does not have a matching mate in the same order (WIGTC-HISEQ:4:1101:1155:2046#ACAGTG/2;0 found instead)


With this input:
tophat --solexa1.3-quals -o /lab/solexa_ploegh/jake/IGHlocus/Bcell_counts -p 8 --segment-length=20 --bowtie1 /lab/solexa_ploegh/jake/IGHlocus/MmusIGH /ACAGTG-s_4_1_sequence.txt /ACAGTG-s_4_2_sequence.txt

Any ideas why? I never had this error before the new releases, using these same datas.
Thanks for anyones thoughts!

Best,
Jake
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Old 06-03-2012, 04:54 PM   #12
wangli
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Maybe the new version is not stable. I utilized V 2.0.0, it works well. You can write to the following email to report the error and for suggestions: tophat.cufflinks@gmail.com
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Old 06-04-2012, 05:16 AM   #13
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Thanks so much, Josien. Greetings from the Ploegh Lab

Jake
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Old 06-11-2012, 06:29 AM   #14
mikhmv
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Default temproary solution

you can try to rename
WIGTC-HISEQ:4:1101:1155:2046#ACAGTG/2;0 to WIGTC-HISEQ:4:1101:1155:2046#ACAGTG/1;0 in the paired file.

I mean make replacement for all read names in the second file to have /1;0 instead of /2;0.

Last edited by mikhmv; 06-11-2012 at 06:31 AM.
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Old 11-22-2012, 01:23 AM   #15
bob-loblaw
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I'm having the same problem now with bowtie2 and tophat v 2.0.3. and I don't have an 0 or 1 at the end of each identifier line? I'm using paired end reads from Illumina HiSeq. Any tips? I figured it might just be one read thats the problem so I removed it but then I got another one.


[2012-11-21 16:45:33] Beginning TopHat run (v2.0.3)
-----------------------------------------------
[2012-11-21 16:45:33] Checking for Bowtie
Bowtie version: 2.0.0.6
[2012-11-21 16:45:33] Checking for Samtools
Samtools version: 0.1.18.0
[2012-11-21 16:45:33] Checking for Bowtie index files
[2012-11-21 16:45:33] Checking for reference FASTA file
Warning: Could not find FASTA file hg19.fa
[2012-11-21 16:45:33] Reconstituting reference FASTA file from Bowtie index
Executing: /usr/local/bowtie2/bowtie2-inspect hg19 > ./tophat_out/tmp/hg19.fa
[2012-11-21 16:48:12] Generating SAM header for hg19
format: fastq
quality scale: phred64 (reads generated with GA pipeline version >= 1.3)
[2012-11-21 16:49:04] Preparing reads
[FAILED]
Error running 'prep_reads'
Error: read #1 (HWI-ST993:228:C0RWJACXX:6:2316:17166:97507) does not have a matching mate in the same order (HWI-ST993:228:C0RWJACXX:6:2316:12084:97378 found instead)
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Old 11-22-2012, 04:16 AM   #16
mikhmv
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Default solution

The help is here:
read #1 (HWI-ST993:228:C0RWJACXX:6:2316:17166:97507) does not have a matching mate in the same order (HWI-ST993:228:C0RWJACXX:6:2316:12084:97378 found instead)

The Red text should be the same for the same position for reads from Forward and Reverse file. If you manually removed one read from one file you need to remove a read with same file name from the second file

Numbers mean the coordinate on illumina slide. Basically you are trying to put in pairs reads with different coordinates.

Last edited by mikhmv; 11-22-2012 at 04:19 AM.
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Old 11-22-2012, 04:21 AM   #17
bob-loblaw
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Quote:
Originally Posted by mikhmv View Post
The help is here:
read #1 (HWI-ST993:228:C0RWJACXX:6:2316:17166:97507) does not have a matching mate in the same order (HWI-ST993:228:C0RWJACXX:6:2316:12084:97378 found instead)

The Red text should be the same for the same position for reads from Forward and Reverse file. If you manually removed one read from one file you need to remove a read with same file name from the second file

Numbers mean the coordinate on illumina slide. Basically you are trying to put in pairs reads with different coordinates.
I already tried removing reads from both files, but I got the same problem with a different read. So basically the reads need to be in the same order in each file then? Is there a way to sort a fastq file based on the coordinates?
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