Tophat error "read #1 does not have a matching mate in the same order"

[wangli]

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.

[harryzs]

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

[wangli]

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?

[harryzs]

Sorry, I have no idea about this.
You may post your question here :

http://seqanswers.com/forums/forumdisplay.php?f=18

[wangli]

Thanks. I got answers from email to [email protected]
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.

[npochet]

I ran into the same error. Do you have any idea when we may expect the new release 2.0.2? Thanks much!

[wangli]

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. [email protected]

[bmejake]

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

[wangli]

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.

[bmejake]

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.

[planseq]

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

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

[wangli]

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: [email protected]

[bmejake]

Thanks so much, Josien. Greetings from the Ploegh Lab

Jake

[mikhmv]

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.

[bob-loblaw]

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)