Tophat prep_reads error

[ega2d]

Has anyone seen this error before? I had Tophat working fine, but wanted to use the newly provided Illumina bowtie_index and I'm now getting this error:

[Thu Aug 4 13:56:11 2011] Beginning TopHat run (v1.3.0)
-----------------------------------------------
[Thu Aug 4 13:56:11 2011] Preparing output location /m/illumina/tophat/
[Thu Aug 4 13:56:11 2011] Checking for Bowtie index files
[Thu Aug 4 13:56:11 2011] Checking for reference FASTA file
[Thu Aug 4 13:56:11 2011] Checking for Bowtie
Bowtie version: 0.12.7.0
[Thu Aug 4 13:56:11 2011] Checking for Samtools
Samtools Version: 0.1.8
[Thu Aug 4 13:56:11 2011] Generating SAM header for /m/ref/genome
[Thu Aug 4 13:56:40 2011] Preparing reads
format: fastq
quality scale: phred33 (default)
[FAILED]
Error retrieving prep_reads info.

OR!!! In two of my samples, I get this error message:

Error: qual length (103) differs from seq length (63) for fastq record !

In case it matters, there is a slice of my pre-maq ill2sanger .txt file and post-maq .fastq file attached.

My apologies if this has been answered before.

[grimmer]

Same thing here. I have chmod 777 and 1TB free on the tophat_out destination, I have rebuilt my reference genome several times, bowtie runs perfectly, and the input files have been successfully aligned by tophat on another machine. Here's my output :

[Wed Jan 4 16:20:51 2012] Beginning TopHat run (v1.3.3)
-----------------------------------------------
[Wed Jan 4 16:20:51 2012] Preparing output location /media/data/grimmer/tophat_out//
[Wed Jan 4 16:20:51 2012] Checking for Bowtie index files
[Wed Jan 4 16:20:51 2012] Checking for reference FASTA file
[Wed Jan 4 16:20:51 2012] Checking for Bowtie
Bowtie version: 0.12.7.0
[Wed Jan 4 16:20:51 2012] Checking for Samtools
Samtools Version: 0.1.18
[Wed Jan 4 16:20:51 2012] Generating SAM header for /media/data/genomes/hg19.ebwt/hg19
[Wed Jan 4 16:20:52 2012] Preparing reads
format: fastq
quality scale: phred33 (default)
[FAILED]
Error retrieving prep_reads info.

[apratap]

Strangely enough I have got this error for the first time ..

[2012-04-30 16:01:30] Preparing reads
[FAILED]
Error running 'prep_reads'
terminate called after throwing an instance of 'int'


-Abhi

Full output below

[2012-04-30 16:01:23] Beginning TopHat run (v2.0.0)
-----------------------------------------------
[2012-04-30 16:01:23] Checking for Bowtie
Bowtie version: 2.0.0.5
[2012-04-30 16:01:23] Checking for Samtools
Samtools version: 0.1.18.0
[2012-04-30 16:01:23] Checking for Bowtie index files
[2012-04-30 16:01:23] Checking for reference FASTA file
Warning: Could not find FASTA file ../../../reference/Chlamy_V5/reference_bowtie2/Chalmy_reinhardtii_110311_v5.fasta.fa
[2012-04-30 16:01:23] Reconstituting reference FASTA file from Bowtie index
Executing: /jgi/tools/bin/bowtie2-inspect ../../../reference/Chlamy_V5/reference_bowtie2/Chalmy_reinhardtii_110311_v5.fasta > tophat_v2_out/tmp/Chalmy_reinhardtii_110311_v5.fasta.fa
[2012-04-30 16:01:29] Generating SAM header for ../../../reference/Chlamy_V5/reference_bowtie2/Chalmy_reinhardtii_110311_v5.fasta
format: fastq
quality scale: phred64 (reads generated with GA pipeline version >= 1.3)
[2012-04-30 16:01:30] Preparing reads
[FAILED]
Error running 'prep_reads'
terminate called after throwing an instance of 'int'

[Siva]

Prep-read errors: Tophat

I have attached the first 20 lines of Illumina sequence reads (sample.txt), these are 100 base single end reads. These are from Illumina's new pipeline (Version 1.8). The sequences are in fastq format. All sequences (raw and pass filter reads) are in a single file. Quality scores are in Sanger FASTQ format. The offset is ascii 33, instead of the previous Illumina Q score offset (ascii 64).

I ran a trial tophat with this culled out sequence.

Command used:

tophat -o ./tophat_out_test_2 -p4 --segment-length 50 --solexa1.3-quals  ../Genome/genome-index P1-test

I get the following error:
...................
format: fastq
quality scale: phred64 (reads generated with GA pipeline version >= 1.3)
[2012-12-05 16:22:18] Preparing reads
[FAILED]
Error running 'prep_reads'
terminate called after throwing an instance of 'int'
...................

I am not sure if this is because of something in the header file or because of the different quality scores. Can any body help me figure this out?

Thanks

[Siva]

Illumina V 1.8 and Solexa quality

In Illumina V 1.8 the reads quality scores are in Sanger Fastq format (off set of ASCII33 and not Phred 64) so using

 --solexa-quals

instead of

--solexa1.3-quals

seems to work! I am still not sure if this is OK though.

Full command that now works:

tophat -o ./tophat_out_test_2 -p4 --segment-length 50 --solexa-quals  ../Genome/genome-index P1-test