Tophat/bowtie 2 errors with Illumina HiSeq

[Tom_4356]

Hi everyone,

I have compiled all the neccesary software, indexed the genome but keep running into issues. I have made the anlaysis before but now I am in a new lab and nothing seems to work. I have three questions after many days of fighting with this.

1) To double check - with Illumina HiSeq the library type in Tophat should be unstranded right? I am hoping it is machine dependent as the samples were prepared and sequenced abroad.

2) Building of index for human genome (or have it done by tophat) from Ensembl toplevel fasta file seems fine and proceeds without errors. However whenever I try to use bowtie2-inspect on it I get this;

bowtie2-inspect: bt2_inspect.cpp:218: void print_ref_sequences(std:stream&, bool, const EList<std::basic_string<char, std::char_traits<char>, std::allocator<char> >, 128>&, const uint32_t*, const std::string&): Assertion `0' failed.

Tried to find more details about this but with no luck. The annotation file from Ensembl created by tophat also does not pass this. Any ideas?

3) Don't know if this is realted or not but every time I try to do the analysis I get this:

[2012-07-27 15:01:01] Beginning TopHat run (v2.0.4)
-----------------------------------------------
[2012-07-27 15:01:01] Checking for Bowtie
Bowtie version: 2.0.0.7
[2012-07-27 15:01:01] Checking for Samtools
Samtools version: 0.1.18.0
[2012-07-27 15:01:01] Checking for Bowtie index files
[2012-07-27 15:01:01] Checking for reference FASTA file
[2012-07-27 15:01:01] Generating SAM header for /home/Genome/HS_Gen
format: fastq
quality scale: phred33 (default)
[2012-07-27 15:01:05] Reading known junctions from GTF file
[2012-07-27 15:01:18] Preparing reads
left reads: min. length=101, max. length=101, 50139110 kept reads (24913 discarde d)
right reads: min. length=101, max. length=101, 50133685 kept reads (30338 discarde d)
[2012-07-27 15:30:58] Creating transcriptome data files..
[2012-07-27 15:34:12] Building Bowtie index from Annotation.fa
[2012-07-27 16:06:01] Mapping left_kept_reads to transcriptome
Annotation with Bowti e2
[FAILED]
Error running bowtie:
Error while flushing and closing output
terminate called after throwing an instance of 'int'

I could download pre-made indexed files from Illumina but I want to know and understand what is wrong. I followed the protocol and everything should be fine.

The comand I use is (simplified):

tophat -p 8 -G /.../Annotation.gtf -o /../A1 --library-type=fr-unstranded /../HS_Gen /.../A1_R1.fastq.gz /.../A1_R2.fastq.gz

Thanks guys for your help.
Tom