Tophat Error

[sbdk82]

I am getting the following error when I use bowtie and bowtie2

Error: Reference sequence has more than 2^32-1 characters!  Please divide the
reference into batches or chunks of about 3.6 billion characters or less each
and index each independently.

[mattia]

What kind of species are you studying?
Maybe the error has been generated due to the reference length.

As the message suggest you:
1) cut the reference in 2 different .fasta files;
2) create a bowtie index for both;
3) align your reads versus the first reference and later the unmapped reads versus the second one;
4) merge your .bam files

[sbdk82]

I am working on Triticum aestivum. I got the reference from Ensemble site ftp://ftp.ensemblgenomes.org/pub/pla..._aestivum/dna/

[sbdk82]

It was working fine when I used Bowtie2 with long index option, but the index files have suffixes .bt21 which are not recognized by tophat. I asked that question in another thread, but someone replied that tophat/2.0.12 also does not support long index.