GATK really does not like alphabetically sorted references. http://gatkforums.broadinstitute.org/discussion/1204/what-input-files-does-t... Does it make sense to rearrange genomes to be sorted in [chr1,chr2, chr3,...] order instead of [chr1,chr10,chr11,...] order before indexing? Lots of tools care about sorting… are there any that must have alphabetical order and not numerical order? Brad -- Brad Langhorst langhorst@neb.com<mailto:langhorst@neb.com>