Construction of genomic libraries from Neurospora crassa with the BigEasyTM v2.0 Linear Cloning System

Introduction The complete genome sequence of the filamentous fungus Neurospora crassa has been difficult to assemble, because a large amount of AT-rich “genomic dark matter” is lost from traditional shotgun, cosmid, and BAC libraries (1-3). This situation is typical of most repeat-rich eukaryotic genomes. Various novel direct sequencing approaches promise to reduce some of these errors. However, preliminary results from 454 pyrosequencing of the Neurospora crassa genome (1, 2) suggest that new sequencing methodology alone will likely not be sufficient to generate finished sequence of even a relatively small eukaryotic genome (~40 Mb), simply because assembly of AT-rich repeats proves to be a major obstacle.