Preparing Fosmid Mate-Paired Libraries Using Cre-LoxP Recombination.

Fosmid end sequencing has been widely utilized in genome sequence assemblies and genome structural variation studies. We have developed a new approach to construct fosmid paired-end libraries that is suitable for Illumina sequencing platform. This approach employs a newly modified fosmid vector (pFosClip) which contains two loxP sites with identical orientation and two inverse Illumina adaptor priming sites flanking the cloning site. DNA prepared from the fosmid library constructed with pFosClip can be treated with the Cre recombinase to remove most of the vector DNA, leaving only 107 bp of the vector sequence with insert DNA. Frequent cutting restriction enzymes and ligase are used to digest the fosmid DNA to small (less than 1 Kb) fragments and recircularize the fosmid ends and all the internal fragments. Finally an inverse PCR step with the Illumina primers is used to enrich the fosmid paired ends (PEs) for sequencing. The advantages of this approach are the following: (1) the circularization of short fragments with sticky ends is efficient; therefore the success rate is higher than other approaches that attempt to join both blunt ends of large fosmid vectors; and (2) the restriction enzyme cutting generates an identifiable junction tag for splitting the paired reads. (3) Multiple restriction enzymes can be used to overcome possible enzyme-cutting bias. Our results have shown that this approach has produced mostly fosmid size (30-40 Kb) pairs from the targeted fungi and plant genomes and has drastically increased the scaffold sizes in the assembled genomes.