Preparation of an 8-kb Mate-Pair Library for Illumina Sequencing

The "mate-pair" approach for dual end sequencing uses long library fragments (1000-10,000 bp) that are circularized by ligation to the ends of a single, common DNA adaptor of known sequence. Once the ends are mated to the adaptor, a final library of fragments that retain the adaptor and only the ends of the original, circularized piece of DNA can be obtained. This protocol provides instructions for preparing an 8-kb paired-end library suitable for sequencing on the Illumina instrument. These are large-insert libraries, and the initial shearing will use various parameters and different instruments to achieve the desired high-size fraction.