P0091 Complementary DNA Shearing and Size-selection Tools for Mate-pair Library Construction

Chris Boles , Sage Science, Beverly, MA
Alex Vira , Sage Science, Beverly, MA
Mate - pair library sequencing is an essential technique for de novo genome sequencing, genome finishing, and analyses of genomic structural variation. Current methods for creating mate-pair libraries are extremely inefficient and time-consuming for two main reasons. First, it is difficult to develop reproducible DNA fragmentation methods that produce narrow size distributions in the range of 2 to 20kb. Second, manual preparative agarose gel electrophoresis is the only size-selection technique currently available for this size range, and it is labor-intensive and irreproducible. Digilab and Sage Science have developed two new products, the Digilab Hydroshear Plus and the Sage Blue Pippin system, that provide a simplified and complementary workflow for optimizing high molecular weight DNA fragmentation and size-selection. The Hydroshear Plus represents the second generation of DNA shearing systems utilizing hydrodynamic force for semi-automated reliable and reproducible fragmentation of genomic DNA into fragments greater then 0.8 Kb. It is the only system in the current market for reliable and consistent preparation of large DNA fragments necessary for genomic library construction and third-generation sequencing. The Sage Blue Pippin system is a new automated preparative electrophoresis system that features an alternating field power supply capable of size-fractionating DNA fragments as large as 50kb. Selected DNA fractions are automatically eluted into a small volume of liquid buffer, ready for enzymatic library construction reactions. We will demonstrate the advantages of the combined Hydroshear Plus/Blue Pippin system by presenting efficient protocols for producing tight DNA fragment distributions in the 8-20 kb size range.