Improvements in Hydrodynamic Shearing Facilitates 50 Kb True Mate Pair Library Construction

Next generation sequencing technologies produce massive quantities of short reads covering entire Gb genomes. A major limitation is the assembly and phasing of genomes, as the current technology cannot resolve repeats, segmentally duplicated sequences, or differentiate between diploid chromosomes.  We have developed a long span mate-pair technology approaching 90% efficiency for 20 kb libraries. De novo assembly of complex genomes should be more efficient with 50 kb true mate pair libraries. This application demands long DNA segments, but few methods are available for reliably generating fragments in excess of 2 kb. Diagenode's Megaruptor® utilizes hydrodynamic shearing to make fragments between 2 kb and 70 kb, with the majority falling in a 2 to 3- fold size distribution. The Megaruptor consists of an automated syringe pump and ceramic valve with a unique disposable shearing assembly called Hydropore. Each Hydropore contains an array of pores rather than a single shearing orifice, eliminating the clogging issues that plague single orifice shearing devices. The disposable Hydropore reduces the possibility of sample cross-contamination. Sample processing is user-friendly due to the intuitive software interface, and two samples can be sheared sequentially. The Megaruptor has been used by several sequencing centers to produce high-quality small insert fragment libraries for Illumina and PacBio platforms. We have used the instrument to prepare 50 kb inserts for a new mate-pair library technology that is >70% efficient.  Reproducibility, size range, and quality of DNA shearing for 50 kb true mate-pair library construction with supporting sequence data will be presented.