These protocols describe methods for size selection of extracted DNA, including a method using our Short Fragment Eliminator (EXP-SFE001) kit to deplete short fragments (<25 kb). Another method described is based on the use of SPRI beads (enriches for molecules above ~1.5-2 kb), the third uses a “size selection buffer” (SSB) to promote precipitation and enrichment of molecules above ~10 kb, and the final method uses the BluePippin from Sage Science (this instrument is capable of enriching for molecules >40 kb, but is highly tuneable to select for molecules with specific lengths <40 kb).

To demonstrate the performance, gDNA was size selected using each method; the BluePippin instrument was set up to enrich for molecules >40 kb and using the Ligation Sequencing Kit, 1 µg of each size selected DNA was prepared for sequencing and the libraries were run on the MinION. A library where no size selection had been performed was also sequenced as control. The data below shows the read length distributions observed and the output generated for each of the libraries. Very little difference in output was observed over the course of the 24 hour sequencing run for SPRI and SSB size selected libraries, compared with the control. However, an accumulation of pores in the unavailable state (see this post for more information) was observed throughout the course of the run for the BluePippin size selected library, so a flow cell wash was performed after ~16 h and more library loaded before recommencing the run.