In brief

The standard protocol of Oxford Nanopore Technologies (ONT) ligation sequencing kit (e.g. SQK-LSK109) uses AMPure beads at DNA repair and end-prep clean up and adapter clean up steps. AMPure beads either shear long fragments DNA or form clump with ultra-High Molecular Weight DNA and High Molecular Weight DNA resulting poor sequencing library preparation. In this method, AMPure beads are replaced by the PEG/NaCl solution and short read eliminator (SRE) XL kit to keep DNA intact during the sequencing library preparation. In addition, loading beads (LB) is also excluded on preparing loading library in order to prevent beads clumping. This protocol is useful to generate long 426 kb ultra-long reads with 7.2 Gb sequence throughput (in MinION) generated in 24 h.


  • This method excludes AMPure beads in ONT ligation sequencing kit library preparation and Loading Beads in library loading steps.

  • Using this method, ligation sequencing kit can generate ultra-long reads (>100 kb) up to 426 kb with significant sequencing throughput (~7 Gb in 24 h in MinION).