ONT library preparation and sequencing

DNA from the Nanobind CBB Big DNA kit and the MagMax Core NA kit for both PAXgene and “purple top” EDTA tubes were combined to create a single sample for Oxford Nanopore Technologies library preparation. Half of this sample was used in the Short Read Eliminator Kit (SS-100-101-01; Circulomics, Inc.) to test the effect of size-selection on read N50, resulting in a size-selected sample. The size-selected and non-size-selected samples were then split between the Rapid Sequencing Kit (SQK-RAD004; Oxford Nanopore Technologies) and the Ligation Sequencing Kit (SQK-LSK109; Oxford Nanopore Technologies) to test the effect of library preparation on read N50, resulting in a total of four unique libraries. Each library was then loaded onto an R9.4.1 flow cell and sequenced in parallel on the ONT GridION platform. It was determined that size-selection did not have the desired effect of increasing read N50, and four additional non size-selected libraries were prepared (two SQK-RAD004 and two SQK-LSK109) to achieve a target depth of at least 20×. The output of all eight flow cells produced a combined total of ∼22.7× depth.