Nebnext multiplex oligos for unique dual index kit

We first generated Oxford Nanopore long reads in house on the MinION device using the SQK-LSK109 library preparation kit followed by sequencing on a R9.4.1 flow cell per the manufacturer's instructions (Oxford Nanopore Technologies). After observing low yield, we sent our remaining two HMW extracts to the UC Davis Core Lab to run on two respective PromethION flow cells. For the first PromethION run a single library was prepared, loaded, and run for 48 hours. After limited yield in that run, for the second PromethION flow cell, the HMW DNA was sheared using a Megaruptor (Diagenode, Inc.) set to a 50 kb target prior to library preparation. Two libraries were made such that the flow cell ran for 24 hours, was flushed with a nuclease wash, and a second library was loaded for the final 24 hours of run time. To generate the Illumina sequencing library, we used the NEBNext Ultra II FS DNA library kit (New England Biolabs, Inc); the initial enzymatic shearing step was accomplished via 10 minutes of incubation at 37ºC, after which we followed the manufacturer's instructions. The library was indexed using NEBNext Multiplex Oligos for Illumina unique dual index kit (New England Biolabs, Inc). This library was run on one lane of a NovaSeq S-Prime flow cell at the Oklahoma Medical Research Foundation Clinical Genomics Center.