design: Set up the end-repair reaction by adding 5 l of the DNA amplicons to 1.75 l of Ultra II End Prep Reaction Buffer and 0.75 l of Ultra II End Prep Enzyme Mix, then topped up with nuclease-free water to make up a 15 l reaction. Incubated at 20C for 5 mins and 65C for 5 mins. Barcode ligation was performed for each sample by adding the following to the above reaction: 2.5 l of specific barcode (EXP_NBD104), 17.5 l of Ultra II Ligation Master Mix and 0.5 l Ligation Enhancer. The reaction was incubated at room temperature for 15 mins and 70C for 10 mins. All barcoded samples were pooled together and cleaned using equal volume of SPRI beads and eluted in 30 l of nuclease-free water. Following clean up, 30 l of the barcoded pool was used to set up an adapter ligation reaction by adding the following: 10 l NEBNext Quick Ligation Reaction Buffer, 5 l ONT AMII adapter mix and 5 l NEBNext Quick T4 DNA Ligase, and incubated at room temperature for 15 mins. The adapter-ligated pool was cleaned using equal volume of SPRI beads and eluted in 15 l of Elution Buffer (SQK-LSK109). 12 l of the final library was used to prepare the loading library.