title: Nanopore Amplicon-Seq of the six HMW Glutenin genes of Triticum aestivum (MC 23)
design: a library was prepared from 23 pooled samples usingthe nanopore native barcoding genomic DNA SQK-NBD110-24 (Oxford NanoporeTechnologies, Oxford, UK), with some modifications in the process of using the NEBNextCompanion Module for Oxford Nanopore Technologies Ligation Sequencing (NewEngland Biolabs, MA, USA). Briefly, ~100 ng of each pooling sample in 4.5 L wasmixed with 0.5 L Native Barcode and 5 L Blunt/TA Ligase Master Mix and incubatedon a Hula mixer for 10 min at room temperature. Purification by Agencourt AMPure XPBeads was performed during phosphorylation. Each of the 12 barcoded samples wasresuspended in 2.7 L of nuclease-free water and transferred to a new LoBind tube foradapter ligation. Then, ~32.5 L pooled and barcoded amplicons were mixed with 10 LNEBNext Quick Ligation Reaction Buffer (5X), 5 L Quick T4 DNA Ligase, and 2.5 LAdapter Mix II (AMII). Adapter Ligation Mix was incubated on a Hula mixer for 10 min atroom temperature. Double washing was performed using 125 L of Short FragmentBuffer (SFB). Incubation was performed in a water bath at 37 C for 10 min, and then for5 min at room temperature. Sequencing was carried out using MinION and a flow cellSQK-LSK109. Basecalling was performed by Guppy (Version 6.3.8).