Nanopore long read sequencing of Anopheles coluzzii ace-1 gene PCR product (817pb)
The amplicons libraries construction was done according the Nanopore protocole NBA_9102_V109_revA_09Jul2020. Two hundred nanograms of DNA are end repaired and dA-tailed using NEBNext End repair/dA-tailing Module (E7546, New England Biolabs, Ipswich, Massachusetts, USA). The samples were then barcoded using EXP-NBD196 (barcodes 196) kits (Oxford Nanopore Technologies, Oxford, UK) and ligation Sequencing Kit 1D SQK-LSK109, (Oxford Nanopore Technologies, Oxford, UK). Barcoded samples were pooled and purified using 0.4 volume of AMPure XP magnetic beads. The AMII sequencing adapter (Oxford Nanopore Technologies, Oxford, UK) was ligated to barcoded pools using Quick T4 DNA ligase (New England Biolabs, Ipswich, Massachusetts, USA) and the sequencing libraries were purified using 0.4 volume of AMPure XP magnetic beads. MinION sequencing was performed as per manufacturers guidelines using R9.4.1 flow cells FLO-MIN106, ONT and controlled using Oxford Nanopore Technologies MinKNOW software version v20.06.5. Flow cells were then transferred to Nanopore MinION Mk1b (Oxford Nanopore Technologies, UK) for Nanopore single molecular sequencing. Protocol extracted from Namias et al. (2022).