Nebnext ultra 2 end repair deoxyribosyladenine da tailing module reagents

DNA libraries for nanopore sequencing were prepared according to the protocol outlined by Oxford Nanopore Technology (ONT) (ACDE_9064_v109_revP_14Aug2019) and quantitated using the Qubit double-stranded DNA (dsDNA) highly selective assay kit (Thermo Fisher Scientific). For all samples, 100 to 200 fmol of amplicon DNA was mixed with NEBNext FFPE DNA repair mix and NEBNext Ultra II end repair/deoxyribosyladenine (dA)-tailing module reagents (New England Biolabs) and incubated at 20°C for 5 min, followed by inactivation at 65°C for 5 min. The end-repaired DNA amplicons were purified with AMPure XP beads (Beckman Coulter) and were barcoded with unique adapter indexes of the Nanopore native barcoding expansion kit (EXP-NBD104) (ONT). The resultant barcoded DNA amplicons were pooled and then loaded into a port on the R9.4.1 flow cell (ONT). Nanopore sequencing data were obtained through an Oxford Nanopore MinION Mk1B device and MinKNOW software (ONT). A brief flow chart of the nanopore sequencing protocol used to obtain HIV-1 genome sequences is illustrated in Fig. S1.

DNA libraries for nanopore sequencing were prepared according to the protocol outlined by Oxford Nanopore Technology (ONT) (ACDE_9064_v109_revP_14Aug2019) and quantitated using the Qubit double-stranded DNA (dsDNA) highly selective assay kit (Thermo Fisher Scientific). For all samples, 100 to 200 fmol of amplicon DNA was mixed with NEBNext FFPE DNA repair mix and NEBNext Ultra II end repair/deoxyribosyladenine (dA)-tailing module reagents (New England Biolabs) and incubated at 20°C for 5 min, followed by inactivation at 65°C for 5 min. The end-repaired DNA amplicons were purified with AMPure XP beads (Beckman Coulter) and were barcoded with unique adapter indexes of the Nanopore native barcoding expansion kit (EXP-NBD104) (ONT). The resultant barcoded DNA amplicons were pooled and then loaded into a port on the R9.4.1 flow cell (ONT). Nanopore sequencing data were obtained through an Oxford Nanopore MinION Mk1B device and MinKNOW software (ONT). A brief flow chart of the nanopore sequencing protocol used to obtain HIV-1 genome sequences is illustrated in Fig. S1.