Nebnext ultra 2 end repair da tailing module

MinION two-dimensional (2D) libraries were constructed targeting inserts >8 kilo-base pair (kbp). A total of 1 µg of DNA was fragmented in a 46 µl volume in a g-TUBE (Covaris) at 6,000 r.p.m. in an Eppendorf 5417 Centrifuge. Sheared DNA was then subjected to a repair step using the NEBNext FFPE DNA Repair Mix (New England Biolabs) and purified with a 1× Hi Prep bead clean-up (GC Biotech). A DNA control was added to the repaired DNA and then end-repaired and A-tailed using the NEBNext Ultra II End Repair/dA-Tailing Module (New England Biolabs), and purified with a 1× Hi Prep bead clean-up; then the AMX and HPA MinION adaptors were ligated using the Blunt/TA Ligase Master Mix. An HP tether was then added and incubated for 10 min at room temperature followed by a further 10 min room temperature incubation with an equal volume of pre-washed MyOne Streptavidin C1 beads (Thermo Fisher Scientific). The library-bound beads were washed twice with bead binding buffer (ONT) before the final library was eluted via a 10 min incubation at 37 °C in the presence of the MinION elution buffer. The final library was then mixed with running buffer, fuel mix and nuclease-free water and loaded onto an R7.3 flow cell according to the manufacturer’s instructions; sequencing data were collected for 48 h.

The 1D gDNA long read selection protocol was used with the SQK-LSK108 kit (Oxford Nanopore Technologies, UK) to prepare MinION-compatible libraries. The DNA shearing step was eliminated from the protocol with the aim of selecting for very long reads. Approximately, 2 µg of E. coli DNA and 2 µg of Salmonella DNA in a total of 100 µL each were added to the NEBNext® Ultra™ II End Repair/dA-Tailing module (New England Biolabs, USA) for end repair and dA-Tailing, following manufacturer’s instructions, and purified using Agencourt AMPure XP beads (Beckman Coulter, USA). Each purified, end-prepped DNA product was barcoded using a separate barcode from the 1D Native barcoding kit (EXP-NBD103, ONT) and following the 1D Native barcoding genomic DNA protocol. The samples were then bead-purified (Beckman Coulter), and equimolar amounts of each barcoded sample were pooled together for a final quantity of 700 ng. Adapters were ligated to the pooled sample using Blunt/TA ligase (New England Biolabs) following the 1D gDNA long read selection protocol. The MinION device was used to sequence the created library on a new FLO-MIN106 R9.4 flow cell^{21 ,22 (link)}. The standard 48 hr 1D sequencing protocol was initiated using the MinKNOW software (ONT, UK). Average quality and coverage of the raw sequencing data were determined using CG-pipeline^{23 (link)}.

The LC-2/ad cell line was obtained from RIKEN BRC, and was cultured as previously described in [17 (link)]. HMW gDNA was extracted from lung cancer cell line, LC-2/ad by using a Smart DNA prep (a) kit (Analykjena). Whole-genome shotgun data were produced from MinION 1D sequencing (SQK-LSK108), MinION 1D^2 sequencing (SQK-LSK308), and MinION Rapid sequencing (SQK-RAD003). For MinION 1D sequencing, 4 μg HMW gDNA was quantified using Tape Station. DNA repair was performed using NEBNext FFPE DNA Repair Mix (M6630, NEB). End-prep was performed using NEBNext Ultra II End Repair/dA-Tailing Module (E7546L, NEB). Adapter ligation was performed using NEBNext Blunt/TA Ligase Master Mix (M0367 L, NEB) and the Ligation Sequencing Kit 1D (SQK-LSK108, Oxford Nanopore Technologies). Libraries were sequenced for 48 h with MinION (R9.5 chemistry, Oxford Nanopore Technologies). For MinION 1D^2 sequencing, the protocol was the same as that for 1D excluding adapter ligation by using Ligation Sequencing Kit 1D^2 (SQK-LSK308, Oxford Nanopore Technologies). The library for MinION Rapid sequencing was prepared according to Sequencing Kit Rapid (SQK-RAD003, Oxford Nanopore Technologies).

Multiplex PCR product was end-repaired using NEBNext® Ultra^™ II End Repair/dA-Tailing Module (New England Biolabs). Each reaction was a mixture of 3 μl NEBNext Ultra II End Prep Enzyme Mix, 7 μl NEBNext Ultra II End Prep Reaction Buffer, 20 μl multiplex PCR products, and 30 μl H2O. End repair was performed on a Eppendorf Mastercycler. Thermal cycling started with the incubation at 20 °C for 30 min and 65 °C for 30 min, with the heated lid set to 80 °C.

Libraries were prepared according to the protocol Genomic DNA by ligation (SQK-LSK109 kit). Genomic DNA fragments (1.5 µg) were repaired and 3′-adenylated with the NEBNext FFPE DNA Repair Mix and the NEBNext® Ultra™ II End Repair/dA-Tailing Module (New England Biolabs, Ipswich, MA, USA). Sequencing adapters provided by ONT (Oxford Nanopore Technologies Ltd, Oxford, UK) were then ligated using the NEBNext Quick Ligation Module (NEB). After purification with AMPure XP beads (Beckmann Coulter, Brea, CA, USA), the library was mixed with the Sequencing Buffer (ONT) and the Loading Bead (ONT) and loaded on MinION or PromethION R9.4.1 flow cells. One PromethION run was performed with Genomic DNA purified with Short Read Eliminator kit (Circulomics, Baltimore, MD, USA) before the library preparation.