Neb blunt ta ligase master mix

Direct cDNA libraries were prepared from the mock and six BoHV-1 p.i samples in three replicates using the ONT’s Direct cDNA Sequencing Kit (SQK-DCS109) according to the manufacturer’s instructions. The first cDNA strand synthesis was performed using Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific) with SSP and VN primers (supplied in the kit) and 100 ng of poly(A) + RNA for each sample. This was followed by the removal of potential RNA contamination using RNase Cocktail Enzyme Mix (Thermo Fisher Scientific), and second strand synthesis using LongAmp Taq Master Mix (New England Biolabs). Double stranded cDNA ends were repaired using NEBNext End repair /dA-tailing Module (New England Biolabs). This was followed by ligation of sequencing adapter employing the NEB Blunt /TA Ligase Master Mix (New England Biolabs). Libraries were barcoded using Native Barcoding (12) Kit (ONT) according to the manufacturer’s instructions.

Nanopore sequencing library preparations using Nanopore Sequencing Kits SQK-MAP005 and SQK-MAP006 were essentially performed as described in the protocols and guidelines provided by Oxford Nanopore Technologies (ONT). Briefly, 1 µg of the genomic DNA isolated from skin lesions was fragmented to an average size of 8–15 kb using g-TUBEs (Covaris). DNA fragments were end-repaired and adenylated using NEBNext Ultra II End-Repair/dA-tailing Module (NEB) followed by cleanup with Ampure XP beads (Beckmann Coulter). Sequencing and hairpin adapters (ONT) were ligated using NEB Blunt/TA Ligase Master Mix (NEB) followed by incubation with the hairpin tether (ONT). Cleanup of libraries was done either with His-Tag beads or MyOne C1 streptavidin beads (Invitrogen) depending on the respective Sequencing Kit and flowcell version. Prepared libraries were eluted in 25 µl of the ONT-supplied elution buffer.
Prior to sequencing, 6 μl of the eluate (pre-sequencing mix), 75 μl running buffer (ONT), 60 µl nuclease free H2O and 4 μl fuel mix (ONT) were combined gently and were immediately loaded onto the prepared MinION flowcells. Sequencing was performed using 48 hr sequencing run scripts with addition of freshly prepared input material to the MinION flowcell every 12 hrs until no further active pores were available anymore.

DNA library preparation for Nanopore sequencing was carried out using Ligation Kit SQK-LSK109 (ONT). Fragmented DNA was repaired and dA-tailed using the NEBNext FFPE DNA Repair Mix and NEBNext Ultra II End Repair/dA-Tailing Module (New England BioLabs). An individual barcode was added to dA-tailed DNA by using the barcoding extension kit EXP-NPB104 in accordance with the ONT protocols with NEB Blunt/TA Ligase Master Mix (New England BioLabs). Each barcoded DNA was pooled in equimolar amounts, and an adapter was attached using the NEBNext Quick Ligation Module (New England BioLabs). The MinION flow cell and reagents were shipped from Bangalore, India, to Nairobi at 4°C. The number of active pores was checked before loading. The samples were pooled using equimolar pooling, the library was loaded into the SpotON flow cell R9.5 (FLO-MIN106), and sequencing was carried out on MinKNOW using the 48-h script.

Approximately 1–3 μg of purified, sheared genomic DNA was library prepped using the following reagents: NEB Oxford Nanopore Companion (New England Biolabs, Cat # E7180S), NEB Blunt/TA Ligase Master Mix (New England Biolabs, Cat # M0367), NEBNext Quick Ligation Reaction Master Mix (New England Biolabs, Cat # B6058), Oxford Ligation Sequencing Kit (Oxford Nanopore Technologies, Oxford, UK, Cat # SQK-LSK109), and the Oxford Native Barcoding Expansion 1–12 (Oxford Nanopore Technologies, Cat # EXP-NBD104). The library was prepared and sequenced according to Oxford Nanopore’s protocol for Ligation Sequencing Kit + Native Barcoding Expansion 1–12. Sequencing was done on a MinION sequencer with v9.4 flow cells (Oxford Nanopore Technolgies, Cat # FLO-MIN106).

For more accurate mapping of the 5′-end of VZV transcripts, random-primer based RT reactions were carried out as a first step of library preparation. For this, poly(A)-selected and Terminator-treated RNA samples and SuperScript IV enzyme (Life Technologies) were used. From the first-strand cDNAs, libraries were prepared according to the modified 1D strand switching cDNA by ligation protocol Ligation Sequencing kit (SQK-LSK108; Oxford Nanopore Technologies). KAPA HiFi DNA Polymerase (Kapa Biosystems) and Ligation Sequencing Kit Primer Mix (part of the 1D Kit) were used to amplify the cDNAs. Next, samples were end-repaired using NEBNext End repair /dA-tailing Module (New England Biolabs), then adapter ligation was carried out using the sequencing adapters supplied with the kit and NEB Blunt/TA Ligase Master Mix (New England Biolabs).

The cDNA library was prepared using the Ligation Sequencing Kit (SQK-LSK108; Oxford Nanopore Technologies) following the modified 1D strand switching cDNA by ligation protocol. Briefly: End repair was carried out on Cap-selected and barcoded samples using NEBNext End repair/dA-tailing Module (New England Biolabs) followed by adapter ligation using adapters (supplied in the kit) and NEB Blunt/TA Ligase Master Mix (New England Biolabs). The cDNA sample was purified between each step using Agencourt AMPure XP magnetic beads (Beckman Coulter), and the library concentration was determined using Qubit 2.0 Fluorometer (through use of the Qubit (ds)DNA HS Assay Kit (Thermo Fisher Scientific). The samples were loaded on R9.4 SpotON Flow Cells.