Nebnext quick t4 dna ligase

The DNA extract of maternal peripheral blood sample was applied to sequence using Ligation Sequencing Kit (SQK-LSK109, Oxford Nanopore Technologies) on PromethION. The DNA sequencing library was prepared according to the manufacturer’s instructions. 2 µg of high-molecular-weight gDNA was used for library construction using NEBNext End Repair/dA-tailing module. The gDNA was first end-repaired and then pured by AMPure XP beads. 60 µl of end-prepped DNA was ligated with an adapter using NEBNext Quick T4 DNA Ligase (NEB, E6056), then purified and quantified after ligation. The prepared DNA library, sequencing buffer, and loading beads was then loaded onto the primed flow cell of PromethION and performed sequencing. After 26 h of the sequencing run, the detected active pore for sequencing was less than 40%, thus we conducted a Flush Buffer washing process and at the same time added more DNA sequencing library, then restarted the sequencing run in the same flow cell (Flow cell ID: PAH18304). The total throughput was around 120Gb fastq data.

For nanopore sequencing via the Oxford Nanopore technology method, purified PCR amplified sequences containing encoded data were subjected to library preparation using the 1D Genomic DNA ligation sequencing kit (SQK-LSK109) from Oxford Nanopore Technologies following the manufacturer’s protocols. Briefly, 0.5 pmol of the double-stranded DNA strands were used as starting material. The DNA was repaired and end-prepped using NEBNext FFPE DNA repair mix (NEB, M6630S) and NEBNext Ultra II End Repair/dA-Tailing Module (NEB, E7546) followed by bead purification using Agencourt AMPure XP beads (Beckman Coulter, A63880) at 1:2 sample to bead ratio. Adapters were then ligated to the end-prepped samples using the NEBNext Quick T4 DNA ligase (NEB, E6056S). The flow cells (R4.2.1) were primed, the sample was loaded onto the priming port of the flow cell and sequenced on the MinION, that generated ~500 K reads/hr. Sequencing was performed using the MinKNOW software (version 18.3.1, Oxford Nanopore Technologies) that converted raw data (in the form of fast5 files) into FastQ files which were used for downstream analysis.

Library preparation for metagenomics MinION sequencing was achieved using Ligation Sequencing Kit (SQK-LSK109) coupled with Native Barcoding Expansion 1–12 kit (EXP-NBD104) for multiplexing of samples, according to manufacturer’s protocol with slight modifications (Oxford Nanopore Technologies, Oxford, U.K.). In brief, 1 µg of DNA from each sample was end-prepped using NEBNext Ultra II End-repair/dA-tailing module (New England Biolabs, Ipswich, MA). Subsequently, each sample was ligated with an unique barcode (EXP-NBD104) along with NEB Blunt/TA Ligase Master Mix (New England Biolabs, Ipswich, MA) for multiplexing. Equimolar amounts of barcoded DNA from each sample were pooled into a single Eppendorf DNA Lobind Tube (Hamburg, Germany). Lastly, adapter ligation of pooled and barcoded DNA was performed using NEBNext Quick T4 DNA Ligase and NEBNext Quick Ligation Reaction Buffer (New England Biolabs, Ipswich, MA). The eluted library was quantified using a Quantus Fluorometer (Promega, Winsconsin, WI, USA). All clean-up steps during library preparation were conducted using Agencourt AMPure XP beads (Beckman Coulter, Indianapolis, IN) according to manufacturer’s protocol.

AMX adapters from ONT Ligation Sequencing Kit (SQL-LSK109) were ligated to the dA-tailed DNA ends using 20 µL of the following adapter mix: 10 µL of NEBNext Quick T4 DNA Ligase (NEB, M2200), 20 µL of Oxford Nanopore Ligation buffer (LNB, ONT, SQL-LSK109) and 3 µL of nuclease-free water. This reaction was incubated for 10 min at room temperature. The ligated sample was purified twice on a magnetic rack using 0.3 × volume of AMPure XP Beads (Becman Coulter, A63881) and 250 µL of long-fragment buffer (ONT, SQL-LSK109). Elution of the ligated sample was performed by adding 13 µL of elution buffer (ONT, SQL-LSK109), incubating for 10 min at room temperature and placing the tube back on the magnetic rack to collect 12 µL of the eluate. The Oxford Nanopore DNA sequencing library was then prepared by adding 25 µL of sequencing buffer (ONT, SQL-LSK109) and 13 µL of loading beads (ONT, SQL-LSK109) to the eluate. Flow cell priming mix was prepared by placing 30 µL of flush tether (ONT, SQL-LSK109) into a tube of flush buffer (ONT, SQL-LSK109).

Nanopore 1D libraries were constructed using 1 μg of amplified cDNA according to the standard ONT SQK-LSK109 protocol. Briefly, cDNA products were end-repaired and dA-tailed using NEBNext Ultra II End Repair/dA-Tailing Module (NEB, #E7546) by incubating at 20 °C for 20 min and 65 °C for 20 min. The cDNA was then purified with 0.7× volumes of SPRIselect beads and eluted in 60 μl of nuclease-free water. Adapter ligation was performed using NEBNext Quick T4 DNA ligase (NEB, #E6056) at room temperature for 10 min. After ligation, libraries were purified using 0.45× volumes of AMPure XP beads (Beckman Coulter, #A63881) and short fragment buffer. The final libraries were loaded onto R9.4.1 flow cells and sequenced on MinION/GridION devices. Sequencing summary statistics, including library information, chemistry, flow cell types, and sequencing output, are detailed in Supplementary Data 15.