Sqklsk109 1d ligation sequencing kit

The cells of isolated strains at the exponential growth stage were collected for total DNA extraction. Total DNA was extracted using DNeasy UltraClean Microbial Kit (QIAGEN, USA) according to the manufacturer’s instructions. DNA for Oxford Nanopore sequencing was purified by 1% agarose gel electrophoresis and Monarch DNA Gel Extraction Kit (NEB, USA).
Oxford Nanopore sequencing and Illumina sequencing were combined to obtain the complete genomes of isolated strains. Oxford Nanopore sequencing library was constructed using the Ligation Sequencing 1D kit (SQK-LSK109) (Oxford Nanopore, USA) according to the manufacturer’s protocol. Qubit® 2.0 Fluorometer (Life Technologies, USA) was used to quantify the constructed library. The library was sequenced using a PromethION (Oxford Nanopore Technologies, UK) with the R9.4 flow cell at NextOmics (Wuhan, China), and the sequencing depth was about 1 Gb.
The genomic Illumina library was constructed using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB, USA) according to the manufacturer’s instructions. The library was sequenced on the Illumina NovaSeq platform (paired-end 150 bp) with a sequencing depth of about 2 Gb at NextOmics (Wuhan, China).

We prepared libraries for Nanopore sequencing per manufacturer’s guidelines (NBE_9065_v109). Amplicons containing fragments between 700 bp and 2500 bp were purified using a 1.0X AMPure XP bead cleanup, and library construction was performed using the SQKLSK109 (1D) Ligation Sequencing Kit (Oxford Nanopore Technologies, ONT) according to manufacturer’s instructions, with some modifications. Briefly, 100 ng purified DNA from a pool of barcoded single cells was subjected to end repair and dA-tailing using the NEB-Next Ultra II End-Repair/dA-tailing Module. Next, we performed a 1X volume AMPure XP bead cleanup and ligated nanopore barcodes to each sample using the 1D Native barcoding kit (EXP-NBD104 / EXP-NBD114) and the Blunt/TA Master Mix (NEB). After a 1X AMPure XP bead cleanup, equimolar amounts of each sample were pooled and nanopore sequencing adapters were ligated to the eluted DNA using the Quick T4 ligase (NEB). The final clean-up of the adaptor-ligated DNA was modified and performed with 0.5X AMPure XP beads. We used a total of 60 ng of the final library to load into a MinION flow cell. We sequenced each flow cell for 10–48 hours and obtained over 10 million reads per run. The computational analysis of long-read single-cell sequencing data is described below.