Rapid sequencing kit sqk rad004

Selected samples from the first replicate of the L. monocytogenes culture enrichment experiment with background microbiota harvested at all time points throughout enrichment in Half Fraser were subjected to quasimetagenomic shotgun sequencing on Flongle flow cells. After MDA and subsequent quantification of DNA using Qubit, libraries were prepared separately for each selected sample using the SQK-RAD004 rapid sequencing kit (Oxford Nanopore Technologies) according to the manufacturer’s instructions, using 3.75 μl of 200 ng DNA as input. Each sample was sequenced on separate FLO-FLG001 Flongle flow cells in a MinION Mk1 sequencing device with MinKNOW UI 4.0.20 software. Reads in fast5 format obtained from MinKNOW were basecalled using guppy v4.2.2 (Oxford Nanopore Technologies) with default settings, including a qscore filtering of 7. PoreChop (75 ) was used to remove adapters. Read quality was assessed using NanoComp (76 (link), 77 ). Sequencing quality metrics are reported in Table S3 in the supplemental material.
Taxonomic classification of the filtered Nanopore reads was performed using the online BugSeq pipeline (78 (link)) and “NCBI nt” index (run 24 March 2021), which runs with a minimum read length of 100 bp and a default low-complexity filter.

Libraries for ONT sequencing were constructed using SQK-RAD004 Rapid Sequencing Kit (Oxford Nanopore Technologies, Oxford, UK) following the manufacturer’s instructions with the exception that the DNA mass input was increased to approximately 1 $\mu$ g. ONT libraries were sequenced using the MinIon system (Oxford Nanopore, Oxford, UK) to produce 4.08 Gbp of sequencing data with an N50 of 9,585 bp. Nanopore long reads were basecalled using Guppy v4.0.15 (–config dna_r9.4.1_450bps_fast.cfg) (Oxford Nanopore Technologies). Reads were then quality filtered to include those with quality scores greater than or equal to seven (approximately 85% basecall accuracy) using NanoFilt v2.7.1 (De Coster et al. 2018 (link)). Filtering resulted in 3.56 Gbp of long-read data, with a read length N50 of 9,799 bp.

The complete genome of the case isolate HKU75^T was sequenced using Illumina and Oxford Nanopore technologies. Genomic DNA was extracted from an overnight culture (37°C) grown on blood agar using a genomic DNA purification kit (Qiagen, Hilden, Germany), as described previously (42 (link)). The Illumina DNA library was prepared using a Nextera XT DNA Sample Prep Kit (Illumina, San Diego, CA, USA) and was sequenced on a NovaSeq 6000 instrument (run type: PE151 bp). The ONT long-read library was prepared with SQK-RAD004 rapid sequencing kit (Oxford Nanopore Technologies, Oxford, UK) according to the manufacturer’s instructions, and sequenced on a MinION sequencer. Illumina reads and Oxford Nanopore MinION reads were assembled by Unicycler/0.4.8 to obtain the whole genome of HKU75^T.