Rapid barcoding sequencing kit sqk rbk004

Genomic DNA was sequenced with Nanopore sequencers (Oxford Nanopore Technologies, UK). The genome structure of HIS019 and HIS471 was confirmed with PacBio sequencing (Pacific Biosciences, USA). For long-read sequencing, genomic DNA (6 mg) was treated with a Short-Read Eliminator Kit XS (Circulomics) to remove fragments < 10 kbp, and libraries were prepared using a Rapid Barcoding Sequencing Kit (SQK-RBK004, Oxford Nanopore Technologies). Sequencing was performed on the MinION (Sample HIS002 and HIS019) and GridION X5 (Sample HIS631, HIS641, HIS016, HIS471) systems using eight R9.4 flow cells. PacBio library construction and sequencing with Sequel II (Pacific Biosciences, USA) was outsourced (Takara Bio, Japan). Illumina paired-end genomic libraries with insert sizes of 300–350 bp were constructed with a Nextera DNA Flex Library Prep Kit (Illumina, USA). The libraries were sequenced with the NextSeq 500/550 Mid Output Kit v2.5 (Illumina, USA) for 151 bp from both ends. Illumina RNA-seq libraries were constructed with the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, USA) for Illumina and sequenced with the NextSeq 500/550 Mid Output Kit v2.5 (Illumina, USA) for 151 bp from both ends.

To resolve organization of the TaeCsTr99 repeats in the wheat genome, nanopore sequencing was conducted on 7DS BAC clones TaaCsp7DS028N04 (28N04) and TaaCsp7DS104G18 (104G18) from the ‘Chinese Spring’ 7DS arm-specific BAC library [40 (link)]. BAC DNA was extracted using alkaline lysis method followed by phenol-chloroform extraction and ethanol precipitation. Finally, the DNA was purified by incubating with 1:1 AMPure XP beads (Beckman Coulter, Miami, FL, USA) for 5 min and eluted into 30 µl 10 mM Tris, pH 8.5. Barcoded sequencing libraries were prepared from 700 ng DNA per BAC clone using Rapid Barcoding Sequencing Kit (SQK-RBK004; Oxford Nanopore Technologies, Oxford, UK) and sequenced together with additional ten clones on the MinION platform (Oxford Nanopore Technologies, Oxford, UK). Raw data were basecalled using Poretools 0.6.0 (https://github.com/arq5x/poretools, accessed on: 30 May 2019), demultiplexed using Porechop 0.2.3 (https://github.com/rrwick/Porechop, accessed on: 30 May 2019) and size-filtered >10 kb, which yielded 315 reads ranging from 10,003 to 101,160 bp, and 62 reads ranging from 10,082 to 149, 812 bp for the clone 28N04 and 104G18, respectively. Selected reads of 99,802 bp and 148,009 bp for 28N04 and 104G18, respectively, spanned the entire lengths of the respective clones.

The bacterial DNA from four blood cultures and six fresh grown isolates (three K. pneumoniae and three E. coli) was isolated and the libraries for Nanopore sequencing were constructed according to previously published protocols [19 (link),20 (link)]. In brief, purified DNA was barcoded using the Rapid Barcoding Sequencing kit SQK-RBK004 (Oxford Nanopore, Oxford, UK) and further purified using an Agencourt AMPure XP system (Beckman Coulter, Brea, CA, USA). Sequencing, data collection and base calling (high accuracy mode) were performed using MinION flow cells (R9.4.1 FLO-MIN106, Oxford Nanopore), MinKNOW software v3.6.5 and Guppy basecaller v3 (ONT), respectively. Human data were discarded, and reads were categorized based on the read quality score as pass (≥5) or fail (<5) by the basecaller. DNA libraries for Illumina sequencing were prepared using Illumina Nextera XT DNA sample preparation kit (Illumina, San Diego, CA, USA). Illumina libraries were sequenced in pair-end mode (2 × 300 bp) using the Illumina MiSeq platform.