Native genomic DNA (gDNA) was sequenced on a MinION R9.4 flowcell [Oxford Nanopore (ONT)]. The sequencing library was prepared using the ONT Rapid Barcoding Sequencing kit (SQK-RBK004) according to the manufacturer’s protocol with the following modification: the gDNA input was increased to 800 ng genomic DNA and the optional SPRI bead cleanup was omitted. The run was allowed to proceed for approximately 6 h.
Minion r9.4 flowcell
Manufactured by Oxford Nanopore
Sourced in United Kingdom
The MinION R9.4 flowcell is a key component of Oxford Nanopore's DNA/RNA sequencing technology. It is designed to host and interface with nanopore sensing elements that detect and record the passage of nucleic acid molecules through the pores. The flowcell provides the necessary fluidics, electronics, and supporting infrastructure to enable real-time, long-read sequencing.
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4 protocols using minion r9.4 flowcell
1
Long-read Sequencing of Genomic DNA
2
Hybrid genome assembly of Lp VT1065
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A draft genome sequence of Lp VT1065 based on Illumina sequencing was previously published [23 ]. Here, the same genome was also sequenced using an Oxford Nanopore Technologies (ONT) MinION R9.4 flow cell (FLO-MIN106D). Genomic DNA was extracted using the MasterPure™ Gram Positive DNA Purification Kit, Lucigen kit. The sequencing library was prepared following the 1D Genomic DNA by ligation SQK-LSK108 protocol provided by ONT. The generated Fast5 files containing the raw signals were base-called using the ONT software Guppy 3.3.2. A hybrid genome assembly was then generated with the Unicycler v0.4.7 pipeline [25 (link)] with default parameters using the short reads from the published Illumina sequence and the long reads generated by the MinION. The assembly was annotated using Prokka 1.14.6 [26 (link)]. The assembled genome was submitted to NCBI to update the earlier submitted draft genome and has the accession number GCA_003049605.2.
3
Complete Genome Sequence of Pseudomonas sp. Leaf58
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As further evidence of the existence of a megaplasmid in Leaf58, we generated a complete genome assembly for this strain (currently found at Figshare [doi:10.6084/m9.figshare.6914033], GenBank accession TBD). After revival from the Baltrus lab stock, a single colony Pseudomonas sp. Leaf58 was picked to an overnight culture in KB media and grown in a shaking incubator at 27 °C. After ∼24 h, DNA was extracted from this culture using a Promega Wizard kit. A rapid sequencing library was created using this DNA, and 169,316 reads (933,937,907 total bp, 5,515 bp average read size) were generated on an Oxford Nanopore MinION R9.4 flowcell using a Rapid sequencing kit (SQK-RAD004) and can be found in the Sequence Read Archive (SRA) (accession SRP157842). Additionally, 100-bp paired end Illumina reads used to generate the original draft genome of this strain were downloaded from the SRA (accession ERR1103815) (Bai et al. 2015 (link)). A complete genome sequence for Pseudomonas sp. Leaf58 was generated by combining these short and long reads in Unicycler (version 0.4.4) (Wick et al. 2017 (link)). This sequence consists of a single chromosome (5,432,868 bp) and the pBASL58 megaplasmid (904,253 bp), both of which were circular according to Unicycler.
4
Colistin-resistant Isolate Sequencing
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DNA was prepared using the Wizard® Genomic DNA Purification Kit (Promega, USA). Paired-end 150-basepair Nextera XT libraries of whole genomic DNA were sequenced on the Illumina NextSeq® 500 platform (Illumina, USA) with a target average coverage of 100-fold was performed on the 39 colistin-resistant Thailand isolates. Twelve mcr-positive isolates were selected for long-read sequencing using high molecular-weight genomic DNA and the rapid barcoding kit (SQK-RBK004) to prepare libraries, which then were sequenced on a MinION R9.4 flow cell (Oxford Nanopore, UK) with a target average coverage of 20-fold to improve the respective draft genome assemblies.
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