Native barcoding expansion 1 12

Manufactured by Oxford Nanopore

Sourced in United Kingdom

Native Barcoding Expansion 1–12 is a set of 12 Native Barcoding kits designed for use with the Oxford Nanopore sequencing platform. The kits enable the preparation of DNA samples for sequencing, allowing for the identification and separation of individual samples within a multiplexed sequencing run.

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Lab products found in correlation

Ligation sequencing kit sqk lsk109, by Oxford Nanopore (3 mentions) Ligation sequencing kit, by Oxford Nanopore (3 mentions) Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions) Qubit 4 fluorometer, by Thermo Fisher Scientific (1 mentions) Nebnext companion module, by New England Biolabs (1 mentions) Minion platform, by Oxford Nanopore (1 mentions) Minion r9.4.1 flow cell, by Oxford Nanopore (1 mentions) Minion, by Oxford Nanopore (1 mentions) Nextera xt dna library preparation kit, by Illumina (1 mentions)

6 protocols using native barcoding expansion 1 12

Nanopore Sequencing of eccDNA and Whole Genomes

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For eccDNA sequencing, library preparation was carried out with 500 ng of cDNA using Native Barcoding Expansion 1–12 (Oxford Nanopore Technologies (Oxford, UK), catalog no. EXP-NBD104), and the Ligation Sequencing Kit SQK-LSK109 (Oxford Nanopore Technologies). Sequencing was performed using MinION equipped with an R9.4.1 flow cell.
For whole-genome sequencing, a fraction of short fragments was removed from 9 µg of total DNA using the Short-Read Eliminator Kit XL (PacBio, SKU 102-208-400), according to the manufacturer’s recommendations. The library was prepared with 1 µg of long fragment-enriched DNA using the Ligation Sequencing Kit SQK-LSK109 (Oxford Nanopore Technologies). Sequencing was carried out using PromethION P2 equipped with an R9.4.1 flow cell for 72 h. Basecalling was done using Guppy 6.4.6 (Oxford Nanopore Technologies, 2019). Adapters were trimmed from reads by Porechop 0.2.4 (Wick, n.d.) with default parameters.

Merkulov P., Serganova M., Petrov G., Mityukov V, & Kirov I. (2024). Long-read sequencing of extrachromosomal circular DNA and genome assembly of a Solanum lycopersicum breeding line revealed active LTR retrotransposons originating from S. Peruvianum L. introgressions. BMC Genomics, 25, 404.

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Native Barcoding for Eccentric DNA

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Library preparation was carried out with 1 μg of eccDNA or genomic DNA using the Native Barcoding Expansion 1–12 (Oxford Nanopore Technologies (Oxford, UK), catalog no. EXP-NBD104) and the Ligation Sequencing Kit SQK-LSK109 (Oxford Nanopore Technologies). Sequencing was performed by MinION equipped with an R9.4.1 flow cell.

Merkulov P., Egorova E, & Kirov I. (2023). Composition and Structure of Arabidopsis thaliana Extrachromosomal Circular DNAs Revealed by Nanopore Sequencing. Plants, 12(11), 2178.

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Nanopore DNA Library Preparation

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Extracted genomic DNA was used for library preparation with NEBNext Companion Module (New England Biolabs, USA) and Ligation Sequencing Kit (Oxford Nanopore Technologies, UK) in accordance with the manufacturer’s protocol. Samples were multiplexed using Native Barcoding Expansion 1–12 (Oxford Nanopore Technologies, UK). Library concentration was measured with the Qubit 4 Fluorometer (Thermo Fisher Scientific, USA) using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, USA). Sequencing was performed on the MinION platform (Oxford Nanopore Technologies, UK) using the R9.4.1 Flow Cell.

Shaidullina E.R., Schwabe M., Rohde T., Shapovalova V.V., Dyachkova M.S., Matsvay A.D., Savochkina Y.A., Shelenkov A.A., Mikhaylova Y.V., Sydow K., Lebreton F., Idelevich E.A., Heiden S.E., Becker K., Kozlov R.S., Shipulin G.A., Akimkin V.G., Lalk M., Guenther S., Zautner A.E., Bohnert J.A., Mardanova A.M., Bouganim R., Marchaim D., Hoff K.J., Schaufler K, & Edelstein M.V. (2023). Genomic analysis of the international high-risk clonal lineage Klebsiella pneumoniae sequence type 395. Genome Medicine, 15, 9.

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Nanopore Sequencing of Arabidopsis Genome

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Library preparation was carried out from 1 μg of DNA using the Native Barcoding Expansion 1–12 (Oxford Nanopore Technologies (Oxford, UK), catalog no. EXP-NBD104) and the Ligation Sequencing Kit SQK-LSK109 (Oxford Nanopore Technologies). Sequencing was performed by MinION equipped with a R10.3 flow cell. The sequencing process was operated by MinKNOW software (v.19.12.5). Basecalling was performed by Guppy (Version 3.2.10). Read mapping was carried out by minimap2 [49 (link)] to TAIR10.1 (https://www.ncbi.nlm.nih.gov/assembly/GCF_000001735.4/ (accessed on 3 November 2021)) genome assembly.

Kirov I., Merkulov P., Dudnikov M., Polkhovskaya E., Komakhin R.A., Konstantinov Z., Gvaramiya S., Ermolaev A., Kudryavtseva N., Gilyok M., Divashuk M.G., Karlov G.I, & Soloviev A. (2021). Transposons Hidden in Arabidopsis thaliana Genome Assembly Gaps and Mobilization of Non-Autonomous LTR Retrotransposons Unravelled by Nanotei Pipeline. Plants, 10(12), 2681.

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Hybrid Genome Sequencing of ATCC33701

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Using ATCC33701 gDNA, the complete genome sequence was obtained by combining sequencing data from both Illumina MiSeq (Illumina, San Diego, CA, USA) and MinION (Oxford Nanopore Technologies, Oxford, UK) sequencers. Illumina sequencing was performed according to the manufacturer’s instructions. Briefly, an index-tagged library was prepared using the Nextera XT DNA Library Preparation Kit (Illumina), and 300-bp paired-end reads were sequenced on an Illumina MiSeq instrument. Nanopore sequencing was also performed according to the manufacturer’s instructions. Briefly, a DNA library was prepared using a Ligation Sequencing Kit and a Native Barcoding Expansion 1–12 (Oxford Nanopore Technologies). The prepared library was subsequently loaded into a MinION flow cell (R9.4.1; Oxford Nanopore Technologies). The MinION sequencing run was performed over 48 h. Base-calling and barcoding were performed using Guppy v2.3.7 (Oxford Nanopore Technologies). Hybrid assembly of the MiSeq and MinION reads was performed using Unicycler v0.4.2 (28 (link)). Complete genomes were annotated using DFAST (https://dfast.nig.ac.jp/).

Suzuki Y., Sakaizawa N., Takai S., Kubota H., Hasegawa N., Sasaki Y, & Kakuda T. (2022). An Autobioluminescent Method for Evaluating In Vitro and In Vivo Growth of Rhodococcus equi. Microbiology Spectrum, 10(3), e00758-22.

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Time-series DNA Extraction and Nanopore Sequencing

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For each enrichment time point (24 to 40 hours in 4 hour intervals), 2 ml aliquots of enrichment were removed and pelletized using a benchtop Centrifuge (Eppendorf 5418 R, NY, USA) at 4000 rpm for 10 min. The pellet was resuspended in 300 ul of TE Buffer. 300 ul of the resuspended cells were loaded on the Maxwell® RSC Instrument (automated DNA extraction instrument, Madison, WI, USA) cartridge for DNA extraction. Genomic DNA was extracted using Maxwell® RSC Cultured Cells DNA Kit (Cat no: AS1260, Madison, WI, USA) on Maxwell RSC instrument following the manufacturer recommended protocol for Gram-positive bacteria.
Sequencing libraries were prepared using the ligation sequencing kit (Cat no: SQK-LSK109, Oxford Nanopore, Oxford, UK), according to the manufacturer's speci cations along with Native Barcoding Expansion 1-12 (Cat no: EXP-NBD104, Oxford Nanopore, Oxford, UK) for multiplexing the samples. The libraries were multiplexed into 2 pools (Pool1: 24hr, 28hr, 32hr: Pool 2: 36hr, 40hr). The libraries were sequenced using GridIon with Flow cell (Cat no: FLO-MIN106, Oxford, UK) following the manufacturer's recommended protocol.

Commichaux S., Javkar K., Ramachandran P., Nagarajan N., Filippis F.D., Chen Y., Reed E., González‐Escalona N., Strain E., Rand H., Pop M., & Ottesen A. (2020). Optimizing source tracking of Listeria monocytogenes with quasimetagenomics and integrated long and short read sequencing.

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