Library preparation with barcode labelling was performed with ~400ng input of genomic DNA using the Rapid Barcoding Kit (RBK004, Oxford Nanopore Technologies, UK) according to manufacturer's instructions. During library preparation, input DNA is fragmented while simultaneously attaching barcodes using a time-efficient transposase-based approach. The final library was loaded onto a R9.4.1 flow cell (FLO-MIN106D, Oxford Nanopore Technologies, UK; alternatively, FLG-0001 cells sharing the architecture with FLO-MIN106D, were used) and whole-genome sequencing was performed for 6 to 24 hours on a MinION Mk 1B device (Oxford Nanopore Technologies, UK). FAST5 files containing the raw data were obtained in real-time using the manufacturer's software MinKNOW (v.1.3.1-v.3.6.0) and transferred to a high-performance computing (HPC) cluster for further analysis. Each flow cell was washed after sequencing (WSH002/WSH003, Oxford Nanopore Technologies, UK) and reused for up to 4 samples. When multiplexing retrospective samples, up to five libraries were sequenced simultaneously and sequencing for up to 24 hours was performed.
Flo min106d
Manufactured by Oxford Nanopore
Sourced in United Kingdom
The FLO-MIN106D is a lab equipment product from Oxford Nanopore. It is a flow cell designed for use with Oxford Nanopore's sequencing platforms. The flow cell is a key component in the sequencing process, providing a controlled environment for the DNA samples to be analyzed.
Automatically generated - may contain errors
Lab products found in correlation
Rapid barcoding kit, by Oxford Nanopore (3 mentions)
Minion, by Oxford Nanopore (3 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Sqk lsk109 kit, by Oxford Nanopore (2 mentions)
Ultrasonicator, by Covaris (2 mentions)
Lsk109, by Oxford Nanopore (2 mentions)
Ampure xp beads, by Oxford Nanopore (2 mentions)
Sqk lsk109, by Oxford Nanopore (2 mentions)
Minion mk1b device, by Oxford Nanopore (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Minion mk1c device, by Oxford Nanopore (1 mentions)
Pcr barcoding kit sqk pbk004, by Oxford Nanopore (1 mentions)
Dna prep kit, by Illumina (1 mentions)
Miseq reagent kit v3, by Illumina (1 mentions)
Rapid sequencing kit, by Oxford Nanopore (1 mentions)
Novaseq 6000 sp reagent kit, by Illumina (1 mentions)
Dneasy ultraclean microbial kit, by Qiagen (1 mentions)
Agencourt ampure xp bead, by Beckman Coulter (1 mentions)
Nebnext ultra 2 end repair da tailing module, by New England Biolabs (1 mentions)
18 protocols using flo min106d
1
Rapid Barcoded Library Prep for Whole-Genome Nanopore Sequencing
2
Long-read Sequencing of Cervical Cancer DNA
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Total DNAs were respectively extracted from a cervical cancer tissue sample and CaSki cells using a QIAamp® DNA Mini Kit (C51304, Qiagen, Hilden, Germany) according to the manufacturer’s protocol and quantified using a Qubit dsDNA HS Assay Kit (Q33230, Thermo Fisher Scientific, Waltham, MA, United States). Two μg of DNA fragments ( >10 kb) was used for library preparation and sequenced using a MinION SQK-LSK109 Oxford nanopore sequencing kit (SQK-LSKSP9, Oxford Nanopore Technologies, Oxford, United Kingdom) according to the manufacturer’s instructions. Briefly, DNA was repaired with FFPE DNA Repair Mix and End repair/dA-tailing Module reagents [E7695, New England BioLabs (NEB), Ipswich, MA] and purified with AMPure XP beads (A63880, Beckman Coulter, United States), washed with 70% ethanol, and eluted with nuclease-free water. Sequencing adapters were added to the 3′ ends of fragmented DNA using Adapter Mix and Quick T4 DNA Ligase with Ligation Buffer (NEB) and purified with AMPure beads. The prepared library was added into a SpotON flow cell (FLO-MIN106D, Oxford Nanopore Technologies) and sequenced using a MinION sequencer Mk1B.
3
Nanopore Sequencing of Wheat Blast Fungus
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Wheat blast, a devastating emerging wheat disease, is caused by the fungus Magnaporthe oryzae Triticum (MoT) (28 (link)). Nanopore long reads from a virulent MoT strain B71 were produced for the de novo genome assembly. B71 nuclear genomic DNA was prepared as described previously (28 (link)). Genomic DNA was subjected to 20 kb size selection using Bluepippin cassette kit BLF7510 with High-Pass Protocol (Sage Science, USA), followed by library preparation with the SQK-LSK109 kit (Oxford Nanopore, UK). Library was loaded to the flow cell FLO-MIN106D (Oxford Nanopore, UK) and sequenced on MinION (Oxford Nanopore, UK). Guppy version 2.2.2 was used to convert Nanopore raw data (fast5) to FASTQ data with default parameters.
4
Low-Coverage Whole-Genome Sequencing Protocol
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Between 200 and 400 ng of genomic tumor DNA of each sample is used for library preparation with barcode labeling using the Rapid Barcoding Kit (SQK-RBK004, Oxford Nanopore Technologies, Oxford, UK) according to the manufacturer’s instructions. Low-coverage whole-genome sequencing (lcWGS) is performed on a Minion Mk1C device (OS ubuntu 18.04) using an R9.4.1 flow cell (FLO-MIN106D, Oxford Nanopore Technologies). Sequencing was performed until the recommended 100M bps (per correspondence with the nanoDx pipeline developer [19 (link)]). Output FAST5 files containing the raw signal data were generated by the manufacturer’s software MinKNOW (v.22.12.5) and the equivalent FASTQ files. They were all transferred to high-performance computing (HPC) clusters for further analysis.
5
MinION Nanopore Sequencing of PCR Products
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DNA concentration of final purified PCR products was determined by Qubit BR (Thermo Fischer) measurement and 12 samples were pooled at equimolar concentrations. Subsequently, 50 femtomol of pooled PCR products were loaded onto a MinION flow cell (FLO-MIN106D) according to the manufacturer’s protocol (PCR Barcoding Kit SQK-PBK004,,Oxford Nanopore Technologies).and sequencing was performed using a MinION device and MinKnow software (MinION Release 19.12.5) for 48 h.
6
Multimodal Sequencing of SARS-CoV-2
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Sequencing libraries were prepared with a tagmentation-based Illumina DNA Prep kit (#20060060; Illumina, San Diego, CA, USA) and run in a NovaSeq 6000 SP Reagent Kit (#20028312; Illumina) flow cell using 2×150 paired-end sequencing. To improve assembly quality, the library from swab 353R, an unpassaged vesicular fluid from a confirmed case, was also run in a MiSeq Reagent Kit v3 (#MS-102-3003; Illumina) flow cell using 2×300 paired-end sequencing. Additionally, sample 353R was also analyzed by single-molecule methods using nanopore sequencing (Oxford Nanopore Technologies, Oxford, UK). For nanopore sequencing, 210 ng of DNA was extracted from swab 353R and used to prepare a sequence library with a Rapid Sequencing Kit (#SQK-RAD114; Oxford Nanopore Technologies); the library was analyzed in an FLO-MIN106D (#FLO-MIN106D; Oxford Nanopore Technologies) flow cell for 25 h. The process rendered 1.12 Gb of filter-passed bases.
7
Metagenomic Sequencing of Environmental Samples
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The extracted DNA was used for both short- and long-read shotgun metagenomic sequencing. For short-read sequencing, the DNA was sheared to 500 bp, on average, using an ultrasonicator (Covaris), and a 24-sample multiplexed library was prepared using an MGIEasy universal DNA library prep set (catalog no. 1000006986; MGI), a circularization kit (catalog no. 1000005259; MGI), and a MGISEQ-2000RS high-throughput sequencing set (catalog no. 1000013857; MGI) with seven cycles of PCR amplification. A 1 × 400 bp single-end sequencing was run using one lane of the MGI DNBSEQ-G400 platform. For long-read sequencing, long DNA molecules were purified using diluted (0.45×) AMPure XP beads, and a sequencing library was prepared using a ligation sequencing kit (LSK-109; Oxford Nanopore). Each of the 24 samples was sequenced by an R9.4.1 flow cell (FLO-MIN106D; Oxford Nanopore) using the Oxford Nanopore GridION platform for 72 h. Base calling was performed using Guppy (v3.2.10; high-accuracy mode).
8
Nanopore Sequencing and Genome Assembly
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DNA was isolated using a DNeasy UltraClean microbial kit (Qiagen, Venlo, The Netherlands). Nanopore sequencing was performed according to protocol SQK-RBK110.96 with Flow Cell version R9.4.1 on a MinION device (FLO-MIN106D; Oxford Nanopore, Oxford, UK), using the super-accurate base-calling method in MinKNOW v22.12.7. Reads were trimmed and downsampled to 200× coverage using filtlong (https://github.com/rrwick/Filtlong , accessed on 6 January 2024) and assembled into circular contigs using Flye v2.9.1 [52 (link)]. Genomes were polished using Medaka v.1.1.0 (https://github.com/nanoporetech/medaka , accessed on 6 January 2024) and Homopolish (https://github.com/ythuang0522/homopolish?tab=readme-ov-file , accessed on 6 January 2024) [53 (link)] and annotated using Prokka v1.14.5 [54 (link)].
9
Nanopore Sequencing Library Preparation
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Sequencing libraries were diluted in elution buffer (QIAGEN 19086) to a concentration corresponding to ∼20 ng of library per sequencing run. MinION flow cells (Oxford Nanopore FLO-MIN106D) were prepared using the Ligation Sequencing Kit (Oxford Nanopore SQK-LSK109). Libraries were then loaded onto the flow cell and sequencing allowed to proceed for 10–20 h depending on library size.
10
Whole Genome Sequencing of Bacterial Isolates using MinION
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MinION (Oxford Nanopore Technologies) was chosen as the LR-NGS platform and WGS was performed on 20 isolates (DNA samples of three isolates were not available). For library preparation, DNA was end-repairelid and A-tailed with NEBNext FFPE DNA Repair Mix and the NEBNext Ultra II End Repair/dA-Tailing Module (New England Biolabs, Ipswich, MA, USA). Ligation of the sequencing adapters was performed using the SQK-LSK109 kit (Oxford Nanopore Technologies) and each sample was barcoded using the EXP-NBD104 kit (Oxford Nanopore Technologies). Each step was followed by purification with Agencourt AMPure XP beads (Beckman Coulter). Libraries were pooled in equimolar amounts and adjusted to a final concentration of 1 pM. Twenty isolates were sequenced per flow cell (FLO-MIN106D; Oxford Nanopore Technologies) using MinKNOW software (ver. 20.10; Oxford Nanopore Technologies).
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