A DNA library for MinION sequencing was prepared based on the protocol for the Rapid Lambda Control Experiment using the Rapid Sequencing Kit (Oxford Nanopore Technologies). The library was loaded onto MinION R9.5 SpotON Flow Cells (Oxford Nanopore Technologies). Base calling was performed by MinKnow. Genome assembly was conducted using Canu v1.6 with the parameter “-nanpore-raw”. The assembled contigs were corrected using short reads obtained from the linked-read library using the HiSeq X platform. The paired-end reads were mapped to the assembled sequences by BWA-0.7.15 with the parameters “mem -M -T 30” (Li and Durbin 2009 (link)). Processing was performed using samtools-1.4 with the steps “view -q 30 -F 0x100” and “view -f 0x2” (Li 2011 (link)). Finally, polymorphisms detected by the Genome Analysis Toolkit HaplotypeCaller with the options “-out_mode EMIT_VARIANTS_ONLY–variant_index_type LINEAR–variant_index_parameter 128000–filter_reads_with_N_cigar” (McKenna et al. 2010 (link)) were incorporated into the assembled sequences using bcftools-1.4 with the option “consensus” (Li 2011 (link)).
Rapid sequencing kit
Manufactured by Oxford Nanopore
Sourced in United Kingdom
The Rapid Sequencing Kit is a laboratory equipment product from Oxford Nanopore. It is designed for rapid DNA or RNA sequencing. The kit includes all the necessary reagents and consumables required for the sequencing process.
Automatically generated - may contain errors
Lab products found in correlation
Minion, by Oxford Nanopore (3 mentions)
Miseq reagent kit v3, by Illumina (2 mentions)
Genomic tip kit, by Qiagen (2 mentions)
Flow cell version flo min107 r9, by Oxford Nanopore (2 mentions)
Guppy v2, by Oxford Nanopore (2 mentions)
Dneasy plant mini kit, by Qiagen (2 mentions)
Nextseq 500, by Illumina (2 mentions)
Dnbseq g400, by MGI Tech (2 mentions)
Minion flow cell, by Oxford Nanopore (2 mentions)
Nextera dna flex library prep kit, by Illumina (2 mentions)
Minion sequencer platform, by Oxford Nanopore (2 mentions)
Novaseq 6000 platform, by Illumina (2 mentions)
Rapid barcoding kit, by Oxford Nanopore (1 mentions)
Sqk rad004, by Oxford Nanopore (1 mentions)
Ultra long dna sequencing kit, by Oxford Nanopore (1 mentions)
Minion r9.4 flow cells, by Oxford Nanopore (1 mentions)
Sqk rbk004, by Oxford Nanopore (1 mentions)
Flo min106d, by Oxford Nanopore (1 mentions)
Novaseq 6000 sp reagent kit, by Illumina (1 mentions)
Dna prep kit, by Illumina (1 mentions)
28 protocols using rapid sequencing kit
1
Hybrid Nanopore and Illumina Genome Assembly
2
Genomic Sequencing of T. caerulea Mycelia
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Genomic DNA sequencing of T. caerulea was carried out with 50 mg mycelia (10 days, in liquid MEP medium at 25°C with shaking at 100 rpm, in the dark) ground under liquid nitrogen. Genomic DNA was extracted as described above. To prepare a library for Nanopore sequencing, 500 ng DNA was processed with a rapid sequencing kit (Oxford Nanopore Technologies) according to the manufacturer’s instructions and sequenced on a MinION flow cell. The genome was assembled using CANU (62 (link), 63 (link)) v.1.9., based on an expected genome size of 50 Mbp. Signal-level reads were indexed against the draft genome using Nanopolish software (64 (link)). After using minimap (65 (link)) and samtools (66 (link)) to sort and map the reads, a consensus sequence was calculated using Nanopolish (67 (link)). Gene models were individually verified with the Augustus algorithm in Aspergillus mode (68 (link)) and compared with BLAST (69 (link)).
3
Targeted Sequencing of HPV16 in Cell Lines
4
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.
5
Genome Assembly of Pisum sativum Line JI128
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DNA was extracted from the leaves of JI128 plants using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Short-read DNA sequencing libraries were prepared according to the manufacturer’s instructions, and sequenced on DNA sequencers, NextSeq 500 (Illumina) and DNBSEQ-G400 (MGI Tech, Shenzhen, China). The genome size of line JI128 was estimated by k-mer distribution analysis using the Jellyfish software (Marcais and Kingsford 2011 (link)).
High-molecular-weight DNA was extracted from JI128 DNA using the Genomic-tip kit (Qiagen), and a long-read sequence library was constructed using the Rapid Sequencing Kit (version SQK-RAD004) (Oxford Nanopore Technologies, Oxford, UK). The library was sequenced with the MinION using flow cell version FLO-MIN107 R9 (Oxford Nanopore Technologies). Base calling from the FAST5 files was performed using Guppy v2.3.5 (Oxford Nanopore Technologies). Long reads were assembled with wtdbg2 v2.2 (Ruan and Li 2020 (link)), and potential sequencing errors in the contigs were corrected only once using Pilon (Walker et al. 2014 (link)). The resulting genome assembly was designated as PSA_r1.0. Assembly completeness was evaluated with the embryophyta_odb10 data using Benchmarking Universal Single-Copy Orthologs (BUSCO) v3.0.2 (Simao et al. 2015 (link)).
High-molecular-weight DNA was extracted from JI128 DNA using the Genomic-tip kit (Qiagen), and a long-read sequence library was constructed using the Rapid Sequencing Kit (version SQK-RAD004) (Oxford Nanopore Technologies, Oxford, UK). The library was sequenced with the MinION using flow cell version FLO-MIN107 R9 (Oxford Nanopore Technologies). Base calling from the FAST5 files was performed using Guppy v2.3.5 (Oxford Nanopore Technologies). Long reads were assembled with wtdbg2 v2.2 (Ruan and Li 2020 (link)), and potential sequencing errors in the contigs were corrected only once using Pilon (Walker et al. 2014 (link)). The resulting genome assembly was designated as PSA_r1.0. Assembly completeness was evaluated with the embryophyta_odb10 data using Benchmarking Universal Single-Copy Orthologs (BUSCO) v3.0.2 (Simao et al. 2015 (link)).
6
Rapid Sequencing Library Construction
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Library construction was performed using a Rapid Sequencing Kit (Oxford Nanopore Technologies, Oxford, UK, Cat. # SQK-RAD004). Before loading on the flow cell, a total of 34 μL of sequencing Buffer and 25.5 μL of loading Beads were added to 12 μL of the DNA libraries and 4.5 μL nuclease free water. After that, priming and loading onto FLO-MIN106 flow cell was performed [15 (link)].
7
Rapid Sequencing Library Construction
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Library construction was performed using Rapid Sequencing Kit (Oxford Nanopore Technologies, Oxford, UK, Cat. # SQK-RAD004). Before loading on the flow cell, a total of 34 μL of sequencing Buffer and 25.5 μL of loading Beads were added to 12 μL of the DNA libraries and 4.5 μL nuclease free water. After that priming and loading onto FLO-MIN106 flow cell were performed.
8
Complete Bacterial Genome Assembly
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Genomic DNA was extracted using the NucleoBond HMW DNA kit (MACHEREY-NAGEL Inc., PA, USA) according to manufacturer’s protocol with the additional step of incubating the previously frozen bacterial pellets (following thawing, a wash with PBS and centrifugation) in 560 µl Tris-EDTA (TE, pH 8.0) containing lysostaphin (1 µg µl−1) prior to the enzymatic lysis step. Genomic DNA (gDNA) was sequenced by Psomagen, Inc. (Rockville, MD) using Illumina HiSeq paired-end with a read length of 100bps. gDNA from the same sample was also sequenced with Oxford Nanopore Technologies FLONGLE Flongle (ONT, United Kingdom) using the Rapid Sequencing Kit (SOK-RAD004) according to the manufacturer’s protocol. Sequencing reads obtained from both platforms were used to create a de novo assembly of the complete genome, as described below in the ‘Bioinformatic analyses’ section.
9
Hybrid Assembly of IncP-6 Plasmids
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Nanopore sequencing was performed according to the manufacturer’s instructions. Briefly, DNA libraries were prepared using a rapid sequencing kit (Oxford Nanopore Technologies), and the prepared library was subsequently loaded into a MinION flow cell (R9.4; Oxford Nanopore Technologies). Raw data (FAST5 files) were base-called, converted to FASTQ format and trimmed of barcode and adapter sequences using Guppy v3.0.3 (Ueno et al., 2003 (link)). Hybrid assemblies of both short and long reads were performed using Unicycler v0.4.8 (Wick et al., 2017 (link)) and annotated with Prokka (1.14.6) (Seemann, 2014 (link)) and RAST server (https://rast.nmpdr.org/ ). Manual annotation of IncP-6 plasmids was additionally performed using National Center for Biotechnology Information (NCBI) BLASTn (Altschul et al., 1990 (link)) and Uniprot databases (Consortium, 2019 (link)). The annotation of ISs was performed using ISFinder (https://isfinder.biotoul.fr/ ) (Siguier et al., 2006 (link)). Final hybrid assemblies were compared to publicly available IncP-6 plasmids using BRIG (Alikhan et al., 2011 (link)), and comparison of plasmids was facilitated using the Artemis Comparison Tool (Carver et al., 2005 (link)).
10
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.
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