The sequencing libraries were prepared using the Native barcoding amplicons protocol with EXP-NBD104 and SQK-LSK109 kits (Oxford Nanopore Technologies) according to Davidov et al. (2020) (link). The 16S rRNA and 18S rRNA gene sequencing libraries were loaded to the same MinION flow cell in two batches: the first included the amplified 16S rRNA and 18S rRNA gene DNA barcodes of replicates 1, 3, and 5 (12 multiplexed libraries in total), and the second included the amplified 16S rRNA and 18S rRNA gene DNA barcodes of replicates 2 and 4 (8 multiplexed libraries in total). The two sequencing runs were separated by a washing step using EXP-WSH002 Kit (Oxford Nanopore Technologies). Each library was loaded onto to the MinION Nanopore Spot-on flow cell (FLO-MIN106D, version R9) and sequenced until reaching ∼7 Giga nucleotides (∼4 M reads). The ITS2 sequencing library was loaded to a new MinION flow cell and sequenced until reaching ∼1.3 Giga nucleotides (∼1 M reads). Base-calling for all libraries were done by the Guppy base calling software 3.3.3, using MinKNOW program with the “high accuracy” option. Raw reads were obtained in FAST5 and FASTq formats from which “pass” quality reads were subjected to further analysis.
Sqk lsk109 kit
Manufactured by Oxford Nanopore
Sourced in United Kingdom, United States
The SQK-LSK109 kit is a laboratory consumable product developed by Oxford Nanopore. It is designed to enable the preparation of DNA or RNA samples for sequencing using Oxford Nanopore's nanopore-based sequencing technology. The kit contains the necessary reagents and components required for the library preparation process prior to sample loading onto an Oxford Nanopore sequencing device.
Automatically generated - may contain errors
Lab products found in correlation
Minion, by Oxford Nanopore (6 mentions)
Nebnext ultra 2 end repair da tailing module, by New England Biolabs (4 mentions)
Ampure xp bead, by Beckman Coulter (4 mentions)
Exp nbd104, by Oxford Nanopore (3 mentions)
Bluepippin cassette kit blf7510, by Sage Science (3 mentions)
Nebnext ffpe dna repair mix, by New England Biolabs (3 mentions)
Promethion sequencer, by Oxford Nanopore (3 mentions)
Bluepippin system, by Sage Science (3 mentions)
Dneasy plant mini kit, by Qiagen (3 mentions)
Flo min106d, by Oxford Nanopore (2 mentions)
Buffer g2, by Qiagen (2 mentions)
Flo min106d flow cell, by Oxford Nanopore (2 mentions)
Agencourt ampure xp bead, by Beckman Coulter (2 mentions)
Nebnext quick ligation module, by New England Biolabs (2 mentions)
Nextflex dna sequencing kit, by PSC Biotech (2 mentions)
Eb buffer, by Qiagen (2 mentions)
Ampure xp buffer, by Beckman Coulter (2 mentions)
Truseq stranded total rna library prep kit, by Illumina (2 mentions)
Minion sequencing device, by Oxford Nanopore (2 mentions)
Nextera library preparation kit, by Illumina (2 mentions)
47 protocols using sqk lsk109 kit
1
Nanopore Sequencing of Microbial Genes
2
Native Barcoding and Sequencing with MinION
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MinION libraries were generated using the EXP-NBD104 (1–12) Native Barcoding and SQK-LSK109 Kits (Oxford Nanopore Technologies, Oxford, UK). Libraries were loaded onto an FLO-MIN106 flowcell on the MinION device (Oxford Nanopore Technologies) and sequenced using MinKNOW 1.15.1 with the standard 48-hour run script.
3
Nanopore Sequencing of H3F3A Mutations
4
Nanopore Sequencing of Paramyxoviruses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Preparation of sequencing libraries was done using the SQK-LSK109 kit (Oxford Nanopore Technologies) according to the manufacturer’s protocol. When sequencing more than one sample per flow cell, the EXP-NBD104 and EXP-NDB114 kits were used for barcoding of individual samples. For each sample, 200‐ng cDNA was used as input and ∼120 fmol library was loaded onto the R.9.4.1 flow cell. Sequencing was performed on a MinION Mk1B or GridION. Basecalling (and demultiplexing) was performed using Guppy v3 and above. Porechop (github.com/rrwick/Porechop) was used to remove sol-Primer sequences added during cDNA generation. To identify reads corresponding to paramyxovirus sequences, a tblastx search of all reads against a custom BLAST database containing a representative sequence of each known orthoparamyxovirus species was done.
5
Nanopore Sequencing of Wheat Blast Fungus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
6
Nanopore and Illumina Sequencing of SARS-CoV-2 Infection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Nanopore WGS of DNA from hESCs after ACE2 transduction and SARS-CoV-2 infection was performed as previously described [27 (link)], except that the SQK-LSK110 kit (Oxford Nanopore Technologies) was used for the construction of the sequencing library instead of the previously used SQK-LSK109 kit [27 (link)]. Sequencing read alignment to the human and SARS-CoV-2 genomes was performed as previously described [27 (link)]. The number of mapped bases was obtained by running samtools stats (version 1.11, http://www.htslib.org/doc/samtools-stats.html ) (accessed on 2 November 2022) [40 (link)] on the aligned SAM file. Sequencing genome coverage was calculated by dividing the mapped base number by the human genome size (3,200,000,000 bp).
Illumina WGS of DNA from Calu3 cells after SARS-CoV-2 infection, as well as sequencing read alignment, was performed as previously described [27 (link)]. Sequencing genome coverage was calculated by multiplying the mapped read–pair number (obtained from the read alignment report) with the read–pair length (2 × 150 bp) and then dividing the result by the human genome size (3,200,000,000 bp).
Illumina WGS of DNA from Calu3 cells after SARS-CoV-2 infection, as well as sequencing read alignment, was performed as previously described [27 (link)]. Sequencing genome coverage was calculated by multiplying the mapped read–pair number (obtained from the read alignment report) with the read–pair length (2 × 150 bp) and then dividing the result by the human genome size (3,200,000,000 bp).
7
High-quality Genomic DNA Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A188 were grown in the greenhouse at 28°C and 23°C day/night, with a photoperiod of 14:10 h (light:dark). Nuclei were isolated from seedling leaves using a modified nucleus isolation protocol [60 (link)] and dissolved in buffer G2 (Qiagen). The lysate was used for DNA isolation with Qiagen DNeasy Plant Mini Kit (Qiagen) following the manufacturer protocol. A188 genomic DNA was size selected for 15–30 kb and above with the BluePippin cassette kit BLF7510 (Sage Science) high-pass-filtering protocol, followed by a library preparation with the SQK-LSK109 kit (Oxford Nanopore). Each DNA library was loaded on an FLO-MIN106D flowcell and sequenced on MinION (Oxford Nanopore). The basecaller Guppy (version 3.4.4) was used to convert FAST5 raw data to FASTQ data with default parameters.
8
Nanopore Sequencing of DNA Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quantification steps were performed using the dsDNA HS assay for Qubit. DNA was end-repaired (New England BioLabs, MA, USA), cleaned with Agencourt AMPure XP Beads (Beckman Coulter, High Wycombe, UK) and dA-tailed (New England BioLabs, MA, USA). The library was prepared from 1400 ng input DNA using the SQK-LSK109 kit (Oxford Nanopore Technologies, Oxford, UK) in accordance with the manufacturer’s protocol.
The library was quantified and prepared for GridION sequencing, using FLO-MIN106 flowcells, MinKNOW v18.12.4, standard 48-h run script with active channel selection enabled, until 2.5 Gb of data was collected from each sample. The mean read length of the sequenced reads was 3944 bps and the mean quality score was 10.45.
The library was quantified and prepared for GridION sequencing, using FLO-MIN106 flowcells, MinKNOW v18.12.4, standard 48-h run script with active channel selection enabled, until 2.5 Gb of data was collected from each sample. The mean read length of the sequenced reads was 3944 bps and the mean quality score was 10.45.
9
Oxford Nanopore long-read sequencing protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The library was prepared according to the following protocol, using the Oxford Nanopore SQK-LSK109 kit. Genomic DNA fragments (4 µg) were repaired and 3′-adenylated with the NEBNext FFPE DNA Repair Mix and the NEBNext® Ultra™ II End Repair/dA-Tailing Module (New England Biolabs, Ipswich, MA, USA). Sequencing adapters provided by Oxford Nanopore Technologies (Oxford Nanopore Technologies Ltd, Oxford, UK) were then ligated using the NEBNext Quick Ligation Module (NEB). After purification with AMPure XP beads (Beckmann Coulter, Brea, CA, USA), half of the library was mixed with the sequencing buffer (ONT) and the loading bead (ONT) and loaded on a PromethION R9.4.1 flow cell. The second half of the library was loaded on the flow cell after a Nuclease Flush using the Flow Cell Wash Kit EXP-WSH003 (ONT) according to the Oxford Nanopore protocol. Reads were basecalled using Guppy version 4.0.1. The nanopore long reads were not cleaned and raw reads were used for genome assembly.
10
Targeted Nanopore Sequencing for Structural Variant Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 1 µg of DNA from the unprocessed spindle biopsy was subjected to library construction using the SQK-LSK109 kit and sequenced on the GridION sequencer with R9.4.1 flow cells (Oxford Nanopore Technologies, Oxford, UK). The adaptive sampling option was enabled in MinKNOW 20.10.6. For targeting, a bed-file and the human genome GRCh38 were used. The intervals were constructed manually, consisting of individual SV breakpoints with 15 kb padding as well as larger regions, e.g., the complete chr9p region. In total, 112 regions comprising 112 Mb were targeted with this approach (Table S2 ). Base calling was done with guppy 5.1.13 and the super-accuracy model. Reads were trimmed with Porechop 0.2.4 [54 (link)] and mapped to GRCh38 with minimap2 2.17 [55 (link)] using the -R, -Y, and the –MD parameters. SNP calling was performed with longshot 0.4.1 [59 (link)], SNPs were filtered for QUAL > 100, and reads were phased with WhatsHap [60 (link)].
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!