DNA libraries for Nanopore sequencing were prepared using the SQK-LSK110 Ligation Sequencing kit (Oxford Nanopore Technologies, UK) according to the manufacturer’s instructions. The libraries were loaded into Nanopore R.9.4.1 chemistry flow cells and sequenced on the MinION Mk1B sequencer (Oxford Nanopore Technologies, UK) using MinKNOW v21.06.13 (https://community.nanoporetech.com/downloads ) software. Raw Nanopore data was basecalled using the “dna_r9.4.1_450bps_sup.cfg” basecalling model with Guppy (v. 5.0.7, https://community.nanoporetech.com/downloads ).
Sqk lsk110 ligation sequencing kit
Manufactured by Oxford Nanopore
Sourced in United Kingdom
The SQK-LSK110 Ligation Sequencing Kit is a laboratory equipment product developed by Oxford Nanopore. It is designed for use with Oxford Nanopore's sequencing platforms. The kit provides the necessary reagents and consumables for performing ligation-based DNA or RNA sequencing.
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Lab products found in correlation
Minion mk1b sequencer, by Oxford Nanopore (1 mentions)
Minion sequencer, by Oxford Nanopore (1 mentions)
Flo min106 flow cell, by Oxford Nanopore (1 mentions)
Agarose gel electrophoresis, by Bio-Rad (1 mentions)
Proteinase k, by Geneaid (1 mentions)
Rnase a, by Serva Electrophoresis (1 mentions)
High sensitive dna chip, by Agilent Technologies (1 mentions)
Ovation ultralow system v2, by Tecan (1 mentions)
Novaseq, by Illumina (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Ds 11 fx spectrophotometer, by DeNovix (1 mentions)
Qubit dsdna br assay kit, by Thermo Fisher Scientific (1 mentions)
Lysozyme, by Geneaid (1 mentions)
Nextera dna flex library prep kit, by Illumina (1 mentions)
Agilent 4200 tapestation system, by Agilent Technologies (1 mentions)
Novaseq 6000 system, by Illumina (1 mentions)
Gridion mk1 device, by Oxford Nanopore (1 mentions)
Minion flow cell, by Oxford Nanopore (1 mentions)
8 protocols using sqk lsk110 ligation sequencing kit
1
Nanopore Sequencing Library Preparation
2
Nanopore Sequencing and Genome Assembly
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The genome was sequenced using the previously isolated HMW gDNA following the Oxford Nanopore Technologies (ONT) Genomic DNA by Ligation protocol for the SQK-LSK110 Ligation Sequencing Kit (Version: GDE_9108_v110_revE_10Nov2020). Sequencing run was performed on a MinION 1B in a R10.3 Flow Cell for 72 h. Basecalling was performed on a NVIDIA Jetson AGX Xavier developer kit using ONT Guppy v5.0.11 and the r9.4.1 HAC model. Reads were corrected, trimmed and assembled using Canu (Koren et al. 2017 (link)) (snapshot v2.2-development + 149 changes (r10258)). Genome size was set to 38 Mb and the correctedErrorRate parameter was set to 0.039 for the assembly step.
Polishing of the genome was performed by mapping high-coverage Nanopore long read data to the assembled genome using minimap2 v2.22 (Li 2018 (link)) and Pilon v1.24 (Walker et al. 2014 (link)). The genome was submitted to GenBank under the accession numbers CP083245-CP083252 and the corresponding BioProject PRJNA756890 and BioSample SAMN20929797 numbers.
Repeat elements were identified with RepeatModeler v2.0.2a and RepeatMasker v4.1.2.p1, and their density across the genome was calculated with the ‘coverage’ function of bedtools v2.30.0.
Polishing of the genome was performed by mapping high-coverage Nanopore long read data to the assembled genome using minimap2 v2.22 (Li 2018 (link)) and Pilon v1.24 (Walker et al. 2014 (link)). The genome was submitted to GenBank under the accession numbers CP083245-CP083252 and the corresponding BioProject PRJNA756890 and BioSample SAMN20929797 numbers.
Repeat elements were identified with RepeatModeler v2.0.2a and RepeatMasker v4.1.2.p1, and their density across the genome was calculated with the ‘coverage’ function of bedtools v2.30.0.
3
Nanopore cDNA library preparation
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Based on the sample molarity and average cDNA transcript length derived from the QC metrics, the sample input volume was calculated and used to progress into nanopore library preparation. The SQK-LSK110 ligation sequencing kit (Oxford Nanopore) was used to generate PromethION long-read cDNA libraries. The final sequencing was run on PromethION flow cell (v9.4.2) with one sample per flow cell by the DNA technology core at UC Davis. The output data was base-called live during the run using base-caller guppy (v5.0.12) in a super-accurate base-calling model.
4
Benchmarking Microbial Species in Metagenomic Samples
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The mock sample was composited with equal amounts of DNA molecules (moles) of six microbial species (6 fmol, 2.08% per species), 3 fmol (1.04%) of E. coli DH5a and 250 fmol DNA molecules of the human genome (86.51%). Two-microgram DNA of the mock sample was subjected to library preparation using MinION with SQK-LSK110 Ligation Sequencing Kit (Oxford Nanopore). Subsequently, the data analysis showed that the mock sample consisted of 2.19% S. epidermidis, 0.44% E. coli DH5a, 0.84% R. mucosa, 1.92% P. aeruginosa, 3.18% S. hominis, 1.30% S. aureus, 1.37% N. gonorrhoeae and 75.57% Homo sapiens. There is a slight deviation in the data analysis result compared with the designed fraction because the measured DNA amount has a deviation in practice (Table S3 ).
5
Whole Genome Sequencing of Trisomy 21 Using Nanopore
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WGS of the patient and her parents was performed using an Oxford Nanopore long-read sequencer. Libraries were prepared using an SQK-LSK110 Ligation Sequencing Kit (Oxford Nanopore Technologies) following the manufacturer’s protocol. Sequencing was carried out on FLO-MIN106 flow cells (Oxford Nanopore Technologies) using a MinION sequencer (Oxford Nanopore Technologies) for 96 h. Seven and one sequencing runs were performed for the patient and each parent, respectively. Reads with a mapping quality ≥ 60 were used for the following analyses. Copy numbers of the chr21 were estimated from the number of mapped reads within 500 bp bins.
6
Extraction and Sequencing of Aci44 Genomic DNA
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Whole genomic DNA (gDNA) of Aci44 was extracted by modified Marmur procedure8 ,25 . Briefly, Aci44 was inoculated in CAMHB and incubated at 37 °C, 200 rpm, overnight. After incubation, Aci44 (3 mL) was harvested and lysed by 10 mg/mL lysozyme (Geneaid, Taiwan). RNA and protein were removed using 0.6 mg/mL RNase A (Serva, Germany) and 0.1 mg/mL proteinase K (Geneaid, Taiwan), respectively. DNA was purified using phenol–chloroform extraction. Quality and quantity of gDNA were determined by 1% agarose gel electrophoresis (Bio-rad, USA), UV spectrophotometer (OD260/280, OD260/230 ratio) (DeNovix DS-11 FX + spectrophotometer, DeNovix, USA), Qubit dsDNA BR assay kit (Invitrogen, USA), and Bioanalyzer using a high-sensitive DNA chip (Agilent, USA). One hundred ng of extracted DNA was used for library preparation using Ovation Ultralow System V2 (Nugen, Switzerland) and sequenced on NovaSeq (Illumina, USA) for short-read sequencing. Nanopore platform was applied for long-read sequencing. One µg of gDNA was used for library preparation by Ligation Sequencing kit (SQK-LSK110, Oxford Nanopore, UK) and sequenced on MinION Nanopore (Oxford Nanopore, UK).
7
Nanopore and Illumina sequencing of DNA samples
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The DNA from sample 18-2804 was used to prepare a 1D ligation library using the Ligation Sequencing Kit SQK-LSK110 according to the manufacturer’s instructions (Oxford Nanopore Technologies, Oxford, UK). ONT libraries were run on a PromethION flowcell (FLO-PRO002) at Future Genomics Technologies BV (Leiden, The Netherlands) using the following settings: basecall model: high-accuracy; basecaller version: Guppy v4.3.4.
Parallel aliquots of both DNA samples (18-2804 and 18-2837) were used to prepare Illumina libraries using the Nextera DNA Flex Library Prep Kit according to the manufacturer’s instructions (Illumina Inc. San Diego, CA, USA). Library quality was measured via electrophoresis in D1000 ScreenTape on an Agilent 4200 TapeStation System (Agilent Technologies Netherlands BV, Amstelveen, The Netherlands). The genomic paired-end (PE) libraries were sequenced with a read length of 2 × 150 nt using the Illumina NovaSeq 6000 system. Image analysis and basecalling were performed by the Illumina pipeline.
Parallel aliquots of both DNA samples (18-2804 and 18-2837) were used to prepare Illumina libraries using the Nextera DNA Flex Library Prep Kit according to the manufacturer’s instructions (Illumina Inc. San Diego, CA, USA). Library quality was measured via electrophoresis in D1000 ScreenTape on an Agilent 4200 TapeStation System (Agilent Technologies Netherlands BV, Amstelveen, The Netherlands). The genomic paired-end (PE) libraries were sequenced with a read length of 2 × 150 nt using the Illumina NovaSeq 6000 system. Image analysis and basecalling were performed by the Illumina pipeline.
8
Nanopore Sequencing of Unique Genomic Samples
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We used approximately 1.5-2 µg of DNA as starting material for library preparation using the Ligation Sequencing Kit (SQK-LSK110; Oxford Nanopore Technologies, Oxford, United Kingdom). Modifications to the manufacturer’s instructions are described in Additional file 3 . Each prepared library was loaded onto a MinION flow cell (R9.4.1; FLOMIN-106D; Oxford Nanopore Technologies) and sequenced on a GridION Mk1 device (Oxford Nanopore Technologies) for 48 h, running MinKNOW v22.03.4. Adaptive sampling was configured using a custom BED file for each sample (see "BED file design " section below) using the Genome Reference Consortium Human Build 37 (GRCh37) as the reference genome [14 ]. We sequenced 30 unique samples (a total of 50 with replicates) on individual flow cells. Inter-run and intra-run replicates were included to test the robustness of our assay (Table 1 and Additional file 1 : Tables S1 and S2).
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