Virus- and vesicle-enriched DNAs were processed using the Nanopore Ligation Sequencing Kit (LSK-109; Oxford Nanopore Technologies, Ltd.) following the manufacturer’s instructions for the processing of high–molecular weight DNA. A total of two virus-enriched libraries were prepared using 2 and 1.5 μg of DNA each for the sequencing runs. All libraries were sequenced on a GridION X5 using FLO-MIN106 (R 9.4.1) flow cells (Oxford Nanopore Technologies, Ltd.). Read base calls were generated from the signal traces using Guppy version 3.0. The sequencing yield for the virus-enriched fraction totaled 31 Gbp, generating reads with an N50 of 37.67 kb (i.e., half of all bases are in reads at least 37,670 bases long). The sequencing yield for the corresponding membrane vesicle–enriched fraction totaled 46 Gbp, with an N50 of 4.6 kbp.
Gridion x5
Manufactured by Oxford Nanopore
Sourced in United Kingdom, United States, China
The GridION X5 is a compact, multi-channel DNA/RNA sequencing device designed for high-throughput genomic analysis. It features five independent flow cell slots, enabling simultaneous operation of up to five sequencing experiments. The GridION X5 uses Oxford Nanopore's proprietary nanopore technology to perform real-time, long-read sequencing of nucleic acid samples.
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Lab products found in correlation
Ampure bead, by Beckman Coulter (6 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (6 mentions)
Nebnext ultra 2 end repair kit, by New England Biolabs (5 mentions)
Dnbseq g400, by MGI Tech (3 mentions)
Minion mk1b, by Oxford Nanopore (3 mentions)
Agilent bioanalyzer, by Agilent Technologies (3 mentions)
Neb quick t4 dna ligase, by New England Biolabs (2 mentions)
Minion, by Oxford Nanopore (2 mentions)
Ligation sequencing kit 1d, by Oxford Nanopore (2 mentions)
Neb blunt ta ligase, by New England Biolabs (2 mentions)
Lsk109, by Oxford Nanopore (2 mentions)
Nextseq 500, by Illumina (2 mentions)
Quick dnatm fecal soil microbe miniprep kit, by Zymo Research (2 mentions)
Flo min106 r 9.4.1 flow cells, by Oxford Nanopore (1 mentions)
Nanopore ligation sequencing kit, by Oxford Nanopore (1 mentions)
Sqk lsk109, by Oxford Nanopore (1 mentions)
Nebnext ultra 2 kit, by New England Biolabs (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Sqk lsk110 kit, by Oxford Nanopore (1 mentions)
Short read sequencing, by Illumina (1 mentions)
40 protocols using gridion x5
1
Nanopore Sequencing of Virus and Vesicle DNAs
2
Next-Generation Sequencing for Metagenomic Profiling
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There are several steps in the NTS analysis, which was described in our previous study (18 (link)). First, bacterial 16S rDNA gene and fungal internal transcribed spacer regions 1 and 2 (ITS1/2) were amplified using corresponding universal primers (21 (link)). Then, amplification products were purified and mixed to be used to construct sequencing libraries. Clinical samples and two Tris-EDTA buffers (no-template control, NTC) were batched in one sequencing library. Next, the library was sequenced using Oxford Nanopore GridION X5 with real-time basecalling enabled (ont-guppy-for-gridion v.1.4.3-1 and v.3.0.3-1; high-accuracy basecalling mode) (22 (link)). After that, the sequencing data were processed to discard low-quality (Q score <7) and undesired length (<200 nt or >2,000 nt). Additionally, an in-house script was used to analyze the output of the basecalling data and generate a real-time taxonomy list of each sample by screening and starting the bioinformatic pipeline when every 4,000 reads passed the base calling process; then, the taxonomy of each read was assigned according to the taxonomic information of the mapped subject sequence. Finally, the interpretation of pathogen detection in the CSF sample was conducted according to a strict set of rules described elsewhere (21 (link)).
3
Hybrid Genome Assembly for Strain KK10
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The complete genome sequence of strain KK10 was previously announced with detailed information on the sequencing method (23 (link)). In brief, genomic DNA of strain KK10 extracted from the cells grown on 20 mM glycerol for 3 days was sequenced employing a hybrid assembly of short-read (DNBSEQ-G400; MGI Tech, Shenzhen, China) and long-read (GridION X5; Oxford Nanopore Technologies, Oxford, UK) sequencing technologies. For the GridION sequencing, a library was created using a ligation sequencing kit (SQK-LSK109; after adapter ligation, >3-kb fragments were enriched). The long-read sequences that were obtained using R9.4.1 flow cells were base-called using Guppy v. 4.0.11 and then trimmed and quality-filtered using Porechop v. 0.2.3 and Filtlong v. 0.2.0 (>1 kb). After trimming and quality filtering of the raw reads obtained from each sequencing platform, the complete genome sequence was determined through de novo assembly using Unicycler v. 0.4.7 (52 (link)) and was validated using Bandage v. 0.8.1 (Fig. S1) (53 (link)). GenSkew v. 1.0 was used to calculate the GC skew and to approximate the origin and terminus of replication.
4
Nanopore Sequencing of End-Repaired DNA
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A total of 1.5 μg of gDNA was end-repaired (NEBnext ultra II end repair kit, New England Biolabs, MA, USA) and purified using 1 × AmPure beads (Beckmann Coulter, USA). Adapter ligation (AMX) was performed at RT (20 °C) for 20 min using NEB Quick T4 DNA Ligase (New England Biolabs, MA, USA). The reaction mixture was purified using 0.6 × AmPure beads (Beckmann Coulter, USA) and sequencing library was eluted in 15 μl of elution buffer provided in the ligation sequencing kit (SQK-LSK109) from Oxford Nanopore Technology (ONT). Sequencing was performed on GridION X5 (Oxford Nanopore Technologies, Oxford, UK) using SpotON flow cell R9.4 (FLO-MIN106) in 48 h sequencing protocol on MinKNOW (version 1.1.20, ONT) with Albacore (v1.1.2)37 live base calling enabled with default parameters.
5
Metagenome Sequencing of Soil Samples
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Metagenomic DNA extracted from the collected soil (MGB-2 and MGB-3) were end-repaired using NEBnext ultra II kit (New England Biolabs, USA), cleaned up with 1x AmPure beads (Beckmann Coulter, USA). Native barcode ligation was performed with NEB blunt/TA ligase using NBD103 and cleaned with 1x AmPure beads. Qubit quantified barcode ligated DNA samples were pooled at an equimolar concentration to attain a 1 μg pooled sample. Adapter ligation (BAM), cleaning of library mix and elution of sequencing library was done as per Kumar et al., 2021 [13 (link)] and was further used for whole-genome sequencing. The whole-genome library was prepared by using a Native Barcoding kit (EXP-NBD103). Barcode sequences are detailed in the (S1 Table ). The sequencing was performed using SpotON flow cell (R9.4) on MinKNOW 2.1 v18.05.5 with a 48 h sequencing protocol [14 (link)] on GridION X5 (Oxford Nanopore Technology (ONT, UK).
6
Transcriptome Analysis of Locust Flight Muscle
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The flight muscle of three independent replicates was collected for tissue preparation. Total RNA extraction was performed using a TRIzol reagent (Invitrogen), and cDNA libraries were prepared in accordance with the protocols of Illumina. Raw data were filtered, corrected, and mapped to locust genome sequence via HISAT software. The gene expression levels were measured using the criteria of reads per kb per million mapped reads. Differentially expressed genes (DEGs) were detected using the EdgeR software. The differences between the treatment and control groups were represented by p-values and FDR. The DEGs with significance levels at FDR <0.05 in each comparison were considered as the candidate target genes (Jiang et al., 2019a (link)). The fastq files of the transcriptome sequence are available at BioProject PRJNA690129. The full-length transcriptome was obtained from Jiang et al., 2019b (link), with NCBI accession number PRJNA517220. Briefly, the full-length transcripts from testes of locusts were enriched by 5’-Cap capturing assay for library preparation; 200 ng of the RNA libraries were loaded on FLO-MIN106 (R9.4) flowcells and were run on a MinION or a GridION X5 (Oxford Nanopore Technologies).
7
Microbiome Profiling of Capsules and Feces
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To determine the composition of the microbiome present in the capsules and faecal samples, DNA extraction was performed using the QIAamp® PowerFecal® Pro DNA kit [16 ]. The DNA was then amplified by PCR using a Biometra T1 Thermocycler T-1 Thermoblock. The composition of the master mix used was the following: 60 mM Tris-SO4, 20 mM (NH4) 2SO4, 2 mM MgSO4, 0.3 mM dNTPs, 3% Glycerol, 0.06% IGEPAL® CA-630, 0.05% Tween20, 125 units/mL LongAmp Taq DNA Polymerase; pH 9.1 at 25 °C and 1.5 µL of each primer, 27f and 1492r, targeting the 16S rRNA. The amplification protocol included 35 cycles as follows: denaturation (95 °C, 10–30 s), annealing (55 °C, 15–60 s) and replication (65 °C, 50 s). Subsequently, amplification products were visualised through gel electrophoresis and purified using the Solid-Phase Reversible Immobilisation (SPRI) technique with magnetic beads [17 (link),18 (link)]. After, a DNA library was built. The samples were then quantified using the Qubit® fluorometer, and the adapters were fixed for subsequent genomic sequencing, which was performed using GridION X5, commercialised by Oxford Nanopore Technologies® [19 ].
8
High-Molecular-Weight DNA Nanopore Sequencing
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1 µg of high molecular weight DNA samples obtained using AllPrep DNA/RNA kit were used for Oxford Nanopore adaptive sequencing. Library preparation was performed using the SQK-LSK110 kit (Oxford Nanopore Technologies, Oxford) following the genomic DNA ligation protocol (https://community.nanoporetech.com/protocols/genomic-dna-by-ligation-sqk-lsk110/ ). Finally, the libraries were loaded separately onto different Nanopore R9.4.1 flow cells (FLO-MIN106), one for sequencing with Adaptive sampling (AS) and one for control sequencing. Both flow cells were run simultaneously on a GridION X5 device (MinKNOW version 21.11.7; Guppy 5.1.13; Oxford Nanopore Technologies) [ONT]) for up to 72 h.
9
Hybrid Assembly of Bacterial Whole Genome
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Genomic DNA was extracted as previously described (10 (link)). Whole-genome sequencing was performed at the Bioengineering Lab. Co. (Kanagawa, Japan) using GridION X5 (Oxford Nanopore Technologies, Oxford, UK) and DNBSEQ-G400 (MGI, Wuhan, China). The complete genome sequence was obtained by hybrid assembly of sequencing data with the Unicycler ver. v.0.4.7 default method. Contig graph was checked by Bandage (v.0.8.1). Completeness of genome assembled was confirmed by CheckM (v.1.1.2). ORFs annotation was analyzed using Prokka v.1.14.5. Default parameters were used for all software. The obtained plasmid sequences were compared to the known C. acnes TP-CU389 pTZC1 sequence (accession no. LC473083 ). A BLAST Ring Image Generator (BRIG; https://brig.sourceforge.net ) was used for comparative analysis with known sequences.
10
Gut Microbiome Profiling in Mice
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Feces samples for sequencing were obtained from barrier-bred mice (8 weeks) and human GM transplanted mice (5 weeks). See supplementary material and methods for detailed information on DNA extraction, PCR amplification of 16S rRNA, and sequencing using the Oxford Nanopore GridION x 5 sequencing platform (Oxford Nanopore Technologies, Oxford, UK).
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