Amplicon sequencing targeting the full length of the 16S rRNA gene was performed using MinION equipped with R9.4.1 flow cell (Oxford Nanopore Technologies, Oxford, UK). A 16S rRNA sequencing library was constructed from 10 µL of extracted DNA using the 16S barcoding kit (Oxford Nanopore Technologies). The library construction was performed according to the manufacturer's instructions except that DNA amplification was carried out using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, MA, USA) with the following thermal cycling conditions: 2 min at 95 °C, 25 cycles of 20 s at 98 °C, 30 s at 60 °C, and 2 min at 72 °C, and 5 min at 72 °C. Sequencing was carried out with Oxford Nanopore's MinKNOW software and basecalls were performed using Guppy (v. 4.3.4) in fast mode using the config file dna_r9.4.1_450bps_fast.cfg. Generated FASTQ files were further analyzed for taxonomic classification using the cloud-based EPI2ME FASTQ 16S workflow with a quality score ≥ 7 for quality filtering.
16s barcoding kit
Manufactured by Oxford Nanopore
Sourced in United Kingdom
The 16S Barcoding Kit is a laboratory equipment product from Oxford Nanopore. It is designed for sequencing the 16S ribosomal RNA gene, which is commonly used for bacterial identification and microbiome analysis.
Automatically generated - may contain errors
Lab products found in correlation
Minion, by Oxford Nanopore (2 mentions)
Longamp taq 2 master mix, by New England Biolabs (2 mentions)
Ampure xp, by Beckman Coulter (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Minknow software, by Oxford Nanopore (1 mentions)
R9.4.1 flow cell, by Oxford Nanopore (1 mentions)
Kapa hifi hotstart readymix, by Roche (1 mentions)
Dneasy powersoil kit, by Qiagen (1 mentions)
Flo min106 flow cell, by Oxford Nanopore (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Qubit 4 fluorometer, by Thermo Fisher Scientific (1 mentions)
Nucleospin dna stool kit, by Macherey-Nagel (1 mentions)
Agencourt ampure xp bead, by Beckman Coulter (1 mentions)
8 protocols using 16s barcoding kit
1
High-Throughput 16S Amplicon Sequencing
2
Nanopore Sequencing of 16S rRNA Genes
3
Full-Length 16S rRNA Sequencing of Insect Larvae
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Five replicates of 20 larvae were considered for 16 S metagenomic analysis. DNAs were extracted using DNeasy PowerSoil kit (Qiagen) by grinding the larvae in the C1 buffer of this kit with a Precellys (VWR).
The Oxford Nanopore Technologies 16 S Barcoding Kit (SQK16S-024) was used to amplify and sequence the full-length 16S ribosomal RNA. Starting with 20–25 ng of extracted DNA, 45 cycles of PCR amplification were performed using New England Biolabs LongAmp Hot Start Taq 2X Master Mix and ONT 16 S barcoded primers (27 F and 1492 R). After purification, the amplicons were quantified and qualified to be mixed equimolarly. Sequencing was performed on Mk1C (MinKNOW 21.10.8) using an R9 flongle and the run initiated with the high-accuracy base calling model (Guppy 5.0.17). The run generated about 429000 reads of which more than 450,000 had a QC > 9. The reads were analyzed with the dedicated EPI2ME 16 S pipeline (v2022.01.07) using the following parameters: minimum score > Q10, minimum length 1000, maximum length 2200, minimum coverage 50%, 5 max target sequence and two different BLAST identity thresholds were tested (85 or 90%). This version used 22,162 sequences of reference coming from the microbial 16 S rRNA NCBI RefSeq database.
The Oxford Nanopore Technologies 16 S Barcoding Kit (SQK16S-024) was used to amplify and sequence the full-length 16S ribosomal RNA. Starting with 20–25 ng of extracted DNA, 45 cycles of PCR amplification were performed using New England Biolabs LongAmp Hot Start Taq 2X Master Mix and ONT 16 S barcoded primers (27 F and 1492 R). After purification, the amplicons were quantified and qualified to be mixed equimolarly. Sequencing was performed on Mk1C (MinKNOW 21.10.8) using an R9 flongle and the run initiated with the high-accuracy base calling model (Guppy 5.0.17). The run generated about 429000 reads of which more than 450,000 had a QC > 9. The reads were analyzed with the dedicated EPI2ME 16 S pipeline (v2022.01.07) using the following parameters: minimum score > Q10, minimum length 1000, maximum length 2200, minimum coverage 50%, 5 max target sequence and two different BLAST identity thresholds were tested (85 or 90%). This version used 22,162 sequences of reference coming from the microbial 16 S rRNA NCBI RefSeq database.
4
Full-Length 16S rRNA Sequencing with ONT MinION
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Full-length V1-V9 sequencing (27 F-AGAGTTTGATCMTGGCTCAG, 1492R -CGGTTACCTTGTTACGACTT) was performed using ONT MinION workflows (Oxford Nanopore Technologies, Oxford, UK). Full length 16S rRNA were amplified using 16S Barcoding Kit (SQK-16S024, Oxford Nanopore Technologies). Amplicons were purified using AMPure® XP beads (Beckman Coulter Diagnostics, USA, CA), quantified using Qubit HS kit (Qiagen), before sequencing on R9.4.1 chemistry (FLO-MIN106) flow cells (Oxford Nanopore Technologies), following manufacturer’s protocol. Basecalling was conducted using the super-accuracy basecalling model with Guppy v6.2.11. Quality score and read length of raw reads were produced using Nanoplot (Table S2 ) (De Coster and Rademakers 2023 (link)).
5
Metagenomic Analysis of Rotten Food Waste
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DNA was extracted from the rotten food waste with NucleoSpin DNA Stool kits (Macherey–Nagel GmbH & Co. KG, Düren, Germany) and quality checked by using a NanoDrop (Thermo Fisher Scientific Inc., MA, USA) and Qubit 4 Fluorometer (Thermo Fisher Scientific Inc.). Sequencing libraries were prepared with a 16S Barcoding Kit (SQK-16S024, Oxford Nanopore Technologies, UK) and sequenced by using MinION (Oxford Nanopore Technologies). The data were demultiplexed by using the adapter trimming tool Porechop34 (link), and Emu35 (link) was used to estimate sequence relative abundance, with default settings. NMDS analysis was performed by using the vegan package in R36 .
6
Nanopore Sequencing of 16S rRNA Gene
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The 16S Barcoding Kit (SQK-RAB204; Oxford Nanopore Technologies, Oxford, United Kingdom) [12 (link)] was used for DNA library preparation. PCR amplification was conducted with LongAmp™ Taq 2× Master Mix (New England Biolabs, Ipswich, MA, USA). Amplification was performed under the following conditions: initial denaturation at 95 °C for 1 min, 25 cycles of 95 °C for 20 s, 55 °C for 30 s, and 65 °C for 2 min, followed by a final extension at 65 °C for 5 min. The PCR product of each sample was cleaned up and concentrated with AMPure XP (Beckman Coulter, Indianapolis, IN, USA). A total of 10 μL purified DNA was used for library preparation. MinION Mk1C sequencing was performed by using R9.4.1 flow cells (ONT) [5 (link), 13 (link)].
7
16S rRNA Amplification and Nanopore Sequencing
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For each sample, ~10 ng of DNA was amplified using specific primers that target the whole 16S rRNA gene (27F 5′‐AGAGTTTGGATCMTGGCTCAG‐3′; 1492R 5′‐GGTTACCTTGTTACGACTT‐3′), as well as subsequent specific barcodes using a 16S Barcoding Kit (SQK‐RAB204; Oxford Nanopore Technologies). After bead purification for removal of excess primers, amplification products were attached to rapid sequencing adapters before being loaded on a MinION flow cell for real‐time sequencing. Samples were analyzed in three separate experiments (RUN1, RUN2C, and RUN3 in barcodes cited in Table S1 ), each containing a mock community sample (more details in Appendix S1 ).
8
Bacterial Diversity Analysis via 16S
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To compare bacteria compositions between samples, a total of 10 ng of bacterial DNA was amplified with the 16S Barcoding Kit (SQK-RAB204; Oxford Nanopore Technologies, Oxford, UK) by PCR as described in the manufacturer's protocol. Post-PCR clean-up was performed using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA) and eluted in 10 μL of 10 mM Tris–HCl (pH 8.0) with 50 mM NaCl.
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