Library preparation was performed using 16S Barcoding Kit 1 – 24 (SQK-16S024) from Oxford Nanopore Technologies (ONT) according to the manufacturer’s protocol with some modifications. The suggested input is 10 ng of genomic DNA in the protocol. Since it is impossible to quantify only bacterial DNA in clinical samples with high human DNA background, the maximum input volume of 15 μl was added instead of 10 ng. The PCR cycle number was also increased from 25 to 35 cycles in order to increase the sensitivity of the assay. A total of 24 barcoded libraries were pooled in equal concentration and sequenced for up to 24 h using the flow cell FLO-MIN106 R9.4.1 with the sequencer GridION on the MinKNOW platform, with super-accuracy basecalling model. To reduce index misassignment, “mid-read barcode filtering” and “barcode both ends” were adopted, and the minimum barcoding score was set to be 85.
16s barcoding kit 1 24
Manufactured by Oxford Nanopore
Sourced in United Kingdom
The 16S Barcoding Kit 1-24 is a laboratory equipment product designed for the preparation of DNA samples for sequencing on Oxford Nanopore Technologies' sequencing platforms. The kit contains reagents and consumables necessary for the library preparation and barcoding of up to 24 samples, enabling parallel processing and analysis of multiple samples simultaneously.
Automatically generated - may contain errors
Lab products found in correlation
Longamp hot start taq 2x master mix, by New England Biolabs (3 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Agencourt ampure xp bead, by Beckman Coulter (2 mentions)
Quantstudio 5 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Qubit 4 fluorometer, by Thermo Fisher Scientific (1 mentions)
Dneasy powersoil pro kit, by Qiagen (1 mentions)
Minion r9.4.1 flow cell, by Oxford Nanopore (1 mentions)
Dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Minion mk1c, by Oxford Nanopore (1 mentions)
Repliqa hifi toughmix, by Quantabio (1 mentions)
E z n a soil dna kit, by Omega Bio-Tek (1 mentions)
Q3000 microvolume spectrophotometer, by Quawell (1 mentions)
Mastercycler gradient, by Eppendorf (1 mentions)
6 protocols using 16s barcoding kit 1 24
1
Optimized 16S Barcoding for Microbiome
2
16S rRNA Sequencing using MinION Nanopore
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16S rRNA amplicon sequencing was performed on a MinION nanopore sequencer (Oxford Nanopore Technologies, Oxford, UK). The amplicon library was prepared using the 16S Barcoding Kit 1-24 (SQK-16S024, Oxford Nanopore Technologies, Oxford, UK). For the PCR amplification and barcoding, 15 ng of template DNA extracted from fecal samples were added to the LongAmp Hot Start Taq 2X Master Mix (New England Biolabs, Ipswich, MA, USA). Initial denaturation at 95 °C was followed by 35 cycles of 20 s at 95 °C, 30 s at 55 °C, 2 min at 65 °C, and a final extension step of 5 min at 65 °C. Purification of the barcoded amplicons was performed using the AMPure XP Beads (Beckman Coulter, Brea, CA, USA) as per Nanopore’s instructions. Samples were then quantified using a Qubit fluorometer (Life Technologies, Carlsbad, CA, USA) and pooled in an equimolar ratio to a total of 50–100 ng in 10 μL. The pooled library was then loaded into a R9.4.1 flow cell and run per the manufacturer’s instructions. MINKNOW software 19.12.5 was used for data acquisition.
3
Nanopore 16S rRNA Amplicon Sequencing
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16S rRNA amplicon sequencing was performed on a MinION nanopore sequencer (Oxford Nanopore Technologies, Oxford, UK). The amplicon library was prepared using the 16S Barcoding Kit 1–24 (SQK-16S024, Oxford Nanopore Technologies, Oxford, UK). For the PCR amplification and barcoding, 15 ng of template DNA extracted from fecal samples, and 30 ng in the case of saliva, were added to the LongAmp Hot Start Taq 2X Master Mix (New England Biolabs, Ipswich, MA). Initial denaturation at 95 °C was followed by 35 cycles of 20 s at 95 °C, 30 s at 55 °C, 2 min at 65 °C, and a final extension step of 5 min at 65 °C. The barcoded amplicons were purified using the AMPure XP beads (Beckman Coulter, Brea, CA) as per Nanopore's instructions. Samples were then quantified using a Qubit fluorometer (Life Technologies, Carlsbad, CA) and pooled in an equimolar ratio to a total of 50–100 ng in 10 μl. The pooled library was then loaded into an R9.4.1 flow cell and run per the manufacturer's instructions. MINKNOW software 19.12.5 was used for data acquisition. The provided primer set includes a recently noted issue with the standard ONT forward primer, which contains three mismatching bases to the family Bifidobacteriaceae and thus fails to amplify microbes of these taxa (Fujiyoshi et al., 2020 (link)).
4
Microbial Mapping of Potato Tuber-Sphere
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Nearly 200 grams of 72 samples obtained from potato tuber-sphere at harvest or after one-month post-harvest storage, from all the individual collection sites (Table S10 ), was used for the microbial mapping of the two different regions. High quality DNA was isolated with the DNeasy PowerSoil Pro Kit (QIAGEN, Carlsbad, USA), following the manufacturer’s instructions and stored at −80°C. Amplification of the 16S rRNA gene was performed using an Applied Biosystems® QuantStudio® 5 Real-Time PCR System (Thermo Fischer Scientific, Waltham, MA, USA), using a LongAmp Hot Start Taq 2x Master Mix (M0533S, New England Biolabs), and 16S barcoded primers.
The 16S Barcoding Kit 1-24 (SQK-16S024, Oxford Nanopore Technologies, UK) was used for sequencing the 16S ribosomal gene and creating the libraries. PCR products were purified with Agecount AMPure XP beads (Beckman Coulter, USA), whilst the quantification was performed using Qubit 4 Fluorometer and the dsDNA HS Assay Kit (Thermo Fisher Scientific, USA). The 72 libraries were created in accordance with the manufacturer’s instructions and loaded on a MinION R9.4.1 flow cell (FLO-MIN106) on the MinION Mk1C (Oxford Nanopore Technologies, UK). For data acquisition, MINKNOW software ver. 1.11.5 (Oxford Nanopore Technologies) was employed.
The 16S Barcoding Kit 1-24 (SQK-16S024, Oxford Nanopore Technologies, UK) was used for sequencing the 16S ribosomal gene and creating the libraries. PCR products were purified with Agecount AMPure XP beads (Beckman Coulter, USA), whilst the quantification was performed using Qubit 4 Fluorometer and the dsDNA HS Assay Kit (Thermo Fisher Scientific, USA). The 72 libraries were created in accordance with the manufacturer’s instructions and loaded on a MinION R9.4.1 flow cell (FLO-MIN106) on the MinION Mk1C (Oxford Nanopore Technologies, UK). For data acquisition, MINKNOW software ver. 1.11.5 (Oxford Nanopore Technologies) was employed.
5
Nanopore-based 16S rRNA Amplicon Sequencing
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Bacterial 16S rRNA gene sequence libraries were prepared using a 16S Barcoding kit 1–24 (Oxford Nanopore Technologies, Oxford, Oxfordshire, UK; SQK-16S024) according to the manufacturer’s instructions. The full-length 16S rRNA gene was amplified using PCR with barcoded nanopore sequence primers, forward primer 27F, 5′-TTTCTGTTGGTGCTGATATTGCAGAGTTTGATCMTGGCTCAG-3′ and reverse primer, 1492R, 5-ACTTGCCTGTCGCTCTATCTTCCGGTTACCTTGTTACGACTT-3′. The amplicons were purified using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA) and equal amounts of amplicons per sample were pooled. The pooled samples were added to a flow cell primer kit (Oxford Nanopore Technologies, EXP-FLP002) and sequenced using a MinION Mk1C sequencer for approximately 8 h. Base calling of nanopore signals was performed using MinKNOW 22.12.5 (ONT) embedded in the Guppy version 6.4.6 pipeline (ONT). Taxonomic assignments were performed using the EPI2ME 16S workflow (ONT). The exclusion criteria for single-nanopore reads were alignment count accuracy <80%, quality score (QC) <8, and read length <1400 >1700 bp.
6
Soil Microbiome 16S rRNA Sequencing
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DNA extraction was performed using 250 mg of soil with E.Z.N.A.® Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) following manufacturer's instructions. Quantification and quality check of the extracts were performed using a Quawell Q3000 microvolume spectrophotometer (Quawell Technology, San Jose, USA). Library preparation was performed using the 16S Barcoding Kit 1-24 (Oxford Nanopore Technologies, Oxford, UK) following manufacturer's instructions. The full-length 16S rRNA bacterial gene was amplified through PCR using the kit's barcoded primers (27F: 5′-AGA GTT TGATCMTGG CTC AG-3′ and 1492R: 5′-CGG TTA CCT TGT TAC GAC TT-3′) allowing multiplexing. Amplification was conducted using repliQa HiFi ToughMix (Quantabio, Beverly, MA, USA) in Eppendorf Mastercycler Gradient (Eppendorf, Hamburg, Germany) at the following conditions: initial denaturation at 95 °C for 1 min followed by 25 cycles of denaturation (95 °C, 20 s), annealing (55 °C, 30 s), and extension (65 °C, 2 min), with a final extension at 65 °C for 5 min. After amplification followed the PCR product purification with Agencourt AMPure XP beads (Beckman Coulter, CA, USA). The concentration of purified DNA amplicons was determined using the microvolume spectrophotometer.
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