ONT Native Barcoding Kit 24 V14

The Native Barcoding Kit 24 V14 (SQK-NBD114.24) is designed for high-accuracy, PCR-free multiplexing of up to 24 DNA samples, enabling efficient use of sequencing resources while preserving native DNA characteristics such as base modifications. It is optimized for use with ONT’s V14 chemistry and R10.4.1 nanopores, providing raw read accuracies of Q20+ (≥99%).

Key Features

1. Multiplexing Capability

• Up to 24 Samples:
  • Each sample is uniquely barcoded, enabling pooling and simultaneous sequencing on a single flow cell.
  • Reduces cost per sample; for example, running 24 samples lowers the cost to ~$28/sample compared to $599 for a single sample.

2. PCR-Free Workflow

• Avoids PCR amplification, preserving:
  • Base modifications for epigenetic studies.
  • Native DNA integrity for long-read sequencing.

3. Optimized for Accuracy and Long Reads

• High Consensus Accuracy:
  • Achieves modal accuracies of Q20+ (≥99%).
• Long Read Potential:
  • Compatible with high molecular weight DNA for long-read sequencing.
  • Avoids unnecessary fragmentation for optimal read lengths.

4. Flexible Input

• DNA Requirements:
  • Genomic DNA (gDNA): 400–1,000 ng per sample.
  • Amplicons: 130 ng/sample (1 kb) or 200 fmol/sample.

5. Compatibility with Upstream Processes

• Supports whole-genome amplification for low input samples (<1 ng).
• Allows size selection using ONT’s Short Fragment Eliminator Kit (EXP-SFE001).

Kit Contents

Plate format                                                            Vial Format

Applications

1. Whole Genome Sequencing:
   • Ideal for studying genomic variants and structural variations.
2. Epigenetic Analysis:
   • Enables detection of base modifications like methylation.
3. Targeted Sequencing:
   • Supports reduced representation methylation multiplex sequencing (RRMS).
4. Pathogen Genomics:
   • Effective for viral genomes (e.g., influenza, SARS-CoV-2).

Workflow

1. DNA Repair and dA-Tailing:
   • Genomic DNA is repaired and dA-tailed using the NEBNext End Repair/dA-Tailing module.
   • Amplicon input skips the DNA repair step.
2. Barcode Adapter Ligation:
   • Unique dT-tailed barcode adapters are ligated to DNA.
3. Pooling:
   • Barcoded samples are pooled and prepared for sequencing.
4. Sequencing:
   • The library is sequenced on ONT devices (e.g., MinION, GridION) using R10.4.1 flow cells.
   • Deconvolution supported by MinKNOW, Guppy, and EPI2ME software.

Protocols

• Ligation sequencing gDNA - native barcoding kit 24 V14
• Ligation sequencing amplicon - native barcoding kit 24 V14
• Ligation sequencing DNA V14 - dual barcoding (SQK-NBD114.24 with EXP-PBC096)
• Ligation sequencing influenza whole genome V14 (SQK-NBD114.24 or SQK-NBD114.96)
• Ligation sequencing gDNA V14 - Adeno-associated virus sequencing (SQK-NBD114.24)
• Ligation sequencing V14 — Human cfDNA multiplex (SQK-NBD114.24
• Ligation sequencing gDNA V14 - reduced representation methylation multiplex sequencing (RRMS) (SQK-NBD114.24)

Storage and Stability

• Shipping Conditions: 2–8°C in temperature-controlled packaging.
• Storage:
  • Room temperature: 1 month (unopened).
  • Refrigerated (2–8°C): 12 weeks (unopened).
• Warranty: 3 months from receipt.

Third-Party Reagents Required

• NEB Blunt/TA Ligase Master Mix (M0367).
• NEBNext FFPE Repair Mix (M6630).
• NEBNext Ultra II End Repair/dA-Tailing Module (E7546).
• NEBNext Quick Ligation Module (E6056).
• Consumables like nuclease-free water, PCR tubes, and ethanol.

Compatibility

Kits:

• Ligation Sequencing Kits (SQK-LSK114, SQK-LSK114-XL).
• Native Barcoding Auxiliary Kit V14 (EXP-NBA114).
• Flow Cell Wash Kit (EXP-WSH004).
• Rapid and PCR Barcoding Kits.

Flow Cells:

• R10.4.1 (FLO-MIN114, FLO-PRO114M, FLO-FLG114).

Devices:

• MinION Mk1B, Mk1C.
• GridION, PromethION.

Advantages

1. Cost Efficiency:
   • Multiplexing significantly reduces sequencing costs per sample.
2. Accuracy and Data Quality:
   • Achieves Q20+ accuracy with V14 chemistry.
3. Scalability:
   • Flexible input options and compatibility with ONT devices enable diverse applications.

The Native Barcoding Kit 24 V14 is a powerful tool for cost-effective, high-accuracy sequencing, making it suitable for genomic research, epigenetic studies, and multiplexed experiments across various applications.