Information
This kit is recommended for users who:
- Wish to multiplex up to 96 samples to reduce price per sample
- Want to achieve modal raw read accuracy of Q20+ (99%) and above
- Want to obtain duplex data
- Want to optimise their sequencing experiment for accuracy and output
- Need a PCR-free method of multiplexing to preserve additional information such as base modifications
- Require control over read length
- Would like to utilise upstream processes such as size selection or whole genome amplification
Please note that to use this kit, you will need to purchase additional 3rd party reagents: see the "3rd Party Materials" tab for more detail.
Barcoding or multiplexing is useful when the amount of data required per sample is less than the total amount of data that can be generated from a single flow cell: it allows a user to pool multiple samples and sequence them together, making more efficient use of the flow cell.
Multiplexing samples onto one flow cell can reduce the cost per sample for a user. Below are some worked examples. Please note, this is an example and pricing depends on the kit purchased.
The Native Barcoding Kit 96 V14 features:
The Native Barcoding Kit 96 V14 is a standalone kit providing 96 unique barcodes to enable PCR-free multiplexing of dsDNA samples such as gDNA and amplicons.
The library preparation method is similar to the Ligation Sequencing Kit protocol; gDNA is repaired (note: DNA repair is skipped when using amplicon input) and dA-tailed using the NEBNext End Repair/dA-tailing module, and then a unique dT-tailed barcode adapter is ligated on the dA-tailed template. Barcoded samples are then pooled together. Each barcode adapter also has a cohesive end and this is used as a hook to ligate to the supplied sequencing adapter.
The kit is optimised to achieve modal sequencing accuracies of over 99% (Q20+) with high output on our latest nanopore: R10.4.1. Our flow cell priming and sequencing reagents have been reformulated to be compatible with this improved Kit 14 adapter and R10.4.1 nanopore.
Kit 14 also includes previous updates such as the higher capture rate of DNA to enable lower flow cell loading amounts, and fuel fix technology, allowing users to run longer experiments without the need for fuel addition during the run.
If your experiment requires long reads, it is recommended to start with full-length gDNA, and fragmentation/shearing is neither advised nor required. Note: If long reads are required, high molecular weight DNA should be used as input. To determine purity, we suggest using the Nanodrop to measure the A260/280 and A260/230 ratios and we recommend that the sample should meet the following criteria:
- A260/280 = 1.8
- A260/230 = 2.0-2.2
The Native Barcoding Kit 96 V14 is compatible with upstream processes such as whole genome amplification (for applications where under 1 ng of sample is available) and size selection (for enrichment of specified fragment lengths, for example, using our Short Fragment Eliminator Kit (EXP-SFE001)). When size selecting, we recommend increasing the amount of input used, as size selection can be a wasteful process.
Deconvolution of barcoded sequencing data is supported by MinKNOW, Guppy and EPI2ME which classify the barcode sequence and sort reads into corresponding folders.
Further considerations:
Starting with lower amounts of input material, or impure samples can affect library preparation efficiency and lower sequencing output. PCR can be used to generate more input material in cases where sample amount is limiting.
Shipping and logistics:
Flow cells and kits are shipped together in an environmentally friendly temperature-controlled shipping box.