This kit is recommended for users who:

• wish to multiplex a high number of samples to reduce price per sample
• need a PCR-free method of multiplexing to preserve additional information such as base modifications
• require control over read length
• are interested in utlising upstream processes such as size selection or whole genome amplifcation
• want to optimise their sequencing experiement or throughput

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:

The Native Barcoding Expansion is an expansion pack providing 96 unique barcodes, to enable PCR-free multiplexing of dsDNA samples such as:

• gDNA - use in combination with Ligation Sequencing kit
• cDNA - use in combination with Direct cDNA Sequencing kit

The Native Barcoding Kit features:

The library preparation method is similar to the Ligation Sequencing Kit protocol; DNA ends are repaired 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 adapters.

The kit is optimised to generate maximum throughput, without the need for PCR. For highest data yields, we recommend starting with a total of 200 fmol of pure input DNA (for two samples, this means starting with 100 fmol of each). Starting with lower amounts of input material, or impure samples can affect library preparation efficiency and lower sequencing throughput.

In order to ensure that 200 fmol of total DNA is used, it is possible to generate the required number of molecules by shearing samples using a Covaris g-TUBE: 1 μg of gDNA sheared to ~8 kb corresponds to 200 fmol. However, if your experiment requires long reads, fragmentation/shearing is neither advised nor required. Note, if long reads are required, then long fragments must be present in the starting material. 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 Expansion 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, using the BluePippin, for example). 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 Guppy and MinKNOW, 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 throughput. PCR can be used to generate more input material in cases where sample amount is limiting. Also, where sample purity is compromised and library preparation efficiency may be reduced, PCR will select for molecules which have been successfully adapted, and generate more of them. The original impurity is lost or diluted. It is often observed that a sample which produces poor sequencing results can be improved by inclusion of PCR (for example, using the PCR Barcoding Kit).

Shipping and logistics:

Flow cells and kits are shipped together in a temperature-controlled shipping box with gel ice packs. Ice packs are stored at 2-8°C, and -20°C ice packs are added to the top of the box (but not in direct contact with product) to maintain the integrity of the shipment.

Products are shipped to customers within the USA and EU Monday to Thursday. Shipments to Canada, Norway, Korea and Japan are expedited Monday to Wednesday; with Australia and New Zealand leaving our warehouses on a Friday. Shipments to the rest of the world are made on Mondays to allow the full working week for packages to arrive.

The delivery charges are calculated when a quote is raised or during checkout. Once an order is made, the delivery ID and delivery information can be tracked in the Store.

Data analysis

Data from a Native Barcoding experiment can be analysed using:

• the Guppy basecalling software
• Barcode demultiplexing in the MinKNOW software

Example protocol for sequencing gDNA with EXP-NBD196 and SQK-LSK109 - for illustration purposes only

Safety Data Sheet for Native Barcoding Expansion (EXP-NBD196)

The Native Barcoding Expansion 96 contains 96 unique barcodes and sufficient reagents for 12 sequencing libraries.

• NEB Blunt/TA Ligase Master Mix (M0367)
• NEBNext® Quick Ligation Reaction Buffer (NEB B6058)
• NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (cat # E7180S)

Alternatively to the NEBNext® Companion Module and the NEBNext® Quick Ligation Reaction Buffer, you can use the three NEBNext® products below:

• NEBNext FFPE Repair Mix (M6630)
• NEBNext Ultra II End repair/dA-tailing Module (E7546)
• NEBNext Quick Ligation Module (E6056)

• Agencourt AMPure XP beads
• Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Cat # 0030129504) with heat seals
• 1.5 ml Eppendorf DNA LoBind tubes
• 0.2 ml thin-walled PCR tubes
• Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
• Freshly prepared 70% ethanol in nuclease-free water

The Native Barcoding Expansion 96 can be used together with:

Kits

• Ligation Sequencing Kit (SQK-LSK109)
• Ligation Sequencing Kit XL (SQK-LSK109-XL)
• Direct cDNA Sequencing Kit (SQK-DCS109)
• Flow Cell Wash Kit (EXP-WSH003)

Flow cells

• FLO-MINSP6
• FLO-MIN106D
• FLO-MIN111
• FLO-PRO002

Devices

• MinION Mk1B
• MinION Mk1C
• GridION Mk1
• PromethION