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Native Barcoding Expansion 1-12 (PCR-free)
EXP-NBD104

A versatile method of preparing barcoded sequencing libraries for applications where high throughput is required

$288.00

Information

This kit is recommended for users who:

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

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:

No barcodes6 barcodes12 barcodes
Flow cell price$500$500$500
Library price$99$99$99
Barcode price-$25$50
Price per sample$599$104$54

The Native Barcoding Expansion 1-12 is an expansion pack, which provides 12 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 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 Native Barcoding Kit features:

FeatureProperty
Preparation time80 minutes
Input requirement1000 ng of double stranded DNA
PCR RequiredNo
FragmentationOptional
Read lengthEqual to fragment length
Read type produced1D
Typical throughput★★★
Associated protocolsNative barcoding genomic DNA (EXP-NBD104 and EXP-NBD114)
Native barcoding amplicons (EXP-NBD104 and EXP-NBD114)
Dual barcoding
Direct cDNA native barcoding
Classic PCR tiling of SARS-CoV-2 virus
Eco PCR tiling of SARS-CoV-2 virus with native barcoding
Automated PCR tiling of SARS-CoV-2 virus with Hamilton NGS STAR 96
Pack size6 reactions
StabilityShipped at 2–8°C

Long-term storage -20°C

Oxford Nanopore Technologies deem the useful life of the product to be 3 months from receipt by the customer.

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 1-12 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 the MinKNOW and Guppy software, 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 at 2–8°C.

Upon receipt, please place the right product in the right long-term storage location.

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.

Workflow

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.

Native barcoding workflow

What's in the box

The Native Barcoding Expansion 1-12 contains 12 unique barcodes and sufficient reagents for six sequencing libraries. EXP-NBD104 kit contents 1

NameAcronymCap colourNo. of vialsFill volume per vial (μl)
Native Barcode 01-12NB01-12White1220
Adapter Mix IIAMIIGreen140

Compatibility

The Native Barcoding Expansion 01-12 can be used together with:

Kits

  • Ligation Sequencing Kit (SQK-LSK109)
  • Direct cDNA Sequencing Kit (SQK-DCS109)
  • Flow Cell Wash Kit (EXP-WSH004)

Flow cells

  • FLO-MINSP6
  • FLO-MIN106D
  • FLO-MIN111
  • FLO-PRO002
  • FLO-FLG001

Devices

  • MinION Mk1B
  • MinION Mk1C
  • Flongle
  • GridION
  • PromethION