This kit is recommended for users who:

• wish to multiplex samples to reduce price per sample
• have a low starting amount of DNA
• require a simple library preparation procedure

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 Rapid PCR Barcoding Kit offers the fastest and simplest method of preparation of barcoded libraries for low quantities of gDNA (1-5 ng), with only ~15 mins of hands-on preparation time.

At the heart of the kit is a transposase which simultaneously cleaves template molecules in each sample and attaches tags, which contain primer binding sites, to the cleaved ends. The kit contains 12 primers which are then used to amplify each sample: each primer contains a barcode and 5’ tag which facilitates the ligase-free attachment of Rapid Sequencing Adapters. Amplified and barcoded samples are then pooled together and Rapid Sequencing Adapters are added to the pooled mix.

The Rapid PCR Barcoding Kit features:

This kit is optimised to offer a simple workflow for those with a limited amount of input material. It has been observed that the fragment length produced after PCR is largely independent of the input fragment length, with the PCR products producing a peak at around 2 kb on an Agilent Bioanalyzer. This corresponds to a read-length of ~2 kb in nanopore sequencing. The length limit is due to the method of library preparation, rather than the processivity limit of the DNA polymerase.

Typical read-length histogram observed when preparing libraries with the Rapid Low Input by PCR Barcoding Kit.

Deconvolution of barcoded sequencing data is supported by the MinKNOW software, the stand-alone Guppy basecaller, and EPI2ME, all of which classify the barcode sequence and sort reads into corresponding folders.

Further considerations:

For users who wish to have greater control over the fragment lengths produced in their PCR reaction, we recommend the Low Input by PCR Barcoding Kit.

As well as being used to generate more material (where starting material is limiting), PCR can be used where sample purity is compromised (which can inhibit library preparation efficiency). PCR selects for molecules which have been successfully adapted, and generates 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. In instances where less than 1 ng of input material is available, we recommend whole genome amplification.

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

The Rapid PCR Barcoding Kit offers the fastest and simplest method of preparation of barcoded libraries for low quantities of gDNA (1-5 ng), with only ~15 mins of hands-on preparation time.

At the heart of the kit is a transposase which simultaneously cleaves template molecules in each sample and attaches tags, which contain primer binding sites, to the cleaved ends. The kit contains 12 primers which are then used to amplify each sample: each primer contains a barcode and 5’ tag which facilitates the ligase-free attachment of Rapid Sequencing Adapters. Amplified and barcoded samples are then pooled together and Rapid Sequencing Adapters are added to the pooled mix.

Example experiment using the Rapid PCR Barcoding Kit (SQK-RPB004) - for illustration purposes only 

Safety Data Sheet for the Rapid PCR Barcoding Kit

Safety Data Sheet for Flow Cell Priming Kit

The Rapid PCR Barcoding Kit contains 12 unique barcodes. There are sufficient barcodes and other reagents to generate six libraries with 12 barcoded samples in each one, which allows for processing a total of 72 samples.

The Flow Cell Priming Kit is also supplied.

Consumables

• Agencourt AMPure XP beads
• 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
• LongAmp Taq 2X Master Mix (e.g. NEB M0287)
• 10 mM Tris-HCl pH 8.5 with 50 mM NaCl

Equipment

• Microfuge
• Timer
• Thermal cycler
• P1000 pipette and tips
• P100 pipette and tips
• P20 pipette and tips
• P10 pipette and tips
• P2 pipette and tips

Optional Equipment

• Agilent Bioanalyzer (or equivalent)
• Qubit fluorometer (or equivalent for QC check)
• Eppendorf 5424 centrifuge (or equivalent)

The Rapid PCR Barcoding Kit can be used together with:

Kits

• RAP Top-up Kit
• Flow Cell Wash Kit (EXP-WSH003)

Flow cells

• FLO-MINSP6
• FLO-MIN106D

Devices

• MinION Mk 1B
• GridION X5