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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:
No barcodes | 6 barcodes | 12 barcodes | |
---|---|---|---|
Flow cell price |
$500 |
$500 |
$500 |
Library price |
$99 |
$99 |
$99 |
Barcode price |
- |
$25 |
$50 |
Price per sample |
$599 |
$104 |
$54 |
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:
Feature | Property |
Preparation time | 15 minutes + PCR |
Input requirement | 1-5 ng gDNA |
PCR Required | Yes |
Fragmentation | Transposase-based |
Read length | ~2 kb |
Read type produced | 1D |
Typical throughput* | ••• |
Associated protocols |
Rapid PCR Barcoding Kit Flow Cell Wash Kit |
Pack size | 6 reactions |
Stability |
Shipped at 2-8° C Long-term storage -20° C |
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.