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Information
This kit is recommended for users who:
- wish to multiplex samples to reduce price per sample
- have a low starting amount of DNA
- want to optimise their sequencing experiment for throughput
- require control over read length
- are interested in cDNA or targeted amplicon sequencing.
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:
| 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 PCR Barcoding Kit is designed to prepare barcoded sequencing libraries when there is a limited amount of starting gDNA available from multiple samples.
The gDNA samples can be fragmented in Covaris g-TUBEs. Sheared ends are repaired and dA-tailed using the NEBNext End Repair/dA-tailing module, and then adapters which contain primer binding sites are ligated onto the prepared ends. The kit contains 12x primer pairs, which are then used to amplify each sample: each primer pair contains a barcode and 5’ tags which facilitate 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 kit involves ~50 mins pre-PCR work. The PCR step is variable (depends on the number of cycles, polymerase speed and template length). The standard protocol suggests starting with 100 ng of gDNA: less can be used, but it may be necessary to increase the number of PCR cycles. The post-PCR work is about 10 mins.
The PCR Barcoding Kit features:
| Feature | Property |
|---|---|
| Preparation time | 60 minutes + PCR |
| Input requirement | <100 ng gDNA |
| PCR Required | Yes |
| Fragmentation | Optional |
| Read length | Equal to fragment length post-PCR |
| Read type produced | 1D |
| Associated protocols |
• PCR Barcoding Kit • Four-primer PCR (SQK-PSK004 or SQK-PBK004) |
| Pack size | 6 reactions |
The fragment length produced after PCR is dependent on the length of the material input into the PCR and is limited by the processivity of the DNA polymerase. This means that superior fragment lengths can be achieved with the PCR Barcoding Kit, compared to the Rapid PCR Barcoding Kit, which is limited to ~2 kb. Therefore this kit offers a solution to those who have limited amounts of starting material (i.e. need to do PCR) but require longer reads than are offered by the Rapid PCR Barcoding Kit.
Typical read length histograms observed when preparing libraries with the PCR Barcoding Kit (A) and the Rapid PCR Barcoding Kit (B).
This kit also offers a method whereby users are able to barcode and tag their own specific amplicons with the same 5’ group, simplifying downstream post-PCR processing. This is done via a four-primer PCR. Users add a 5’ tail to their own primer sequences and this acts a priming site for a set of barcoded “outer” primers (supplied in the PCR Barcoding Kit) and these primers contain the 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters.
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:
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 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. In instances where less than 1 ng of input material is available, we recommend whole genome amplification.
Shipping and logistics:
Flow cells and kits are shipped together in an environmentally friendly temperature-controlled shipping box.
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 PCR Barcoding Kit is designed to prepare barcoded sequencing libraries when there is a limited amount of starting gDNA available from multiple samples.
The gDNA samples can be fragmented in Covaris g-TUBEs. Sheared ends are repaired and dA-tailed using the NEBNext End Repair/dA-tailing module, and then adapters which contain primer binding sites are ligated onto the prepared ends. The kit contains 12x primer pairs which are then used to amplify each sample: each primer pair contains a barcode and 5’ tags which facilitate 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 kit involves ~50 mins pre-PCR work. The PCR step is variable (depends on the number of cycles, polymerase speed and template length). The standard protocol suggests starting with 100 ng of gDNA: less can be used, but it may be necessary to increase the number of PCR cycles. The post-PCR work is about 10 mins.
This kit also offers a method whereby users are able to barcode and tag their own specific amplicons with the same 5’ group, simplifying downstream post-PCR processing. This is done via a four-primer PCR. Users add a 5’ tail to their own primer sequences and this acts a priming site for a set of barcoded “outer” primers (supplied in the PCR Barcoding Kit) and these primers contain the 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters.
Protocols
• PCR Barcoding Kit
• Four-primer PCR (SQK-PSK004 or SQK-PBK004)
To access the protocols, you will need to register for a Nanopore Community account.
