PCR sequencing kits enable the preparation of genomic DNA for nanopore sequencing from as little as 100 ng DNA using PCR.
- Low input amounts — as little as 100 ng DNA
- High sequencing yields
- Control over read length
- Multiplex ≤12 samples with the PCR Barcoding Kit
The PCR Sequencing Kit and PCR Barcoding Kit are designed to prepare sequencing libraries when there is a limited amount of starting gDNA available.
The gDNA is fragmented in a Covaris g-TUBE. The 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. An amplification step follows using primers that contain 5' tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters.
The kits involve approximately 50 minutes of 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 takes approximately 10 minutes.
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 Sequencing Kit or PCR Barcoding Kit, compared to the Rapid PCR Barcoding Kit, which is limited to ~2 kb. Therefore these kits offer 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.
These kits also offer a method whereby users are able to 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 generic 'outer' primers (supplied with the kits) and these primers contain the 5' tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters.
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.
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 kit contains 12× primer pairs, which are 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. Deconvolution of barcoded sequencing data is supported by the MinKNOW and Guppy software provided with MinION sequencing devices, which classify the barcode sequence and sort reads into corresponding folders.
Sample multiplexing for the PCR Barcoding Kit is ≤12.
Delivery information: Flow cells and kits are shipped together at 2 to 8 °C. Upon receipt, please place the product in a long-term storage location at ‒20 °C. Oxford Nanopore Technologies deem the useful life of the product to be three months from receipt by the customer.
Caution: Oxford Nanopore Technologies products are not intended for use for health assessment or to diagnose, treat, mitigate, cure, or prevent any disease or condition.