PCR Barcoding Kit
PCR Barcoding Kit
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.
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.
Key features:
Prep time | PCR + 60 minutes |
Input amount | 100 ng dsDNA |
Read length | = fragment length post-PCR |
Typical throughput | 2-3+ Gb in 6 hours, 8+ Gb in 48 hours per flow cell on MinION/GridION |
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.
