PCR-based, PCR-free and rapid barcoding for nanopore sequencing libraries

Oxford Nanopore’s barcoding kits allow users to pool and sequence multiple libraries or amplicons in a single sequencing run, making more efficient use of the run. Barcoded libraries can then be pooled prior to sequencing, or libraries can be loaded sequentially onto the flow cell. There are currently three versions of the kit available, in which barcodes are added to ligation-based libraries by PCR or ligation, and to rapid libraries as part of the transposition reaction (Figs. 1a, 1b and 1c, respectively). We have also recently released a 4-primer PCR protocol, which allows users to barcode their own locus-specific PCRs and to attach adapters using our rapid-attachment chemistry.

96 PCR barcodes are currently available. The barcodes segregate cleanly (Fig. 2a), with >96% of reads having a barcode identified, and >99.7% of those reads having the correct barcode called. We have increased the number of available PCR-free ‘native’ barcodes from 24 to 96 and we are also in the process of expanding the number of PCR-free ‘rapid’ barcodes to 96. Nanopore data is generated in real time, and runs can be stopped as soon as sufficient data has been obtained, allowing a different library to be loaded onto the same flow cell. Our wash kit removes ~99.9% of strands from the previous library (Figs. 2b-c). Flow cells can be stored after washing, which is useful for times when not all samples are available for library prep at the start of a run.

To attach barcodes to locus-specific amplicons, our previous protocol required two consecutive PCR reactions. We have shortened this protocol substantially (Fig. 3a). The new protocol relies on a single four-primer PCR: locus-specific primers are used, which are tailed with barcodes and universal sequences. Rapid-attachment universal primers are also included in the PCR, resulting in tailed amplicons to which rapid adapters can be attached. Only successfully amplified nested PCR products are capable of receiving sequencing adapters. This final step takes approximately 10 minutes (Fig. 3b). Here we show the results of performing this protocol on the human CO1 gene, with nanopore reads aligned to the NCBI reference (Fig. 3c).

When looking at PCR amplicons or plasmids, only a small amount of data is needed. Here, it would be helpful to be able to sequence larger numbers of templates on a single flow cell. To accommodate this, we have developed PCR-based (Fig. 4a) and PCR-free dual barcoding protocols. We tested the concept by taking swabs from a variety of environments and growing bacteria from each environment on a separate agar plate (Fig. 4b). We performed 16S PCR on each colony, and barcoded each set of amplicons with an inner, colony-specific, barcode. We then made a pool of amplicons for each plate and labelled each pool with an outer, plate- specific, barcode. Fig. 4c shows the counts of each barcode combination after splitting.