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


Date: 30th November 2017

PCR-based and PCR-free barcoding strategies for nanopore sequencing libraries, with 96 verified barcodes, and sequential loading of sequencing libraries onto a single reusable flow cell.

Fig. 1 PCR-based, PCR-free and rapid barcoding workflows, shown for gDNA templates

PCR-based and PCR-free barcoding for amplicons, cDNA or genomic DNA

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 flowcell. 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.

Fig. 2 a) 96-plex barcoding of E. coli amplicons b) and c) sequential loading of libraries

Sequential loading of barcoded libraries onto a single flowcell, using our wash kit

96 PCR barcodes are currently available. These barcodes segregate cleanly (Fig. 2a), with 90% of reads having a barcode identified, and 98.8% of those reads having the correct barcode called. We are also increasing the number of available PCR-free ‘native’ barcodes from 12 to 24. Nanopore data is generated in real time, and runs can be stopped as soon as sufficient data has been obtained. A different library can then be loaded onto the same flowcell. However, strands from the previous library may still remain (Fig. 2b). Our wash kit removes the previous library, and cross-contamination is avoided by barcoding (Fig. 2c). The flowcell can be stored after washing, which is useful for when not all samples are available for library preparation at the start of a run.

Fig. 3 Four-primer barcoding a) workflow b) Bioanalyzer trace c) CO1 sequence alignment

Four-primer PCR for rapid PCR barcoding and attachment of sequencing adapters

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 nested 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 results of performing this protocol on the human CO1 gene, with nanopore reads aligned to the NCBI reference (Fig. 3c).

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Fig. 4 Dual barcoding a) workflow for PCR libraries b) test case c) results after splitting barcodes

Dual barcoding allows greater numbers of libraries to be pooled in a sequencing run

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 flowcell. To accommodate this, we are developing 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.

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