Fig. 2 Identification of species using 16S PCR with the rapid amplicon kit, and the rapid whole-genome amplification (WGA) protocol
In situations where the amount of available sample DNA is limited, or where there is a low level of pathogen DNA mixed with a high level of host DNA, and we wish to identify the pathogen, it can be helpful to amplify the target organism by PCR. For bacterial species identification, the 16S ribosomal RNA gene can be amplified from all bacteria non-specifically, without amplifying eukaryotic host DNA, or viruses. As with the whole-genome species ID approach shown in Fig. 1, we have found bead-beating to lyse cells rapidly, yielding DNA with a sufficiently high fragment length for amplification of the 1.5 kb 16S gene. There is no need to purify the extracted DNA before PCR. Fast polymerases are available which can process 1 kb of template in around 30 seconds, meaning that 16S PCR can be performed on a standard thermocycler in 25 minutes. If PCR is performed using Oxford Nanopore’s modified primers, sequencing adapters can be attached rapidly following amplification, by chemical ligation. This reaction tends to be more robust than enzymatic ligation, so again, there is no need to perform a clean-up step before adapter attachment. Finally, the sequencing library is loaded onto a MinIONTM flowcell and the run started. Using the 16S analysis workflow, species identities are returned in almost real time, making it possible to go from cells to identification in under 40 minutes (Fig. 2). Alternatively, if all the genomes present in a small amount of sample are to be sequenced and analysed, it is possible to amplify the sample by WGA. This can generate sufficient DNA for sequencing in 90 minutes, starting with picogram quantities of input. Following this reaction, adapters are added using the rapid genomic DNA library kit, and the resulting library is loaded and sequenced. The WIMP workflow can then be used to identify species from the resulting whole genome data, with the first results being generated in under 100 minutes from the start of the workflow.