Rapid sequencing of plasmids and resistance gene detection

Multi‑drug resistant bacteria are an increasing threat to global public health. Many AMR genes are located on plasmids; however, the sequencing of plasmids using traditional short‑ read technologies is hindered by the presence of high levels of repetitive DNA. Bacterial plasmids can also span 100s of kilobases¹, adding a further level of complexity to accurate assembly.

The application of short‑read approaches generates incomplete, fragmented plasmid assemblies that are often impossible to differentiate from genomic sequence. Understanding the location of AMR genes (i.e., plasmid or genome) can allow more informed epidemiological tracking and provide new insights into evolution and the transmission mechanisms underlying the development of antibiotic resistance^2,3,4.

Researchers at the National Institutes of Health, USA, are now applying nanopore sequencing to deliver improved plasmid assemblies for rapid AMR characterisation⁴.

The team first sequenced plasmid DNA from a well‑characterised, carbapenem‑ resistant Klebsiella pneumoniae isolate using a ligation‑based library preparation kit (Oxford Nanopore). Assembly of the long nanopore reads provided a consensus accuracy of 99%.

The accuracy could be increased to 99.9% through polishing with reads derived from short‑read technology; however, the researchers stated that, for potential future clinical applications, they favoured nanopore‑only reads due to the more rapid turnaround time.

All three known plasmids in the isolate were 98% covered by the assemblies and all AMR genes were accurately identified. To assess the minimal turnaround times for comprehensive resistance gene identification, the researchers sequenced plasmid DNA from a second K. pneumoniae isolate using the transposon‑based Rapid Sequencing Kit (Oxford Nanopore).

Using this strategy, it was possible to deliver full antimicrobial gene detection in under 6 hours⁴.

The researchers reported that:

"[the] use of plasmid DNA enabled lower depth sequencing, and assemblies sufficient for full antimicrobial resistance gene annotation were obtained with as few as 2000-5000 reads that can be acquired in 20 minutes of sequencing"⁴.

Nanopore sequencing of plasmid DNA for AMR profiling has also been demonstrated by researchers at the Shenzhen Research Institute in China, who utilised barcoding for cost‑effective, multiplexed analysis of 12 multidrug resistant (MDR) bacterial strains. Their work allowed the generation of 20 complete plasmids (and 1 near complete plasmid) in a single 8‑hour sequencing run. Of note, one >90 kb plasmid was fully represented by a single read, which therefore required no assembly.

The researchers also point out that multiple sequencing runs are possible on a single nanopore flow cell within 48 hours of starting the first run. Based on their research, they believe 3 runs could be accommodated on a single flow cell, each lasting for 8, 10 and 12 hours respectively. In combination with barcoding, the researchers state that it might be possible to sequence 36 samples in total, leading to significant reduction in the cost of producing complete plasmid sequences.

This case study is taken from the Microbiology white paper.