Completing bacterial genome and plasmid assemblies

The challenge of delivering complete genome assemblies with traditional short‑ read sequencing technologies is well documented1,2. According to Ryan Wick at The University of Melbourne, Australia:

"This has major implications for tracking the spread of mobile genetic elements, including those which carry antimicrobial resistance determinants"3.

As most genomes assemblies have been created using short‑ read technology, there are thousands of bacterial isolates with incomplete genome sequences that can now be enhanced by combining long‑read nanopore data using a hybrid assembly strategy.

In order to address the challenge of cost‑ effectively completing large number of genome assemblies, Ryan devised a low‑ coverage, multiplex, nanopore sequencing strategy, where up to 12 isolates could be combined in a single run4. In addition, he developed the Unicycler4 assembler to overcome the limitations encountered with existing hybrid assemblers when dealing with low‑coverage reads and circular bacterial chromosomes.

Applying this strategy to Klebsiella pneumoniae the team were able to deliver complete bacterial chromosomes and plasmids, with highly accurate base calling (Figure 7). Furthermore,

the low‑depth, multiplex method allowed 12 bacterial isolates to be sequenced using nanopore technology for just $80 per sample3.

Commenting on these results, Ryan stated:

"Now we can see where genes are relative to each other, we can see which are on plasmids and which are on the chromosome, and this is extremely useful for tracking horizontal gene transfer"4.

Figure: A hybrid analysis strategy using the Unicycler assembly tool to combine data derived from short‑read sequencing technology with long read nanopore data (28x depth) allowed complete and accurate assembly of the K. pneumoniae isolate INF177. Figure courtesy of Ryan Wick, University of Melbourne, Australia.

This case study is taken from the Microbiology white paper.

  1. Tyson, J.R. et al. MinION‑based long‑read sequencing and assembly extends the Caenorhabditis elegans reference genome. Genome Res. 28(2):266‑274 (2018).
  2. Duc Cao, M. et al. Scaffolding and completing genome assemblies in real‑time with nanopore sequencing. Nature Communications 8 (2017).
  3. Wick, R.R., Judd, L.M., Gorrie, C.L. and Holt, K.E. Completing bacterial genome assemblies with multiplex MinION sequencing. bioRxiv 160614 (2017).
  4. Wick, R. Hybrid Nanopore and Illumina assembly: working towards the perfect bacterial genome. Presentation. [Accessed: 15 February 2018].