Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads

Abstract

The Illumina DNA sequencing platform generates accurate but short reads, which can be used to produce accurate but fragmented genome assemblies. Pacific Biosciences and Oxford Nanopore Technologies DNA sequencing platforms generate long reads that can produce complete genome assemblies, but the sequencing is more expensive and error-prone. There is significant interest in combining data from these complementary sequencing technologies to generate more accurate "hybrid" assemblies. However, few tools exist that truly leverage the benefits of both types of data, namely the accuracy of short reads and the structural resolving power of long reads. Here we present Unicycler, a new tool for assembling bacterial genomes from a combination of short and long reads, which produces assemblies that are accurate, complete and cost-effective. Unicycler builds an initial assembly graph from short reads using the de novo assembler SPAdes and then simplifies the graph using information from short and long reads. Unicycler uses a novel semi-global aligner to align long reads to the assembly graph. Tests on both synthetic and real reads show Unicycler can assemble larger contigs with fewer misassemblies than other hybrid assemblers, even when long-read depth and accuracy are low. Unicycler is open source (GPLv3) and available at github.com/rrwick/Unicycler.

Fig 1. Key steps in the Unicycler pipeline.

Fig 2. Simulated short-read assemblies: Errors.

Misassembly and small-error (mismatches and indels) rates for assemblies of simulated short-read sets, summarising results across all reference genomes and replicate tests (total 360 per assembler).

Fig 3. Simulated short-read assemblies: NGA50.

NGA50 for assemblies of simulated short-read sets, summarising results across all reference genomes and replicate tests (total 360 per assembler).

Fig 4. Simulated hybrid assemblies: Errors.

Error rates for hybrid assemblies of simulated short-read and long-read sets, summarising results across all reference genomes and replicate tests (total 2520 per assembler).

Fig 5. Simulated hybrid assemblies: NGA50 against long-read depth.

Mean NGA50 values for hybrid assemblies of simulated read sets. Mean values were calculated across all read lengths, read accuracies and replicate tests for each reference genome (210 hybrid-read sets each); the top panel shows mean values for all 12 reference genomes (2520 hybrid-read sets). Horizontal dashed lines indicate the N50 size of the reference genome. For the bacterial genomes, this is the size of their only chromosome; for Saccharomyces, it is the size of chromosome XIII, an intermediate-sized replicon in the genome.

Fig 6. Simulated hybrid assemblies: Read length and accuracy.

NGA50 values segregated by read length and read accuracy. These plots summarise results across all reference genomes and replicate tests, but only include the tests of 8x long-read depth. For read lengths, the p-value is from a two-tailed t-test. For read accuracies, the p-value is from a one-way ANOVA test.

Fig 7. E. coli K-12 assemblies: NGA50 against long-read depth.

Mean NGA50 values for hybrid assemblies of real E. coli read sets, summarised across 20 replicate tests at each depth. Top panel shows mean values for all six long-read sets.

Fig 8. Klebsiella pneumoniae INF274 assembler comparison.

Final assemblies of Klebsiella pneumoniae INF274 produced by Unicycler, SPAdes, HGAP and Canu. The contigs/graph of the assembly are shown on the left, coloured by replicon. The read depth plot of plasmid 1’s contig is shown on the right. Low read depth at the ends of the contig is indicative of start-end overlap.

Fig 9. Klebsiella pneumoniae INF125 ONT assemblies over sequencing time.

Assembly metrics of K. pneumoniae INF125 produced by Unicycler, SPAdes, npScarf and miniasm over a four-hour period of sequencing. Miniasm assemblies contain error rates comparable to that of the raw reads and are therefore excluded from the error rate plots.