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

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.

Wick RR ，Judd LM ，Gorrie CL ，Holt KE ... -  《-》

Comparison of long-read sequencing technologies in the hybrid assembly of complex bacterial genomes.

Illumina sequencing allows rapid, cheap and accurate whole genome bacterial analyses, but short reads (<300 bp) do not usually enable complete genome assembly. Long-read sequencing greatly assists with resolving complex bacterial genomes, particularly when combined with short-read Illumina data (hybrid assembly). However, it is not clear how different long-read sequencing methods affect hybrid assembly accuracy. Relative automation of the assembly process is also crucial to facilitating high-throughput complete bacterial genome reconstruction, avoiding multiple bespoke filtering and data manipulation steps. In this study, we compared hybrid assemblies for 20 bacterial isolates, including two reference strains, using Illumina sequencing and long reads from either Oxford Nanopore Technologies (ONT) or SMRT Pacific Biosciences (PacBio) sequencing platforms. We chose isolates from the family , as these frequently have highly plastic, repetitive genetic structures, and complete genome reconstruction for these species is relevant for a precise understanding of the epidemiology of antimicrobial resistance. We assembled genomes using the hybrid assembler Unicycler and compared different read processing strategies, as well as comparing to long-read-only assembly with Flye followed by short-read polishing with Pilon. Hybrid assembly with either PacBio or ONT reads facilitated high-quality genome reconstruction, and was superior to the long-read assembly and polishing approach evaluated with respect to accuracy and completeness. Combining ONT and Illumina reads fully resolved most genomes without additional manual steps, and at a lower consumables cost per isolate in our setting. Automated hybrid assembly is a powerful tool for complete and accurate bacterial genome assembly.

De Maio N ，Shaw LP ，Hubbard A ，George S ，Sanderson ND ，Swann J ，Wick R ，AbuOun M ，Stubberfield E ，Hoosdally SJ ，Crook DW ，Peto TEA ，Sheppard AE ，Bailey MJ ，Read DS ，Anjum MF ，Walker AS ，Stoesser N ，On Behalf Of The Rehab Consortium ... -  《Microbial Genomics》

Benchmarking hybrid assembly approaches for genomic analyses of bacterial pathogens using Illumina and Oxford Nanopore sequencing.

We benchmarked the hybrid assembly approaches of MaSuRCA, SPAdes, and Unicycler for bacterial pathogens using Illumina and Oxford Nanopore sequencing by determining genome completeness and accuracy, antimicrobial resistance (AMR), virulence potential, multilocus sequence typing (MLST), phylogeny, and pan genome. Ten bacterial species (10 strains) were tested for simulated reads of both mediocre- and low-quality, whereas 11 bacterial species (12 strains) were tested for real reads. Unicycler performed the best for achieving contiguous genomes, closely followed by MaSuRCA, while all SPAdes assemblies were incomplete. MaSuRCA was less tolerant of low-quality long reads than SPAdes and Unicycler. The hybrid assemblies of five antimicrobial-resistant strains with simulated reads provided consistent AMR genotypes with the reference genomes. The MaSuRCA assembly of Staphylococcus aureus with real reads contained msr(A) and tet(K), while the reference genome and SPAdes and Unicycler assemblies harbored blaZ. The AMR genotypes of the reference genomes and hybrid assemblies were consistent for the other five antimicrobial-resistant strains with real reads. The numbers of virulence genes in all hybrid assemblies were similar to those of the reference genomes, irrespective of simulated or real reads. Only one exception existed that the reference genome and hybrid assemblies of Pseudomonas aeruginosa with mediocre-quality long reads carried 241 virulence genes, whereas 184 virulence genes were identified in the hybrid assemblies of low-quality long reads. The MaSuRCA assemblies of Escherichia coli O157:H7 and Salmonella Typhimurium with mediocre-quality long reads contained 126 and 118 virulence genes, respectively, while 110 and 107 virulence genes were detected in their MaSuRCA assemblies of low-quality long reads, respectively. All approaches performed well in our MLST and phylogenetic analyses. The pan genomes of the hybrid assemblies of S. Typhimurium with mediocre-quality long reads were similar to that of the reference genome, while SPAdes and Unicycler were more tolerant of low-quality long reads than MaSuRCA for the pan-genome analysis. All approaches functioned well in the pan-genome analysis of Campylobacter jejuni with real reads. Our research demonstrates the hybrid assembly pipeline of Unicycler as a superior approach for genomic analyses of bacterial pathogens using Illumina and Oxford Nanopore sequencing.

Chen Z ，Erickson DL ，Meng J 《BMC GENOMICS》

Polishing the Oxford Nanopore long-read assemblies of bacterial pathogens with Illumina short reads to improve genomic analyses.

Oxford Nanopore sequencing has been widely used to achieve complete genomes of bacterial pathogens. However, the error rates of Oxford Nanopore long reads are high. Various polishing algorithms using Illumina short reads to correct the errors in Oxford Nanopore long-read assemblies have been developed. The impact of polishing the Oxford Nanopore long-read assemblies of bacterial pathogens with Illumina short reads on improving genomic analyses was evaluated using both simulated and real reads. Ten species (10 strains) were selected for simulated reads, while real reads were tested on 11 species (11 strains). Oxford Nanopore long reads were assembled with Unicycler to produce a draft assembly, followed by three rounds of polishing with Illumina short reads using two polishing tools, Pilon and NextPolish. One round of NextPolish polishing generated genome completeness and accuracy parameters similar to the reference genomes, whereas two or three rounds of Pilon polishing were needed, though contiguity remained unchanged after polishing. The polished assemblies of Escherichia coli O157:H7, Salmonella Typhimurium, and Cronobacter sakazakii with simulated reads did not provide accurate plasmid identifications. One round of NextPolish polishing was needed for accurately identifying plasmids in Staphylococcus aureus and E. coli O26:H11 with real reads, whereas one and two rounds of Pilon polishing were necessary for these two strains, respectively. Polishing failed to provide an accurate antimicrobial resistance (AMR) genotype for S. aureus with real reads. One round of polishing recovered an accurate AMR genotype for Klebsiella pneumoniae with real reads. The reference genome and draft assembly of Citrobacter braakii with real reads differed, which carried blaCMY-83 and fosA6, respectively, while both genes were present after one round of polishing. However, polishing did not improve the assembly of E. coli O26:H11 with real reads to achieve numbers of virulence genes similar to the reference genome. The draft and polished assemblies showed a phylogenetic tree topology comparable with the reference genomes. For multilocus sequence typing and pan-genome analyses, one round of NextPolish polishing was sufficient to obtain accurate results, while two or three rounds of Pilon polishing were needed. Overall, NextPolish outperformed Pilon for polishing the Oxford Nanopore long-read assemblies of bacterial pathogens, though both polishing strategies improved genomic analyses compared to the draft assemblies.

Chen Z ，Erickson DL ，Meng J 《-》

Genome assembly using Nanopore-guided long and error-free DNA reads.

Long-read sequencing technologies were launched a few years ago, and in contrast with short-read sequencing technologies, they offered a promise of solving assembly problems for large and complex genomes. Moreover by providing long-range information, it could also solve haplotype phasing. However, existing long-read technologies still have several limitations that complicate their use for most research laboratories, as well as in large and/or complex genome projects. In 2014, Oxford Nanopore released the MinION® device, a small and low-cost single-molecule nanopore sequencer, which offers the possibility of sequencing long DNA fragments. The assembly of long reads generated using the Oxford Nanopore MinION® instrument is challenging as existing assemblers were not implemented to deal with long reads exhibiting close to 30% of errors. Here, we presented a hybrid approach developed to take advantage of data generated using MinION® device. We sequenced a well-known bacterium, Acinetobacter baylyi ADP1 and applied our method to obtain a highly contiguous (one single contig) and accurate genome assembly even in repetitive regions, in contrast to an Illumina-only assembly. Our hybrid strategy was able to generate NaS (Nanopore Synthetic-long) reads up to 60 kb that aligned entirely and with no error to the reference genome and that spanned highly conserved repetitive regions. The average accuracy of NaS reads reached 99.99% without losing the initial size of the input MinION® reads. We described NaS tool, a hybrid approach allowing the sequencing of microbial genomes using the MinION® device. Our method, based ideally on 20x and 50x of NaS and Illumina reads respectively, provides an efficient and cost-effective way of sequencing microbial or small eukaryotic genomes in a very short time even in small facilities. Moreover, we demonstrated that although the Oxford Nanopore technology is a relatively new sequencing technology, currently with a high error rate, it is already useful in the generation of high-quality genome assemblies.

Madoui MA ，Engelen S ，Cruaud C ，Belser C ，Bertrand L ，Alberti A ，Lemainque A ，Wincker P ，Aury JM ... -  《BMC GENOMICS》