Comparison of De Novo Assembly Strategies for Bacterial Genomes.

(1) Background: Short-read sequencing allows for the rapid and accurate analysis of the whole bacterial genome but does not usually enable complete genome assembly. Long-read sequencing greatly assists with the resolution of complex bacterial genomes, particularly when combined with short-read Illumina data. However, it is not clear how different assembly strategies affect genomic accuracy, completeness, and protein prediction. (2) Methods: we compare different assembly strategies for , which causes Glässer's disease, characterized by fibrinous polyserositis and arthritis, in swine by using Illumina sequencing and long reads from the sequencing platforms of either Oxford Nanopore Technologies (ONT) or SMRT Pacific Biosciences (PacBio). (3) Results: Assembly with either PacBio or ONT reads, followed by polishing with Illumina reads, facilitated high-quality genome reconstruction and was superior to the long-read-only assembly and hybrid-assembly strategies when evaluated in terms of accuracy and completeness. An equally excellent method was correction with Homopolish after the ONT-only assembly, which had the advantage of avoiding hybrid sequencing with Illumina. Furthermore, by aligning transcripts to assembled genomes and their predicted CDSs, the sequencing errors of the ONT assembly were mainly indels that were generated when homopolymer regions were sequenced, thus critically affecting protein prediction. Polishing can fill indels and correct mistakes. (4) Conclusions: The assembly of bacterial genomes can be directly achieved by using long-read sequencing techniques. To maximize assembly accuracy, it is essential to polish the assembly with homologous sequences of related genomes or sequencing data from short-read technology.

Zhang P ，Jiang D ，Wang Y ，Yao X ，Luo Y ，Yang Z ... -  《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》

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》

Completion of draft bacterial genomes by long-read sequencing of synthetic genomic pools.

Illumina technology currently dominates bacterial genomics due to its high read accuracy and low sequencing cost. However, the incompleteness of draft genomes generated by Illumina reads limits their application in comprehensive genomics analyses. Alternatively, hybrid assembly using both Illumina short reads and long reads generated by single molecule sequencing technologies can enable assembly of complete bacterial genomes, yet the high per-genome cost of long-read sequencing limits the widespread use of this approach in bacterial genomics. Here we developed a protocol for hybrid assembly of complete bacterial genomes using miniaturized multiplexed Illumina sequencing and non-barcoded PacBio sequencing of a synthetic genomic pool (SGP), thus significantly decreasing the overall per-genome cost of sequencing. We evaluated the performance of SGP hybrid assembly on the genomes of 20 bacterial isolates with different genome sizes, a wide range of GC contents, and varying levels of phylogenetic relatedness. By improving the contiguity of Illumina assemblies, SGP hybrid assembly generated 17 complete and 3 nearly complete bacterial genomes. Increased contiguity of SGP hybrid assemblies resulted in considerable improvement in gene prediction and annotation. In addition, SGP hybrid assembly was able to resolve repeat elements and identify intragenomic heterogeneities, e.g. different copies of 16S rRNA genes, that would otherwise go undetected by short-read-only assembly. Comprehensive comparison of SGP hybrid assemblies with those generated using multiplexed PacBio long reads (long-read-only assembly) also revealed the relative advantage of SGP hybrid assembly in terms of assembly quality. In particular, we observed that SGP hybrid assemblies were completely devoid of both small (i.e. single base substitutions) and large assembly errors. Finally, we show the ability of SGP hybrid assembly to differentiate genomes of closely related bacterial isolates, suggesting its potential application in comparative genomics and pangenome analysis. Our results indicate the superiority of SGP hybrid assembly over both short-read and long-read assemblies with respect to completeness, contiguity, accuracy, and recovery of small replicons. By lowering the per-genome cost of sequencing, our parallel sequencing and hybrid assembly pipeline could serve as a cost effective and high throughput approach for completing high-quality bacterial genomes.

Derakhshani H ，Bernier SP ，Marko VA ，Surette MG ... -  《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 《-》

Are we there yet? Benchmarking low-coverage nanopore long-read sequencing for the assembling of mitochondrial genomes using the vulnerable silky shark Carcharhinus falciformis.

Whole mitochondrial genomes are quickly becoming markers of choice for the exploration of within-species genealogical and among-species phylogenetic relationships. Most often, 'primer walking' or 'long PCR' strategies plus Sanger sequencing or low-pass whole genome sequencing using Illumina short reads are used for the assembling of mitochondrial chromosomes. In this study, we first confirmed that mitochondrial genomes can be sequenced from long reads using nanopore sequencing data exclusively. Next, we examined the accuracy of the long-reads assembled mitochondrial chromosomes when comparing them to a 'gold' standard reference mitochondrial chromosome assembled using Illumina short-reads sequencing. Using a specialized bioinformatics tool, we first produced a short-reads mitochondrial genome assembly for the silky shark C. falciformis with an average base coverage of 9.8x. The complete mitochondrial genome of C. falciformis was 16,705 bp in length and 934 bp shorter than a previously assembled genome (17,639 bp in length) that used bioinformatics tools not specialized for the assembly of mitochondrial chromosomes. Next, low-pass whole genome sequencing using a MinION ONT pocket-sized platform plus customized de-novo and reference-based workflows assembled and circularized a highly accurate mitochondrial genome in the silky shark Carcharhinus falciformis. Indels at the flanks of homopolymer regions explained most of the dissimilarities observed between the 'gold' standard reference mitochondrial genome (assembled using Illumina short reads) and each of the long-reads mitochondrial genome assemblies. Although not completely accurate, mitophylogenomics and barcoding analyses (using entire mitogenomes and the D-Loop/Control Region, respectively) suggest that long-reads assembled mitochondrial genomes are reliable for identifying a sequenced individual, such as C. falciformis, and separating the same individual from others belonging to closely related congeneric species. This study confirms that mitochondrial genomes can be sequenced from long-reads nanopore sequencing data exclusively. With further development, nanopore technology can be used to quickly test in situ mislabeling in the shark fin fishing industry and thus, improve surveillance protocols, law enforcement, and the regulation of this fishery. This study will also assist with the transferring of high-throughput sequencing technology to middle- and low-income countries so that international scientists can explore population genomics in sharks using inclusive research strategies. Lastly, we recommend assembling mitochondrial genomes using specialized assemblers instead of other assemblers developed for bacterial and/or nuclear genomes.

Baeza JA ，García-De León FJ 《BMC GENOMICS》