# Factors Affecting the Quality of Bacterial Genomes Assemblies by Canu after Nanopore Sequencing
## 1 Introduction  
During the past few years, approaches to whole-genome sequencing (WGS) have been evolving [1]. Since the application of low-throughput first-generation Sanger sequencing, which is definitely expensive and time-consuming, several strategies have been developed [2]. Among these, next-generation sequencing (NGS) systems, such as IonTorrent, Illumina, or Roche 454, represent low-cost, high-throughput (1.2-6000 Gb) alternatives, but are limited by the reduced length of the produced reads (about 50-600 bp) [3,4]. Indeed, short reads often result in an inability to resolve repeat genome regions, leading to misassemblies, gaps, and difficulties in detecting structural variations (SVs), making reconstruction of the original molecules challenging to achieve [3,5,6]. Such an issue has been overcome by the introduction of third-generation sequencing systems, developed by PacBio and Oxford Nanopore Technologies (ONT), able to produce long reads (hundreds to thousands of kb) in a real-time process, resulting in high throughput (20 Gb to 6 Tb), time savings, and cost reductions [3,5,6]. However, long-read sequencing usually presents higher error rates compared to NGS systems (about 15%), although the ONT base-calling tools have been constantly updating to improve accuracy [7; 8]. Some authors have followed a hybrid approach in de novo assembly, by using short reads from NGS to correct errors in the long third-generation sequences [9]. Another common approach is to align the reads against a reference genome, an option that is nevertheless not applicable when a high-quality reference is not available in databases [10]. On the other hand, a weighted bioinformatic analysis might be supportive of error correction, leading to good results [11]. A key point in gaining an accurate final sequence is the assembly of the reads. In this sense, a hierarchical strategy is one of the best options, consisting of multiple rounds of read overlapping and correction prior to performing assembly [11; 12]. Several assemblers are available for this purpose, both commercial and open source. Commercial software is intended as user-friendly, stand-alone suites that group several bioinformatic tools and are characterized by a highly accessible graphic interface [13]. Despite the great benefits in terms of ease of use, these suites, often designed as closed systems, do not offer the same flexibility as open-source tools, which allow fine adjustments to processing parameters in order to adapt to specific requirements or user needs. Among the open-source assemblers, Canu is one of the most accurate hierarchical tools [14; 15]. Specifically intended for third-generation sequencing systems, Canu performs correction, trimming (removing adapters and breaking chimeras), and assembly of reads in consecutive steps, while also offering the advantages of a lower runtime and coverage requirements, therefore improving assembly continuity [12]. This software integrates the MinHash Alignment Process (MHAP), conceived specifically to handle repeats among long-sequencing reads [12; 16]. Usually, assemblers work by comparing individual \(k\)-mers to identify potential overlaps, resulting in a high computational cost, and by ignoring highly repetitive \(k\)-mers, thus increasing the probability to discard some correct overlaps [16]. Conversely, Canu adopts a probabilistic algorithm by applying the term frequency and the inverse document frequency to weight and compare compressed MinHash sketches, reducing the chance to select repetitive \(k\)-mers for overlapping [12]. Additionally, Canu is one of the most configurable assemblers, with hundreds of adjustable parameters, allowing to perfectly fit each assembly to specific necessities [15]. This feature is particularly useful when challenging sequencing is performed, for instance when bacterial genomes are under investigation, due to some critical features of prokaryotic organisms, such as high plasticity, presence of plasmids, circular replicons with no defined start/end point [7; 15; 17].