# Factors Affecting the Quality of Bacterial Genomes Assemblies by Canu after Nanopore Sequencing
## 4 Discussion
Those genomes are characterized by adaptive modifications (i.e., repeats, insertions or deletions, single nucleotide polymorphisms, inversions) as a result of prophage insertion and/or excision, transposition events, or rearrangements with mobile genetic elements as plasmids, bacteriophages, and transposons [29]. Usually, long-read sequencing systems can overcome the issue of repeated elements, as their reads may be larger than repeats. Anyway, all those modifications may lead to gaps and misassemblies in de novo assembly. Considering that the largest contig breaks detected in the present investigation are associated with genomic prophage islands, this is another hint about the capability of third-generation sequencing to provide a comprehensive picture of the genomic composition of bacteria. It is noteworthy that the serovar automated prediction returned different results, according not only to the contig quality, but also to the software version, since only SeqSero2 succeeded in determining the serovar Bovismorbificans, and only when the contigs from S2 were computed. On the other hand, some researchers complain about the longer computational time required for Canu in contrast to other assemblers (personal communications). The Canu documentation warns that the adjustment of some parameters may hugely increase the computational time. However, with a medium-performance personal computer, the assembly took about 13 h with S2, thus confirming that the tools are quite fast in reading assembling. Nevertheless, a reduction in computational time was observed by using MinHash, along with a lower RAM demand [30]. Instead, they always require several rounds of polishing, which obviously extend the final computational time, despite some Authors obtained good assemblies with only one round of polishing after running of Canu [12]. In contrast, no assembly was obtained when Flye was used as an assembler. This was probably due to the different algorithms adopted by the two software. While Canu is a hierarchical tool with a probabilistic approach to the alignments of _k_-mers, Flye accurately bridges the disjointigs obtained in the initial steps of the computing process [21]. Despite being less demanding if compared with other assemblers (e.g., Bowtie, BWA, and SOAP2), it still requires high memory usage and, consequently, high RAM availability [31]. Moreover, some authors found Flye, when used in association with Pilon for the hybrid approach, more inaccurate, with respect to Canu, for producing bacterial genome sequences [32]. All those considering, the nanopore sequencing with flongle cells may be inexpensive and time-saving and may provide high-quality results, if some conditions are strictly observed. Among those, the good enough quality of the purified DNA, the functional efficiency of flowcells, and a proper setting of the assembly software cannot be superseded. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app12063110/s1](https://www.mdpi.com/article/10.3390/app12063110/s1)]([https://www.mdpi.com/article/10.3390/app12063110/s1](https://www.mdpi.com/article/10.3390/app12063110/s1)), Table S1: Results of sequence similarity search for BLAST. Only the first match for each search has been reported; Results S1: Serotype prediction results obtained with SeqSero and SeqSero2. **Author Contributions:** Conceptualization, N.P. and A.S.; methodology, A.S., N.P. and C.F.M. ; software, A.S., N.P. and C.F.M. ; validation, C.C., A.C., R.S. and F.A. ; formal analysis, C.C. and C.F.M. ; investigation, A.S., R.S. and C.C. ; resources, A.C.; data curation, A.S., N.P. and F.A. ; writing--original draft preparation, A.S.; writing--review and editing, N.P. ; supervision, A.C. and F.A. ; project administration, A.C. and F.A. All authors have read and agreed to the published version of the manuscript. This research received no external funding. Not applicable.