# Factors Affecting the Quality of Bacterial Genomes Assemblies by Canu after Nanopore Sequencing
## 4 Discussion  
The long-read sequencing technology, such as the ONT MinION one, is achieving optimal results in sequencing bacterial genomes [7]. Although long reads significantly reduce the problem of fragmentation in genome assemblies, contiguity might not be completely ensured. In this sense, the quality of experiments may be severely affected by several factors. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Setting** & **Starting Point * (bp)** & **Ending Point * (bp)** & **Gap Length (bp)** & **Total Gap Length (bp)** \\ \hline \multirow{8}{*}{1} & & 1,166,439 & 1,197,280 & 30,841 & \\  & & 1,785,616 & 1,794,274 & 8658 & \\  & & 2,114,982 & 2,115,257 & 275 & \\  & & 2,242,947 & 2,248,793 & 5846 & 63,995 \\  & & 2,826,781 & 2,827,299 & 518 & \\  & & 2,851,663 & 2,865,122 & 13,459 & \\  & & 3,289,256 & 3,293,654 & 4398 & \\ \hline \multirow{8}{*}{2} & & 1,166,439 & 1,197,280 & 30,841 & \\  & & 1,786,652 & 1,786,988 & 336 & \\  & & 1,789,133 & 1,790,178 & 1045 & \\  & & 2,114,982 & 2,115,257 & 275 & \\  & & 2,826,781 & 2,827,299 & 518 & \\  & & 2,851,663 & 2,865,122 & 13,459 & \\  & & 2,851,663 & 2,865,122 & 13,459 & \\ \hline \multirow{8}{*}{3} & & 1,166,439 & 1,197,280 & 30,841 & \\  & & 1,785,619 & 1,791,839 & 6220 & \\  & & 2,114,982 & 2,115,257 & 275 & \\  & & 2,241,964 & 2,248,793 & 6829 & 62,538 \\  & & 2,826,781 & 2,827,299 & 518 & \\  & & 2,851,663 & 2,865,122 & 13,459 & \\  & & 3,289,256 & 3,293,652 & 4396 & \\ \hline \multirow{8}{*}{6} & & 1,166,439 & 1,197,280 & 30,841 & \\  & & 1,785,633 & 1,793,621 & 7988 & \\  & & 2,002,542 & 2,008,321 & 5779 & \\  & & 2,114,982 & 2,115,257 & 275 & \\  & & 2,241,967 & 2,248,789 & 6822 & 73,118 \\  & & 2,826,781 & 2,827,299 & 518 & \\  & & 2,851,663 & 2,865,122 & 13,459 & \\  & & 3,288,371 & 3,293,632 & 5261 & \\  & & 4,236,632 & 4,238,807 & 2175 & \\ \hline \hline \end{tabular}  
* The starting and ending points are relative to the chromosome reference sequence NZ_CP073715.  
\end{table} 
Table 5: Overview of the gaps in the contigs aligned to the chromosome. Among these, the quality of the DNA to be included in the library is pivotal. In particular, high molecular weight DNA is fundamental to generate longer, and the here-reported findings strongly support this evidence, considering that the sequencing runs obtained from the two samples characterized by high fragmentation showed a low N50 read length, which made it impossible to proceed with the assembly step. Another crucial element is the quality of flow cells, in terms of pores available for sequencing. In fact, the worst results were obtained using a flow cell with 33 available pores, probably too few to generate adequate data to be assembled, at least alone. It should be underlined that the manufacturer clearly recommends using only flow cells with more than 50 pores, also offers an efficient warranty policy for cells not complying with optimal features upon arrival to the customer's facility. Interestingly, the N50 read length obtained from the low-quality cell was only slightly lower than those from the first one, but the overall number of reads was sensibly reduced. This confirms that the number of pores is related to the throughput of the run, while the DNA integrity affects the read length. Therefore, only the combination of high-quality DNA and efficient cells may ensure optimal results. Nonetheless, particular attention must be always paid to the downstream analysis of the output of the run, especially if de novo assembly is required. In that case, the production of a contig representative of the entire molecule with a coverage high enough to reduce at the minimum the sequencing errors would be desirable. Among strengths, the direct analysis of the potential resistance gene set harbored by a strain has been often appreciated [28]. Despite the fact that the here-analyzed did not provide the specific resistance gene, the analysis was carried out rapidly and accurately, since the results obtained using the ARMA workflow matched with those from the assembled sequences.