# Factors Affecting the Quality of Bacterial Genomes Assemblies by Canu after Nanopore Sequencing
## 3 Results
### De novo Assembly and Quality Assessment  
Data about de novo assembly and QUAST quality assessment against the reference genome are reported in Table 4. \begin{table}
\begin{tabular}{c c c c c} \hline
**Flow Cell ID** & **Available Pores** & **Reads Generated** & **Estimated Bases** & **Read Length N50 (Bases)** \\ \hline AFT434 & 75 & 116,980 [1] & 74,930,000 [2] & 873 \\ AFT530 & 33 & 21 & 9630 & 600 \\ AFT593 & 75 & 156,760 [1] & 584,660,000 [2] & 6610 \\ \hline \end{tabular}  
* Data from the MinKNOW report, approximated to the nearest ten. 2 Data from MinKNOW, approximated to the ten-thousandth digit.  
\end{table} 
Table 2: Overview of sequencing. \begin{table}
\begin{tabular}{c c c c} \hline
**Flow Cell ID** & **AFT434** & **AFT530** & **AFT593** \\ \hline Number of reads & 82,690.0 & 11,693.0 & 112,928.0 \\ Number of bases & 42,470,043.0 & 6,349,058.0 & 369,469,949.0 \\ Read length N50 & 651.0 & 805.0 & 5573.0 \\ Median read length & 322.0 & 308.0 & 2185.0 \\ Median read quality & 11.3 & 10.7 & 11.2 \\ Q5 & 82,690 (100.0\%) 42.5 Mb & 11,693 (100.0\%) 6.3 Mb & 112,928 (100.0\%) 369.5 Mb \\ Q7 & 82,690 (100.0\%) 42.5 Mb & 11,693 (100.0\%) 6.3 Mb & 112,928 (100.0\%) 369.5 Mb \\ Q10 & 69,215 (83.7\%) 37.0 Mb & 8662 (74.1\%) 4.9 Mb & 95,936 (85.0\%) 316.0 Mb \\ Q12 & 26,384 (31.9\%) 17.7 Mb & 2132 (18.2\%) 1.4 Mb & 27,779 (24.6\%) 85.5 Mb \\ Q15 & 435 (0.5\%) 0.5 Mb & 34 (0.3\%) 0.0 Mb & 58 (0.1\%) 0.1 Mb \\ \hline \end{tabular}  
* Data about de novo assembly and QUAST quality assessment against the reference genome are reported in Table 4.  
\end{table} 
Table 3: Quality of the basecalled reads obtained with NanoPlot. Canu failed in assembling the reads generated from AFT434 and AFT530 (settings S4 and S5) with default parameters due to the extremely high percentage of reads shorter than 1000 bp (90.143% for S4 and 87.9244% for S5). The best results overall were achieved with setting S2, in which Canu parameters were more finely adjusted. The S2 assembly generated only two contigs (instead of seven for both S1 and S3, and eight for S6), one of them very close in length to the reference chromosome size. Likewise, S2 exhibited the highest coverage (77.16X), but only minor variations were observed among the other succeeded experiments. Two misassemblies in only one contig were reported by QUAST in all the settings, with the total number of mismatches ranging from 990 (S1) to 1439 (S6), and indels from 340 (S1) to 759 (S6). Even in this case, the worst results have been observed in the S6 experiment, which consisted of the results of only one sequencing run. The unaligned contig length was longer in S1 and S3 (183,520 and 184,317, respectively), and shorter in S2 and S6 (153,  
Figure 4: Comparison of the sequences from setting S3 against the reference genome. The contigs are detailed above the query line. Red and blue lines indicate forward and reverse matches, respectively. Figure 3: Comparison of the sequences from setting S2 against the reference genome. The contigs are detailed above the query line. Red and blue lines indicate forward and reverse matches, respectively. The contig S2 tig0001, obtained with setting S2, was alignable with the reference genome, and it almost completely covered the \(S\). Bovismorbificans chromosome. For S3, three contigs (S3 tig0001, S3 tig0011, and S3 tig0007) were aligned to the chromosome, with the other four being unaligned. For S6, only one contig (S6 tig0015) remained unaligned to the reference chromosome. The shortest number and length of gaps were observed in S2, while the longest was in S6. Overall, only the S2 tig0001 extended along the whole chromosome of \(S\). Bovismorbificans, with the exception of some minor gaps. Gaps retrieved in contigs obtained from all settings are reported in Table 5 as detected by QUAST. The total number of gaps varied among the settings, with 6 gaps in S2 (aggregate length 46,474 bp), 7 gaps in S1 (63,995 bp), 7 gaps in S3 (62,538 bp), and 10 gaps in S6 (73,118 bp).