# Factors Affecting the Quality of Bacterial Genomes Assemblies by Canu after Nanopore Sequencing
## 4 Discussion
Long-read sequencing systems have been proven to be superior in reaching high quality in the de novo assembly, but some drawbacks might be observed. In fact, Canu, considered the best solution for the de novo assembly of long reads, might fail to produce high-quality contigs if not properly and finely tuned. Even not considering the assembly of the low-quality outputs (S4 and S5), when Canu was launched with default parameters, the assembly quality was lower in terms of N50 length (3,162,557 for S3, 1,040,624 for S6), number of contigs (seven for S3 and eight for S6) and gap number and length (7 gaps with a total 62,538 bp length for S3, and 10 gaps with a total 73,118 bp length). Several strategies have been addressed to prevent the failure in reaching full coverage of a bacterial chromosome. The hybrid assembly has been of such approach, using short reads to fill gaps in the scaffold generated by MinION assemblies [9]. In the present study, the overall output has been enriched by combining the long, high-quality reads of the AFT593 run with the shorter ones from the AFT434 and AFT530 runs. Even with the default setting of Canu, such enrichment has improved, despite marginally, the quality of the contig (S3) if compared to the contig only deriving from the best run (S6). Better results have been gained when Canu parameters have been adjusted. In setting S1 only one parameter was adjusted, namely the \"correctedErrorRate\" (the allowed difference in an overlap between two corrected reads; with a default value of 0.144 for Nanopore reads). This value was slightly decreased to 0.12, after considering that coverage for all the settings was more than 72X, as suggested by the Canu developers for high coverage datasets (>60X). In this case, the assembly quality was much better than those obtained in S6, but compatible with setting S3 (N50 length of 3,162,545 bp, seven contigs produced, and seven gaps with a total 63,995 bp length). More parameters were adjusted in S2. In particular, the \"corErrorRate\" value (defined as \"do not use overlaps with error rate higher than this when computing corrected reads\" in the Canu documentation available at [[https://canu.readthedocs.io/en/latest/parameter-reference.html](https://canu.readthedocs.io/en/latest/parameter-reference.html)]([https://canu.readthedocs.io/en/latest/parameter-reference.html](https://canu.readthedocs.io/en/latest/parameter-reference.html)), latest accessed on 18 February 2022) was increased from 0.30 to 0.40 to improve the corrected coverage; \"corMhapSensitivity\" (namely, the \"coarse sensitivity level, based on read coverage\") was set to \"high\", despite the developers suggested using it for low coverages; \"corMinCoverage\" (\"limit read correction to only overlaps longer than this\") was brought to 0 to correct as many reads as possible; \"corOutCoverage\" (which controls how much coverage in corrected reads is generated) was set to 100. Conversely, the \"correctedErrorRate\" was left default in this setting. That setting (S2) allowed obtaining the best assembly quality. In fact, N50 length resulted in 4,670,990 bp, and only two contigs were generated, long 104,427 bp and 4,670,990 bp, respectively. The BLAST search showed that the shorter contig showed homology with a plasmid of \(S\). Bovismorbificans, while the largest corresponded to the chromosome of the same Salmonella serovar. Artemis ACT showed that such a contig covered the entire \(S\). Bovismorbificans chromosome, without evidencing other rearrangements than the six detected gaps. Three of those gaps were probably due to the actual lack of such regions in the sequenced molecule since they corresponded to prophage genomic islands within the reference sequence. Therefore, the S2 setting was found to be the most appropriate also in terms of number and length of gaps, since only six gaps were detected, with a low probability of being artifactual. In fact, most of the contig breaks in the bacterial DNA molecules are due to the intrinsic plasticity of microbial genomes.