Abstract
Pseudoalteromonas haloplanktis TAC125 is among the most commonly studied bacteria adapted to cold environments. Aside from its ecological relevance, P. haloplanktis has a potential use for biotechnological applications. Due to its importance, we decided to take advantage of next generation sequencing (Illumina) and third generation sequencing (PacBio and Oxford Nanopore) technologies to resequence its genome. The availability of a reference genome, obtained using whole genome shotgun sequencing, allowed us to study and compare the results obtained by the different technologies and draw useful conclusions for future de novo genome assembly projects. We found that assembly polishing using Illumina reads is needed to achieve a consensus accuracy over 99.9% when using Oxford Nanopore sequencing, but not in PacBio sequencing. However, the dependency of consensus accuracy on coverage is lower in Oxford Nanopore than in PacBio, suggesting that a cost-effective solution might be the use of low coverage Oxford Nanopore sequencing together with Illumina reads. Despite the differences in consensus accuracy, all sequencing technologies revealed the presence of a large plasmid, pMEGA, which was undiscovered until now. Among the most interesting features of pMEGA is the presence of a putative error-prone polymerase regulated through the SOS response. Aside from the characterization of the newly discovered plasmid, we confirmed the sequence of the small plasmid pMtBL and uncovered the presence of a potential partitioning system. Crucially, this study shows that the combination of next and third generation sequencing technologies give us an unprecedented opportunity to characterize our bacterial model organisms at a very detailed level.
Highlights
Cold environments covering most of the Earth, harbour a vast diversity of cold-adapted organisms[1]
The Oxford Nanopore Technologies (ONT) sequencing library was prepared without size selection and produced much longer reads than the size selected Pacific Biosciences (PacBio) library
We aligned the reads to the reference genome (NC_007481.1, NC_007482.1) and evaluated the read quality based on sequence alignments
Summary
Cold environments covering most of the Earth, harbour a vast diversity of cold-adapted organisms[1]. The method applied to generate the P. haloplanktis TAC125 reference genome, whole genome shotgun sequencing using Sanger sequencing technology, has enabled the sequencing of many genomes, including those of human and mouse. It is expensive, labour-intensive and time-consuming[25]. Third generation sequencing technologies, such as Oxford Nanopore Technologies (ONT) real-time direct DNA/RNA sequencing and Pacific Biosciences (PacBio) Single Molecule, Real-Time (SMRT) Sequencing, can produce extremely long reads (20 kb and even longer) and are more suitable for generating highly continuous genomes[27,28] These technologies open new doors in microbial genomics and enable a broad range of microbial studies[29]. The findings can be instrumental for all researchers who are planning de novo genome sequencing projects using these newer technologies
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