High quality 3C de novo assembly and annotation of a multidrug resistant ST-111 Pseudomonas aeruginosa genome: Benchmark of hybrid and non-hybrid assemblers

Abstract

Genotyping methods and genome sequencing are indispensable to reveal genomic structure of bacterial species displaying high level of genome plasticity. However, reconstruction of genome or assembly is not straightforward due to data complexity, including repeats, mobile and accessory genetic elements of bacterial genomes. Moreover, since the solution to this problem is strongly influenced by sequencing technology, bioinformatics pipelines, and selection criteria to assess assemblers, there is no systematic way to select a priori the optimal assembler and parameter settings. to assembly the genome of Pseudomonas aeruginosa strain AG1 (PaeAG1), short reads (Illumina) and long reads (Oxford Nanopore) sequencing data were used in 13 different non-hybrid and hybrid approaches. PaeAG1 is a multiresistant high-risk sequence type 111 (ST-111) clone that was isolated from a Costa Rican hospital and it was the first report of an isolate of P. aeruginosa carrying both blaVIM-2 and blaIMP-18 genes encoding for metallo-β-lactamases (MBL) enzymes. To assess the assemblies, multiple metrics regard to contiguity, correctness and completeness (3C criterion, as we define here) were used for benchmarking the 13 approaches and select a definitive assembly. In addition, annotation was done to identify genes (coding and RNA regions) and to describe the genomic content of PaeAG1. Whereas long reads and hybrid approaches showed better performances in terms of contiguity, higher correctness and completeness metrics were obtained for short read only and hybrid approaches. A manually curated and polished hybrid assembly gave rise to a single circular sequence with 100% of core genes and known regions identified, >98% of reads mapped back, no gaps, and uniform coverage. The strategy followed to obtain this high-quality 3C assembly is detailed in the manuscript and we provide readers with an all-in-one script to replicate our results or to apply it to other troublesome cases. The final 3C assembly revealed that the PaeAG1 genome has 7,190,208 bp, a 65.7% GC content and 6,709 genes (6,620 coding sequences), many of which are included in multiple mobile genomic elements, such as 57 genomic islands, six prophages, and two complete integrons with blaVIM-2 and blaIMP-18 MBL genes. Up to 250 and 60 of the predicted genes are anticipated to play a role in virulence (adherence, quorum sensing and secretion) or antibiotic resistance (β-lactamases, efflux pumps, etc). Altogether, the assembly and annotation of the PaeAG1 genome provide new perspectives to continue studying the genomic diversity and gene content of this important human pathogen. Genotyping methods and genome sequencing are indispensable to reveal genomic structure and evolution of bacterial clones with high resolution 1. In this sense, production of large amounts of short sequencing data from genomes (reads) has been facilitated by continuous advances in Next Generation Sequencing (NGS) technologies.

From Pan-genome to the Genomic Context of the Two Integrons of ST-111 Pseudomonas Aeruginosa AG1: A VIM-2-carrying Old-acquaintance and a Novel Imp-18-carrying Integron

“…Varying pro les of the presence/absence of the 57 PaeAG1 genomic islands are found in the ST-111 strains and related genomes A comparative analysis based on sequence alignment was run in order to determinate the presence and frequency of the PaeAG1 genomic islands in other phylogenetically related strains. Genomic islands locus were previously predicted (12). We rst represented the distribution of the genomic islands along the PaeAG1 genome, as presented in Fig.…”

“…Carbapenem resistance in PaeAG1 was demonstrated to be explained by activity of two MBLs (VIM-2 and IMP-18) (11), each gene harbored in two independent class 1 integrons (11,12). With the aim of describing the genomic context associated with the two integrons, we characterized the architecture of genomic regions de ned by GIC VIM−2 and GIC IMP−18 .…”

“…This strain was the rst report of a P. aeruginosa isolate carrying both VIM-2 and IMP-18 genes encoding for metallo-β-lactamases (MBLs) enzymes, both with carbapenemase activity (11). As shown in our previous works, including the genome assembly (GenBank CP045739) (12), these genes belong to two independent class 1 integrons, each contained in one out the 57 predicted genomic islands of PaeAG1 (11,12). Other elements such as six phages, mobile genetic elements and some virulence factors are also harbored in genomic islands.…”

“…Since the two integrons are absent in the reference strain Pae-PAO1, an alignment of the genomic regions (BLASTn) and another of amino acid (AA) sequences (BLASTp) were used to identify the limits of the complete inserted region in PaeAG1. The two speci c inserted regions were composed of two or more genomic islands in a row, as obtained in our previous study (grouped or with overlapping regions) (12). Thus, regions were called GIC VIM−2 (genomic island cluster containing VIM-2-carrying integron) and GIC IMP−18 (genomic island cluster harboring the IMP-18-carrying integron).…”

“…The 57 PaeAG1 genomic islands were previously identi ed using IslandViewer v4 (www.pathogenomics.sfu.ca/islandviewer/), as we reported recently (12). Genomic islands regions (".bed" le) were downloaded from the same platform and sequences (".fasta" format) were obtained using the getfasta function in bedtools software v2.…”