Hybrid <em>De Novo</em> Genome Assembly for the Generation of Complete Genomes of Urinary Bacteria using Short- and Long-read Sequencing Technologies

Abstract

Complete genome sequences provide valuable data for the understanding of genetic diversity and unique colonization factors of urinary microbes. These data may include mobile genetic elements, such as plasmids and extrachromosomal phage, that contribute to the dissemination of antimicrobial resistance and further complicate treatment of urinary tract infection (UTI). In addition to providing fine resolution of genome structure, complete, closed genomes allow for the detailed comparative genomics and evolutionary analyses. The generation of complete genomes de novo has long been a challenging task due to limitations of available sequencing technology.Paired-end Next Generation Sequencing (NGS) produces high quality short reads often resulting in accurate but fragmented genome assemblies. On the contrary, Nanopore sequencing provides long reads of lower quality normally leading to errorprone complete assemblies. Such errors may hamper genome-wide association studies or provide misleading variant analysis results. Therefore, hybrid approaches combining both short and long reads have emerged as reliable methods to achieve highly accurate closed bacterial genomes. Reported herein is a comprehensive method for the culture of diverse urinary bacteria, species identification by 16S rRNA gene sequencing, extraction of genomic DNA (gDNA), and generation of short and long reads by NGS and Nanopore platforms, respectively. Additionally, this method describes a bioinformatic pipeline of quality control, assembly, and gene prediction algorithms for the generation of annotated complete genome sequences. Combination of bioinformatic tools enables the selection of high quality read data for hybrid genome assembly and downstream analysis. The streamlined approach for the hybrid de novo genome assembly described in this protocol may be adapted for the use in any culturable bacteria.

Inter-species diversity and functional genomic analyses of closed genome assemblies of clinically isolated, megaplasmid-containing Enterococcus raffinosus Er676 and ATCC49464

“…Er676 was isolated by plating urine on chromogenic agar (Chromagar Orientation, BD) and picking well-isolated colonies with enterococcal morphology. For species identification, single colonies were selected and subjected to PCR and Sanger sequencing of the conserved 16S rRNA gene as described previously [19,20]. Sequences were queried against the nr/ nt database using megablast (blast v2.10.0) and the species E. raffinosus was confirmed [21].…”

“…In preparation for sequencing, genomic DNA was isolated from overnight cultures of Er676 and ATCC49464 grown in Brain Heart Infusion (BHI) broth at 37 °C using the DNeasy Blood and Tissue kit (Qiagen). To facilitate lysis, 20 mg ml −1 lysozyme and 450 kUml −1 mutanolysin were included in the lysis buffer [19,22]. DNA was quantified via a Qubit fluorometer and quality and integrity were assessed by agarose gel electrophoresis.…”

“…DNA was quantified via a Qubit fluorometer and quality and integrity were assessed by agarose gel electrophoresis. Next-generation sequencing was performed as previously described using both Illumina NextSeq 500 and Oxford Nanopore Technologies (ONT) MinION following library preparation [19].…”

“…Hybrid genome assembly was performed as described previously [19]. Briefly, raw sequence read data were subject to quality assessment and trimming using CLC Genomics Workbench v12.0.3, NanoStats v1.2.0 and NanoFilt v2.6.0 [23].…”