Abstract
Brucella melitensis is a key etiological agent of brucellosis and has been increasingly subject to characterization using sequencing methodologies. This study aimed to investigate and compare short-read, long-read, and hybrid assemblies of B. melitensis. Eighteen B. melitensis isolates from Southern Israel were sequenced using Illumina and the Oxford Nanopore (ONP) MinION, and hybrid assemblies were generated with ONP long reads scaffolded on Illumina short reads. Short reads were assembled with INNUca with SPADes, long reads and hybrid with dragonflye. Abricate with the virulence factor database (VFDB) and in silico PCR (for the genes BetB, BPE275, BSPB, manA, mviN, omp19, perA, PrpA, VceC, and ureI) were used for identifying virulence genes, and a total of 61 virulence genes were identified in short-read, long-read, and hybrid assemblies of all 18 isolates. The phylogenetic analysis using long-read assemblies revealed several inconsistencies in cluster assignment as compared to using hybrid and short-read assemblies. Overall, hybrid assembly provided the most comprehensive data, and stand-alone short-read sequencing provided comparable data to stand-alone long-read sequencing regarding virulence genes. For genomic epidemiology studies, stand-alone ONP sequencing may require further refinement in order to be useful in endemic settings.
Highlights
Eighteen B. melitensis isolates from brucellosis cases recovered in blood culture from patients treated at the Soroka University Medical Center, Beer Sheva, Southern Israel were retrieved from the National Brucellosis Reference Laboratory (Kimron Veterinary Institute, Beit Dagan, Israel)
For the investigation of specific genes in other clinically-relevant organisms, more research is needed, as while this study found that the Oxford Nanopore (ONP) platform was consistent in identifying virulence genes, recent studies have noted that ONP technology had inconsistent performance regarding the detection of antimicrobial resistance genes in Gram-negative bacteria when compared to Illumina technology [4,9,59]
This study aimed to investigate long-read sequencing and hybrid sequencing of clinical B. melitensis isolates, with the specific intention to compare short read-based, long read-based and hybrid assemblies in order to recommend practicable workflow for future clinical use
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Microbial genomics analysis is widely being recognized as a potentially useful method to diagnose difficult-to-detect organisms and provide real-time surveillance for outbreaks [1,2]. Traditional short-read sequencing methodologies have drawbacks in terms of contig length and turnaround time. Short-read sequencing inevitably results in gaps in the assembly, and the gaps present in short read-only assemblies are a concern as genes present within that gap may be missed, and assemblies on the edge of a contig (next to the gap) may be of lower quality than assemblies in the middle of the contig
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