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Article ; Online: Assembling the perfect bacterial genome using Oxford Nanopore and Illumina sequencing.

Wick, Ryan R / Judd, Louise M / Holt, Kathryn E

2023 Volume 19, Issue 3, Page(s) e1010905

Abstract: A perfect bacterial genome assembly is one where the assembled sequence is an exact match for the organism's genome-each replicon sequence is complete and contains no errors. While this has been difficult to achieve in the past, improvements in long-read ...

Abstract	A perfect bacterial genome assembly is one where the assembled sequence is an exact match for the organism's genome-each replicon sequence is complete and contains no errors. While this has been difficult to achieve in the past, improvements in long-read sequencing, assemblers, and polishers have brought perfect assemblies within reach. Here, we describe our recommended approach for assembling a bacterial genome to perfection using a combination of Oxford Nanopore Technologies long reads and Illumina short reads: Trycycler long-read assembly, Medaka long-read polishing, Polypolish short-read polishing, followed by other short-read polishing tools and manual curation. We also discuss potential pitfalls one might encounter when assembling challenging genomes, and we provide an online tutorial with sample data (github.com/rrwick/perfect-bacterial-genome-tutorial).
MeSH term(s)	Animals ; Nanopores ; High-Throughput Nucleotide Sequencing ; Genome, Bacterial/genetics ; Oryzias ; Technology
Language	English
Publishing date	2023-03-02
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2193340-6
ISSN	1553-7358 ; 1553-734X
ISSN (online)	1553-7358
ISSN	1553-734X
DOI	10.1371/journal.pcbi.1010905
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Article ; Online: Improved Genome Sequence of Australian Methicillin-Resistant Staphylococcus aureus Strain JKD6159.

Wick, Ryan R / Judd, Louise M / Monk, Ian R / Seemann, Torsten / Stinear, Timothy P

Microbiology resource announcements

2023 Volume 12, Issue 2, Page(s) e0112922

Abstract: Staphylococcus aureus strain JKD6159 represents a prominent community-acquired methicillin-resistant S. aureus (MRSA) clone in Australia. Here, we report an improved assembly of the original S. aureus JKD6159 genome sequence. By using deep sequencing ... ...

Abstract	Staphylococcus aureus strain JKD6159 represents a prominent community-acquired methicillin-resistant S. aureus (MRSA) clone in Australia. Here, we report an improved assembly of the original S. aureus JKD6159 genome sequence. By using deep sequencing with multiple technologies combined with carefully curated assembly and polishing, we believe the assembly to contain zero errors.
Language	English
Publishing date	2023-01-18
Publishing country	United States
Document type	Journal Article
ISSN	2576-098X
ISSN (online)	2576-098X
DOI	10.1128/mra.01129-22
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Article ; Online: Tissue-associated and vertically transmitted bacterial symbiont in the coral Pocillopora acuta.

Maire, Justin / Tsang Min Ching, Sarah Jane / Damjanovic, Katarina / Epstein, Hannah E / Judd, Louise M / Blackall, Linda L / van Oppen, Madeleine J H

The ISME journal

2024 Volume 18, Issue 1

Abstract: Coral microhabitats are colonized by a myriad of microorganisms, including diverse bacteria which are essential for host functioning and survival. However, the location, transmission, and functions of individual bacterial species living inside the coral ... ...

Abstract	Coral microhabitats are colonized by a myriad of microorganisms, including diverse bacteria which are essential for host functioning and survival. However, the location, transmission, and functions of individual bacterial species living inside the coral tissues remain poorly studied. Here, we show that a previously undescribed bacterial symbiont of the coral Pocillopora acuta forms cell-associated microbial aggregates (CAMAs) within the mesenterial filaments. CAMAs were found in both adults and larval offspring, suggesting vertical transmission. In situ laser capture microdissection of CAMAs followed by 16S rRNA gene amplicon sequencing and shotgun metagenomics produced a near complete metagenome-assembled genome. We subsequently cultured the CAMA bacteria from Pocillopora acuta colonies, and sequenced and assembled their genomes. Phylogenetic analyses showed that the CAMA bacteria belong to an undescribed Endozoicomonadaceae genus and species, which we propose to name Candidatus Sororendozoicomonas aggregata gen. nov sp. nov. Metabolic pathway reconstruction from its genome sequence suggests this species can synthesize most amino acids, several B vitamins, and antioxidants, and participate in carbon cycling and prey digestion, which may be beneficial to its coral hosts. This study provides detailed insights into a new member of the widespread Endozoicomonadaceae family, thereby improving our understanding of coral holobiont functioning. Vertically transmitted, tissue-associated bacteria, such as Sororendozoicomonas aggregata may be key candidates for the development of microbiome manipulation approaches with long-term positive effects on the coral host.
MeSH term(s)	Animals ; Anthozoa/microbiology ; Phylogeny ; RNA, Ribosomal, 16S/genetics ; Bacteria/genetics ; Metagenome ; Gammaproteobacteria/genetics ; Coral Reefs ; Symbiosis
Chemical Substances	RNA, Ribosomal, 16S
Language	English
Publishing date	2024-02-15
Publishing country	England
Document type	Journal Article
ZDB-ID	2406536-5
ISSN	1751-7370 ; 1751-7362
ISSN (online)	1751-7370
ISSN	1751-7362
DOI	10.1093/ismejo/wrad027
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Article ; Online: Recovery of small plasmid sequences via Oxford Nanopore sequencing.

Wick, Ryan R / Judd, Louise M / Wyres, Kelly L / Holt, Kathryn E

Microbial genomics

2021 Volume 7, Issue 8

Abstract: Oxford Nanopore Technologies (ONT) sequencing platforms currently offer two approaches to whole-genome native-DNA library preparation: ligation and rapid. In this study, we compared these two approaches for bacterial whole-genome sequencing, with a ... ...

Abstract	Oxford Nanopore Technologies (ONT) sequencing platforms currently offer two approaches to whole-genome native-DNA library preparation: ligation and rapid. In this study, we compared these two approaches for bacterial whole-genome sequencing, with a specific aim of assessing their ability to recover small plasmid sequences. To do so, we sequenced DNA from seven plasmid-rich bacterial isolates in three different ways: ONT ligation, ONT rapid and Illumina. Using the Illumina read depths to approximate true plasmid abundance, we found that small plasmids (<20 kbp) were underrepresented in ONT ligation read sets (by a mean factor of ~4) but were not underrepresented in ONT rapid read sets. This effect correlated with plasmid size, with the smallest plasmids being the most underrepresented in ONT ligation read sets. We also found lower rates of chimaeric reads in the rapid read sets relative to ligation read sets. These results show that when small plasmid recovery is important, ONT rapid library preparations are preferable to ligation-based protocols.
MeSH term(s)	Bacteria/genetics ; Bacteria/isolation & purification ; DNA, Bacterial ; Gene Library ; High-Throughput Nucleotide Sequencing/methods ; Nanopore Sequencing ; Nanopores ; Plasmids/genetics ; Sequence Analysis, DNA ; Whole Genome Sequencing/methods
Chemical Substances	DNA, Bacterial
Language	English
Publishing date	2021-08-06
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2835258-0
ISSN	2057-5858 ; 2057-5858
ISSN (online)	2057-5858
ISSN	2057-5858
DOI	10.1099/mgen.0.000631
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Article ; Online: Kaptive 2.0: updated capsule and lipopolysaccharide locus typing for the

Lam, Margaret M C / Wick, Ryan R / Judd, Louise M / Holt, Kathryn E / Wyres, Kelly L

Microbial genomics

2022 Volume 8, Issue 3

Abstract: The outer polysaccharide capsule and lipopolysaccharide (LPS) antigens are key targets for novel control strategies ... ...

Abstract	The outer polysaccharide capsule and lipopolysaccharide (LPS) antigens are key targets for novel control strategies targeting
MeSH term(s)	Genomics ; Humans ; Klebsiella ; Klebsiella Infections/epidemiology ; Klebsiella pneumoniae/genetics ; Lipopolysaccharides
Chemical Substances	Lipopolysaccharides
Language	English
Publishing date	2022-03-20
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2835258-0
ISSN	2057-5858 ; 2057-5858
ISSN (online)	2057-5858
ISSN	2057-5858
DOI	10.1099/mgen.0.000800
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Article ; Online: Epidemiology and genomic analysis of Klebsiella oxytoca from a single hospital network in Australia.

Stewart, James / Judd, Louise M / Jenney, Adam / Holt, Kathryn E / Wyres, Kelly L / Hawkey, Jane

BMC infectious diseases

2022 Volume 22, Issue 1, Page(s) 704

Abstract: Background: Infections caused by Klebsiella oxytoca are the second most common cause of Klebsiella infections in humans. Most studies have focused on K. oxytoca outbreaks and few have examined the broader clinical context of K. oxytoca.: Methods: ... ...

Abstract	Background: Infections caused by Klebsiella oxytoca are the second most common cause of Klebsiella infections in humans. Most studies have focused on K. oxytoca outbreaks and few have examined the broader clinical context of K. oxytoca. Methods: Here, we collected all clinical isolates identified as K. oxytoca in a hospital microbiological diagnostic lab across a 15-month period (n = 239). Whole genome sequencing was performed on a subset of 92 isolates (all invasive, third-generation cephalosporin resistant (3GCR) and non-urinary isolates collected > 48 h after admission), including long-read sequencing on a further six isolates with extended-spectrum beta-lactamase or carbapenemase genes. Results: The majority of isolates were sensitive to antimicrobials, however 22 isolates were 3GCR, of which five were also carbapenem resistant. Genomic analyses showed those identified as K. oxytoca by the clinical laboratory actually encompassed four distinct species (K. oxytoca, Klebsiella michiganensis, Klebsiella grimontii and Klebsiella pasteurii), referred to as the K. oxytoca species complex (KoSC). There was significant diversity within the population, with only 10/67 multi-locus sequence types (STs) represented by more than one isolate. Strain transmission was rare, with only one likely event identified. Six isolates had extended spectrum beta-lactamase (bla Conclusion: Whilst antimicrobial resistance was rare, the resistance plasmids were similar to those found in other Enterobacterales, demonstrating that KoSC has access to the same plasmid reservoir and thus there is potential for multi-drug resistance. Further genomic studies are required to improve our understanding of the KoSC population and facilitate investigation into the attributes of successful nosocomial isolates.
MeSH term(s)	Anti-Bacterial Agents/pharmacology ; Drug Resistance, Multiple, Bacterial ; Genomics ; Hospitals ; Humans ; Inosine Monophosphate ; Klebsiella Infections/epidemiology ; Klebsiella Infections/microbiology ; Klebsiella oxytoca/genetics ; Klebsiella pneumoniae ; Microbial Sensitivity Tests ; Plasmids/genetics ; beta-Lactamases/genetics
Chemical Substances	Anti-Bacterial Agents ; Inosine Monophosphate (131-99-7) ; beta-Lactamases (EC 3.5.2.6)
Language	English
Publishing date	2022-08-24
Publishing country	England
Document type	Journal Article
ZDB-ID	2041550-3
ISSN	1471-2334 ; 1471-2334
ISSN (online)	1471-2334
ISSN	1471-2334
DOI	10.1186/s12879-022-07687-7
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Article ; Online: High-performance enrichment-based genome sequencing to support the investigation of hepatitis A virus outbreaks.

Zufan, Sara E / Mercoulia, Karolina / Kwong, Jason C / Judd, Louise M / Howden, Benjamin P / Seemann, Torsten / Stinear, Timothy P

Microbiology spectrum

2023 Volume 12, Issue 1, Page(s) e0283423

Abstract: Importance: This proof-of-concept study introduces a hybrid capture oligo panel for whole-genome sequencing of all six human pathogenic hepatitis A virus (HAV) subgenotypes, exhibiting a higher sensitivity than some conventional genotyping assays. The ... ...

Abstract	Importance: This proof-of-concept study introduces a hybrid capture oligo panel for whole-genome sequencing of all six human pathogenic hepatitis A virus (HAV) subgenotypes, exhibiting a higher sensitivity than some conventional genotyping assays. The ability of hybrid capture to enrich multiple targets allows for a single, streamlined workflow, thus facilitating the potential harmonization of molecular surveillance of HAV with other enteric viruses. Even challenging sample matrices can be accommodated, making them suitable for broad implementation in clinical and public health laboratories. This innovative approach has significant implications for enhancing multijurisdictional outbreak investigations as well as our understanding of the global diversity and transmission dynamics of HAV.
MeSH term(s)	Humans ; Hepatitis A virus/genetics ; Hepatitis A/epidemiology ; Whole Genome Sequencing ; Disease Outbreaks ; Chromosome Mapping
Language	English
Publishing date	2023-11-29
Publishing country	United States
Document type	Journal Article
ZDB-ID	2807133-5
ISSN	2165-0497 ; 2165-0497
ISSN (online)	2165-0497
ISSN	2165-0497
DOI	10.1128/spectrum.02834-23
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Article ; Online: Functional potential and evolutionary response to long-term heat selection of bacterial associates of coral photosymbionts.

Maire, Justin / Philip, Gayle K / Livingston, Jadzia / Judd, Louise M / Blackall, Linda L / van Oppen, Madeleine J H

mSystems

2023 Volume 8, Issue 6, Page(s) e0086023

Abstract: Importance: Symbiotic microorganisms are crucial for the survival of corals and their resistance to coral bleaching in the face of climate change. However, the impact of microbe-microbe interactions on coral functioning is mostly unknown but could be ... ...

Abstract	Importance: Symbiotic microorganisms are crucial for the survival of corals and their resistance to coral bleaching in the face of climate change. However, the impact of microbe-microbe interactions on coral functioning is mostly unknown but could be essential factors for coral adaption to future climates. Here, we investigated interactions between cultured dinoflagellates of the Symbiodiniaceae family, essential photosymbionts of corals, and associated bacteria. By assessing the genomic potential of 49 bacteria, we found that they are likely beneficial for Symbiodiniaceae, through the production of B vitamins and antioxidants. Additionally, bacterial genes involved in host-symbiont interactions, such as secretion systems, accumulated mutations following long-term exposure to heat, suggesting symbiotic interactions may change under climate change. This highlights the importance of microbe-microbe interactions in coral functioning.
MeSH term(s)	Animals ; Anthozoa/genetics ; Coral Reefs ; Hot Temperature ; Adaptation, Physiological ; Bacteria/genetics
Language	English
Publishing date	2023-11-01
Publishing country	United States
Document type	Journal Article
ISSN	2379-5077
ISSN (online)	2379-5077
DOI	10.1128/msystems.00860-23
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Article ; Online: Performance of neural network basecalling tools for Oxford Nanopore sequencing.

Wick, Ryan R / Judd, Louise M / Holt, Kathryn E

Genome biology

2019 Volume 20, Issue 1, Page(s) 129

Abstract: Background: Basecalling, the computational process of translating raw electrical signal to nucleotide sequence, is of critical importance to the sequencing platforms produced by Oxford Nanopore Technologies (ONT). Here, we examine the performance of ... ...

Abstract	Background: Basecalling, the computational process of translating raw electrical signal to nucleotide sequence, is of critical importance to the sequencing platforms produced by Oxford Nanopore Technologies (ONT). Here, we examine the performance of different basecalling tools, looking at accuracy at the level of bases within individual reads and at majority-rule consensus basecalls in an assembly. We also investigate some additional aspects of basecalling: training using a taxon-specific dataset, using a larger neural network model and improving consensus basecalls in an assembly by additional signal-level analysis with Nanopolish. Results: Training basecallers on taxon-specific data results in a significant boost in consensus accuracy, mostly due to the reduction of errors in methylation motifs. A larger neural network is able to improve both read and consensus accuracy, but at a cost to speed. Improving consensus sequences ('polishing') with Nanopolish somewhat negates the accuracy differences in basecallers, but pre-polish accuracy does have an effect on post-polish accuracy. Conclusions: Basecalling accuracy has seen significant improvements over the last 2 years. The current version of ONT's Guppy basecaller performs well overall, with good accuracy and fast performance. If higher accuracy is required, users should consider producing a custom model using a larger neural network and/or training data from the same species.
MeSH term(s)	Klebsiella pneumoniae ; Nanopores ; Neural Networks, Computer ; Sequence Analysis, DNA/methods ; Software
Language	English
Publishing date	2019-06-24
Publishing country	England
Document type	Evaluation Study ; Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2040529-7
ISSN	1474-760X ; 1465-6906
ISSN (online)	1474-760X
ISSN	1465-6906
DOI	10.1186/s13059-019-1727-y
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Article: Genomic Diversity and Antimicrobial Resistance of Haemophilus Colonizing the Airways of Young Children with Cystic Fibrosis.

Watts, Stephen C / Judd, Louise M / Carzino, Rosemary / Ranganathan, Sarath / Holt, Kathryn E

mSystems

2021 , Page(s) e0017821

Abstract: Respiratory infection during childhood is a key risk factor in early cystic fibrosis (CF) lung disease progression. Haemophilus influenzae and Haemophilus parainfluenzae are routinely isolated from the lungs of children with CF; however, little is known ... ...

Abstract	Respiratory infection during childhood is a key risk factor in early cystic fibrosis (CF) lung disease progression. Haemophilus influenzae and Haemophilus parainfluenzae are routinely isolated from the lungs of children with CF; however, little is known about the frequency and characteristics of Haemophilus colonization in this context. Here, we describe the detection, antimicrobial resistance (AMR), and genome sequencing of H. influenzae and
Language	English
Publishing date	2021-08-31
Publishing country	United States
Document type	Journal Article
ISSN	2379-5077
ISSN	2379-5077
DOI	10.1128/mSystems.00178-21
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