Nanopore long reads enable the first complete genome assembly of a Malaysian <i>Vibrio parahaemolyticus</i> isolate bearing the pVa plasmid associated with acute hepatopancreatic necrosis disease

Han Ming Gan; Christopher M Austin

doi:10.12688/f1000research.21570.1

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Research Article

Nanopore long reads enable the first complete genome assembly of a Malaysian Vibrio parahaemolyticus isolate bearing the pVa plasmid associated with acute hepatopancreatic necrosis disease

[version 1; peer review: 1 approved with reservations, 1 not approved]

Han Ming Gan ^1-3, Christopher M Austin^1-3

PUBLISHED 16 Dec 2019

Author details Author details

¹ Centre for Integrative Ecology, School of Life and Environmental Sciences,, Deakin University, Geelong, Victoria, 3220, Australia
² Deakin Genomics Centre, Deakin University, Geelong, Victoria, 3220, Australia
³ School of Science, Monash University Malaysia, Petaling Jaya, Selangor, 47500, Malaysia

Han Ming Gan
Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Resources, Software, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Christopher M Austin
Roles: Funding Acquisition, Supervision, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Pathogens gateway.

This article is included in the Nanopore Analysis gateway.

Abstract

Background: The genome of Vibrio parahaemolyticus MVP1, isolated from a Malaysian aquaculture farm with shrimp acute hepatopancreatic necrosis disease (AHPND), was previously sequenced using Illumina MiSeq and assembled de novo, producing a relatively fragmented assembly. Despite identifying the binary toxin genes in the MVP1 draft genome that were linked to AHPND, the toxin genes were localized on a very small contig precluding proper analysis of gene neighbourhood.
Methods: The genome of MVP1 was sequenced on Nanopore MinION to obtain long reads to improve genome contiguity. De novo genome assembly was performed using long-read only assembler followed by genome polishing and hybrid assembler.
Results: Long-read assembly produced three complete circular MVP1 contigs: chromosome 1, chromosome 2 and the pVa plasmid encoding pirAB^vp binary toxin genes. Polishing of the long-read assembly with Illumina short reads was necessary to remove indel errors. Complete assembly of the pVa plasmid could not be achieved using Illumina reads due to identical repetitive elements flanking the binary toxin genes leading to multiple contigs. These regions were fully spanned by the Nanopore long-reads resulting in a single contig. Alignment of Illumina reads to the complete genome assembly indicated there is sequencing bias as read depth was lowest in low-GC genomic regions. Comparative genomic analysis revealed a gene cluster coding for additional insecticidal toxins in chromosome 2 of MVP1 that may further contribute to host pathogenesis pending functional validation. Scanning of publicly available V. parahaemolyticus genomes revealed the presence of a single AinS-family quorum-sensing system that can be targeted for future microbial management.
Conclusions: We generated the first chromosome-scale genome assembly of a Malaysian pirAB^Vp-bearing V. parahaemolyticus isolate. Structural variations identified from comparative genomic analysis provide new insights into the genomic features of V. parahaemolyticus MVP1 that may be associated with host colonization and pathogenicity.

Keywords

Vibrio parahaemolyticus, Nanopore, Illumina, quorum-sensing, shrimp, Acute Hepatopancreatic Necrosis Disease

Corresponding author: Han Ming Gan

Competing interests: No competing interests were disclosed.

Grant information: The author(s) declared that no grants were involved in supporting this work.

Copyright: © 2019 Gan HM and Austin CM. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Gan HM and Austin CM. Nanopore long reads enable the first complete genome assembly of a Malaysian Vibrio parahaemolyticus isolate bearing the pVa plasmid associated with acute hepatopancreatic necrosis disease [version 1; peer review: 1 approved with reservations, 1 not approved]. F1000Research 2019, 8:2108 (https://doi.org/10.12688/f1000research.21570.1) First published: 16 Dec 2019, 8:2108 (https://doi.org/10.12688/f1000research.21570.1) Latest published: 16 Dec 2019, 8:2108 (https://doi.org/10.12688/f1000research.21570.1)

Introduction

Vibrio parahaemolyticus is a marine gram-negative Enterobacteriaceae that has been recognized as an important pathogen affecting commercially-relevant shrimp species such as the giant tiger prawn (Penaeus monodon) and the Pacific white shrimp (Litopenaeus vannamei)^1–3. In recent years, V. parahaemolyticus has been linked to acute hepatopancreatic necrosis disease (AHPND)^4,5. An AHPND-causing V. parahaemolyticus isolate is thought to produce a homolog of the insecticidal Photorhabdus insect-related (Pir) binary toxin that induces prolonged damage to the shrimp tissue². AHPND preferentially affects shrimp post-larvae or juveniles with nearly 100% mortality rate within 30–35 days post-stocking⁶. Studies suggest that AHPND originated from China in 2009 and subsequently spread to South East Asia⁷. In Malaysia, AHPND was first reported in 2011 where it still persists in shrimp farms⁸.

The implementation of effective microbial management in aquaculture requires genomic surveillance data that can provide accurate information regarding the geographical origin, virulence factors and antibiotic resistance of a sequenced microbial strain⁹. In Malaysia, this remains challenging due to the paucity of available genomic resources for the relevant pathogens. To date, a majority of the microbial genomic studies in Malaysia consist of single draft genome reports mostly focusing on clinical pathogens without substantial comparative genomics^10–13. However, recent years have seen a modest increase in the number of studies with more comprehensive sequencing dataset and analysis^14–16. For example, Yan et al. sequenced and assembled the draft genomes of 40 Malaysian V. parahaemolyticus isolates associated with shrimp aquaculture and performed comparative genomics of more than Asian 100 V. parahaemolyticus genomes from public databases. In-silico multi-locus sequencing typing and phylogenomic analyses indicate that several Malaysian V. parahaemolyticus isolates belong to previously undescribed sequence types and genomic lineages¹⁷ and recommended further studies be undertaken.

The pirAB^vp genes coding for AHPND-causing binary toxins are localized on a 70-kb pVa plasmid². Surprisingly, despite the small size of the plasmid relative to the chromosomal genome, a complete de novo assembly of the pVa plasmid using Illumina-only short reads has not been achieved to date. Recent studies suggested that this is due to the presence of repetitive elements on the pVa plasmid that are longer than the Illumina read length¹⁸. As a result, pirAB-containing contigs that were assembled from Illumina reads are generally short with limited gene content, precluding detailed analysis of the pirAB gene neighborhood and their stability in the plasmidome¹⁹. V. parahaemolyticus isolate MVP1 is one of the first pirAB^Vp -harboring V. parahaemolyticus isolated from Malaysia to have its genome sequenced¹¹. Isolate MVP1 was previously sequenced on an Illumina MiSeq followed by de novo genome assembly using the SPAdes assembler. While the pirAB^vp genes could be identified in the draft genome, the assembly was problematic as these were localized on a short contig flanked by small fragments of a transposase gene.

Recent years have seen the democratization of long-read sequencing enabled by Nanopore technology. In contrast to another popular long-read sequencing platform, PacBio, Nanopore sequencing requires minimal lab footprint and very low capital investment. Importantly, it is the first sequencing technology that allows native sequencing, thus eliminating sequencing bias associated with the activity of Taq-polymerase. Long reads have been utilized for de novo genome assembly in two distinct ways. First, long reads can be used directly in genome assembly followed by polishing with Illumina short reads to improve consensus accuracy²⁰. An alternative approach utilizes long-reads to reorder and link contigs that were initially assembled from Illumina reads^21,22. While the final assembly produced with this hybrid approach is highly accurate, the contiguity of the assembly is dependent on the quality of the initial Illumina assembly²³.

Leveraging on the availability of Illumina dataset for isolate MVP1, we aimed to improve on the original genome assembly¹¹ through the addition of 40× Nanopore read coverage. We first performed a Nanopore-led assembly using the long-read Flye assembler²⁴ followed by polishing with Illumina reads. In addition, we generated a hybrid assembly using Unicycler assembler²³. Although both methods produced a complete circular pVa plasmid sequence with intact pirAB^vp, complete chromosome assembly was only achieved with the Nanopore-led Flye assembly. This superior assembly provides new insight into the genomic features of V. parahaemolyticus MVP1 and will allow a greater understanding of host pathogenicity through comparative genomic analysis with selected V. parahaemolyticus isolates.

Methods

Nanopore sequencing

Sample collection, gDNA isolation and Illumina sequencing of V. parahaemolyticus isolate MVP1 have been previously reported¹¹. For Nanopore sequencing, 1 μg of unfragmented gDNA was processed using the now obsolete Nanopore SQK-NSK007 library preparation kit. Sequencing was subsequently performed on an R9 flowcell attached to a MinION device for 48 hours. Base-calling of the produced raw fast5 files used Guppy v3.1 (high accuracy mode; requires registration to Oxford Nanopore Technology community site).

De novo genome assembly and genome polishing

Illumina-only assembly and hybrid assembly incorporating high-quality (q>7) Nanopore long reads were performed using Unicycler v.0.4.7²³. Only contigs equal or larger than 1,000 bp were retained for subsequent analyses. A long-read only de novo assembly was also performed with Flye v2.4.2 utilizing similar Nanopore sequencing data²⁴. Illumina polishing of the Flye assembly used the unicycler polish tool in Unicycler v.0.4.7 that performed multiple rounds of bwa-mem-based Illumina read alignment to the assembly followed by polishing with Pilon v1.22²⁵. Genome completeness was assessed with BUSCO v3 (Gammaproteobacteria odb9) based on the whole proteome from each genome assembly that was predicted using Prodigal v 2.6.3 (default setting)^26,27.

Comparative Genomic analysis and protein homology modeling

Automated annotation of the whole-genome was carried out using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP)²⁸. The translated coding sequences generated from PGAP were subject to additional functional annotation using InterProScan. Intra-chromosomal structural variations and sequence similarity were visualized with BLAST ring image generator (BRIG) using the blastN (e-value = 0.001 cutoff). Sequencing depth was also calculated in BRIG based on the BAM alignment file generated from the bwa-mem mapping of Illumina paired-end reads to the whole-genome²⁹. Structural modeling of the putative insecticidal toxin proteins identified in MVP1 used the online Phyre2 webserver³⁰.

Identification and phylogenetic analysis of LuxM, OpaM and AinS proteins

Vibrio proteins containing the InterPro signature IPR035304, corresponding to the AinS-family N-acyl-homoserine-lactone autoinducer synthase, were downloaded from the UniProt database³¹ on the 7^th of November 2019 and clustered at 98% amino acid identity cutoff using cd-hit v4.6³². The protein clusters were subsequently aligned with MAFFT v7.31³³ (”--maxiterate 1000 –localpair” setting) followed by maximum likelihood tree construction using FastTree v2.1³⁴. Calculation and visualization of pairwise protein sequence divergence used SDT v1.2³⁵. In addition, to confirm the absence of the LuxI-type autoinducer synthase homolog in the Vibrio parahaemolyticus proteomes, additional domain search specifically for PF00765 (LuxI autoinducer synthase domain) was performed followed by subsequent InterProScan validation as previously described³⁶.

Results and discussion

Chromosomal assembly enabled by Nanopore long reads

A total of 231 megabases of Nanopore data contained in 30,315 reads (N50 = 10,908 bp) were produced from a single Nanopore MINION run on the discontinued R9 flowcell. The base-called Nanopore fastq file has been deposited in the SRA database under the accession code SRX6759854. An initial Unicycler assembly using previously generated Illumina reads (SRX6759853) yielded a relatively fragmented genome (198 contigs; N50 length = 54,109 bp). Supplementing the assembler with approximately 40 x coverage of Nanopore long reads led a substantial improvement in the Unicycler assembly contiguity (9 contigs; N50 length = 1,738,848 bp). Of the 9 assembled contigs, one of them was flagged as “complete” with a contig length of 70kb. On the other hand, the Flye assembly using only Nanopore reads produced three contigs all flagged as “complete and circular”. Despite generating the most contiguous assembly, the initial Flye assembly has the worst BUSCO score with 37.4% BUSCO single-copy and 45.1% fragmented BUSCO genes (Table 1). Polishing of the Flye assembly with Illumina paired-end reads restored the genome completeness to a level that is comparable to the hybrid and Illumina-only assemblies (Table 1 and Extended data: Supplemental File 1). The high percentage of fragmented genes in long-read-only assembly is usually a result of erroneous frameshifts from indel sequencing errors in the long reads²⁰. Although higher coverage of long read may lead to an increase the consensus accuracy; its accuracy will unlikely match that of an Illumina-polished assembly due to Nanopore-specific systematic errors. As a result, polishing of long-read assembly using Illumina short reads can be considered as a cost-effective but less convenient approach for producing highly accurate and contiguous microbial genome assembly.

Table 1. Statistics and BUSCO completeness assessment of the Vibrio parahaemolyticus MVP1 assembly.

Assembler (input data)	Genome Length (bp)	GC%	# contigs	N50 (bp)	BUSCO (gammaproteobacteria_odb9, n=452)
Assembler (input data)	Genome Length (bp)	GC%	# contigs	N50 (bp)	Single-copy	Duplicated	Fragmented	Missing
Unicycler (Illumina)	5,167,326	45.47%	198	54,109	98.9	0.9	0.2	0
Unicycler (Hybrid)	5,378,063	45.34%	9	1,738,848	98.9	0.9	0.2	0
Flye (Nanopore)	5,405,284	45.23%	3	3,456,517	37.4	0	45.1	17.5
Flye+Pilon (Nanopore+Illumina)	5,389,211	45.30%	3	3,446,010	99.1	0.7	0.2	0

Reduced Illumina sequencing depth in several low-GC genomic regions

The alignment of Illumina reads to the final chromosomal assembly revealed substantial lower sequencing depths in genomic regions with low GC-content (<30%) (Figure 1). Some of these regions are unique to MVP1 and may risk being overlooked in the Illumina-only assembly at lower sequencing coverage. For example, less than 5x sequencing depth was observed in the region 3,240 to 3,280 kb that contain various putative carbohydrate metabolism genes that are unique to isolate MVP1 in this current genomic comparison. The incorporation of PCR during library preparation is known to reduce the representation of high-AT genomic regions due to the polymerase amplification bias towards GC-balanced fragments³⁷. Reduced Illumina sequencing depth in high-AT genomic regions affecting biological interpretation has also been reported in several crustacean mitogenomic libraries as both the phylogenetically informative control region and 16S rRNA genes are relatively AT-rich^38,39. Depending on the amount of DNA available for sequencing, a few modifications to the Illumina library preparation step can be considered, such as PCR-free preparation for sample with high starting DNA (> 100 ng) and reduced PCR cycle coupled with higher (>100× genome coverage) sequencing depth for samples with low starting DNA.

Figure 1. Genomic comparisons of pVa plasmid-harboring Vibrio parahaemolyticus.

Left, V. parahaemolyticus MVP1 chromosome 1 compared against five other V. parahaemolyticus chromosome 1. Right, V. parahaemolyticus MVP1 chromosome 2 compared against five other V. parahaemolyticus chromosome 2. The innermost rings show GC content while the second innermost rings show Illumina sequencing depth along the genome (blue). The remaining rings indicate BLAST comparisons of other V. parahaemolyticus genomes against MVP genome assembly. Arrows indicate genes of interest and were labeled with the predicted protein names.

Multiple localization of IS5 family transposase genes in the complete genomes of pVa-carrying V. parahaemolyticus

In the Illumina-only assembly, the pVa plasmid was assembled into three contigs (Contig24: 63.4 kb, Contig164: 3.4kb; Contig198: 1 kb). Contig198 contains a 921-bp gene that codes for an intact IS5 family transposase and could be aligned equally well to two genomic regions flanking the pirAB^Vp genes in the complete plasmid assembly (Figure 2). This observation can be explained by the inability of Illumina reads to fully span the repeats thus causing them to be collapsed into a single contig. As a result, the localization of pirAB^Vp on the pVa plasmid cannot be confirmed from an Illumina-only assembly. In contrast the entire pirAB^Vp genes and their flanking transposase genes were fully covered by a number of Nanopore long reads enabling the complete assembly of the pVa plasmid in addition to supporting the localization of pirAB^Vp genes on the pVa plasmid. Nanopore read depth compared to Illumina, is relatively even across the aligned genomic region, which is consistent with the absence of PCR bias in the Nanopore library preparation.

Figure 2. Alignment of Illumina-assembled contigs, Illumina and Nanopore reads to the annotated Vibrio parahaemolyticus MVP1 pVa plasmid sub-region containing the pirAB^Vp genes.

Direction of arrow in the annotation indicates transcription orientation. Blue and red arrows in the Nanopore read alignment indicate forward and reverse strands, respectively.

The minor structural variations previously reported in pirAB^Vp -containing region suggest that these genes are not stably maintained and are prone to transposition mediated by the flanking IS5 family transposase genes⁴⁰. Interestingly, the localization of this IS5 family transposase gene is not specific to just the pVa plasmid. Local nucleotide similarity search of this transposase gene against the complete MVP genome revealed three additional perfect hits (100% query coverage and 100% nucleotide identity) in each of the chromosomal genome (Figure 1). The localization of these additional IS5 family transposase in both chromosome 1 and chromosome 2 further raises the possibility of the IS5 transposase-flanked pirAB^Vp genes being integrated into the chromosomal region^41,42.

Structural variations in the MVP1 genome

Chromosome 2 of Isolate MPV1 consists of a 160 kb genomic region that is mostly absent in five out of six pVa plasmid-harboring V. parahaemolyticus isolates included in this comparative genomic analysis. Functional annotation of the genes located in this region revealed two gene clusters that may further contribute to host pathogenicity in addition to the binary Pir-like toxins on the pVa plasmid. The first gene cluster located from 1,822,499 to 1,836,895 bp in chromosome 2 consists of three relatively large genes (Gene Locus Tag: BSR23_26145, BSR23_26150 and BSR23_26155) coding for another type of putative insecticidal toxins. Functional annotation using InterProScan revealed the presence of protein domains commonly associated with the toxin-complex (Tc) toxins (Figure 3). In addition, Phyre2 protein modeling also indicated their high structural homology to their respective homologous toxin components (Extended data: Supplemental Files 2–4). In Xenorhabdus nematophilus, the three toxin components formed a native toxin complex and the ingestion of this complex led to growth inhibition of two different insect larvae, Helicoverpa zea and Heliothis virescens⁴³. In addition, further tests showed that the native toxin complex is capable of binding to solubilized gut membranes of H. zea larvae in addition to inducing pore formation in black lipid membranes⁴³. Given the functional resemblance of such toxins to the Pir-like binary toxins, the presence of all three genes coding for the complete set of insecticidal Tc toxins in V. parahaemolyticus may contribute to mortality without AHPND lesions in shrimps, as previously reported⁴⁴.

Figure 3. InterProScan identification of conserved protein domains in the putative toxin complex family proteins coded by three linked genes located on Chromosome 2 of Vibrio parahaemolyticus MVP1.

Interestingly, isolate MVP1 also harbor a fuc operon that is not commonly present in members of this species (Extended data: Supplemental Table 1). The presence of this operon in MVP1 translates into the genomic potential for the internalization of fucose for incorporation into the capsular polysaccharides or bacterial glycoproteins. While it is also possible that the fuc operon may contribute to improved utilization of fucose as an energy source, Williams et al. did not observe a greater capacity to utilize fucose in an EMS-causing V. parahaemolyticus isolate 13-028/A3⁴⁵.

High prevalence and divergence of the LuxM/OpaM/AinS family autoinducer synthesis proteins among Vibrio spp

NCBI annotation of MVP1 proteome revealed the presence of only the LuxM/OpaM-type AHL synthase. An in-house HMMsearch for the LuxI autoinducer synthase domain (PFAM signature: PF00765) in the MVP1 proteome also did not reveal significant hits, indicating that MVP1 employs a single system for the biosynthesis of AHL signal (Extended Data: Supplemental File 5). Additional UniProt query search of “PF00765 AND Vibrio parahaemolyticus” on 14^th November 2019, showed only one positive hit across all currently available V. parahaemolyticus proteomes. However, further investigation of the positive hit, AAY51_09590, revealed that this belonged to a Citrobacter spp. previously misclassified as V. parahaemolyticus⁴⁶. In contrast, V. harveyi was shown to utilize at least two quorum-sensing systems e.g. LuxI that synthesizes 3-oxo-C6-HSL and the AinS that synthesizes C8-HSL⁴⁷. Despite being classified as members of the same family for sharing the similar protein domain signature, AinS exhibited a substantially lower pairwise amino acid divergence (~ 30%) in comparison to LuxM and OpaM. By rooting the LuxM/OpaM phylogenetic tree with the AinS from Aliivibrio fischeri as the outgroup, we observed a strongly supported monophyletic cluster consisting of the functionally validated LuxM and OpaM in addition to their homologs from other Vibrio spp (Figure 4A). The pairwise amino acid identity among members in this LuxM/OpaM clade averages around 50% (Figure 4B), consistent with the relatively short branch length among members of the clade.

Figure 4. Phylogenetic analysis and sequence divergence of the AinS-like family proteins in Vibrio spp.

(A) Maximum likelihood tree depicting the evolutionary relationships of AinS-like family protein clusters among Vibrio spp. Number after the “=” sign in each tip label indicates the number of proteins represented by the cluster. Node were colored according to the SH-like local support values and branch lengths indicate number of substitutions per site. (B) Pairwise identity matrix of the identified AinS-like family proteins in Vibrio spp.

In Indian white shrimps (Fenneropenaeus indicus), co-injection of a pathogenic V. parahaemolyticus strain DHAP1 and purified recombinant AHL lactonase, AiiA, reduced Vibrio viable counts and biofilm development in the shrimp intestine, suggesting the role of AHL-mediated quorum-sensing system in host colonization⁴⁸. While the presence of intact opaM in several sequenced V. parahaemolyticus isolates supports their ability to accumulate AHL and engage in quorum-sensing activity, this is best validated using a simple biosensor or LC/MS approach that can not only verify the AHL producing phenotype but also provide insight into the structural diversity of AHL signals^49–51. Furthermore, elucidating the role of opaM in mediating host colonization and pathogenesis among AHPND-causing V. parahaemolyticus via transposon mutagenesis will be instructive.

Conclusions

Using approximately 40× genome coverage of Nanopore long reads, we produced the first chromosome-scale genome assembly of a Malaysian pirAB^Vp-bearing V. parahaemolyticus isolate. Although the genome completeness of the initial Flye assembly was relatively poor, polishing of the genome assembly with Illumina reads improved its completeness to a level that is comparable to a high-quality microbial genome assembly. Structural variations identified from genomic comparisons provide new insights into the genomic features of V. parahaemolyticus MVP1 that may be associated with host colonization and pathogenicity.

Data availability

Underlying data

Vibrio parahaemolyticus strain MVP1 chromosome 1, complete sequence, Accession number CP043421: https://www.ncbi.nlm.nih.gov/nuccore/CP043421

Vibrio parahaemolyticus strain MVP1 chromosome 2, complete sequence, Accession number CP043422: https://www.ncbi.nlm.nih.gov/nuccore/CP043422

Vibrio parahaemolyticus strain MVP1 plasmid pVa, complete sequence, Accession number CP043423: https://www.ncbi.nlm.nih.gov/nuccore/CP043423

Raw Illumina reads and basecalled Nanopore Reads have also been deposited in NCBI Sequence Read Archive under the BioProject PRJNA355061.

Extended data

Zenodo: Nanopore long reads enable the first complete genome assembly of a Malaysian Vibrio parahaemolyticus isolate bearing the pVa plasmid associated with acute hepatopancreatic necrosis disease, http://doi.org/10.5281/zenodo.3568485⁵².

This project contains the following extended data:

Supplemental File 1: Main genome assemblies (Unpolished Flye assembly, Polished Flye assembly, Unicycler Hybrid Assembly and Unicycler Illumina-only assembly) generated in this study for comparison and their BUSCO output.
Supplemental File 2: Phyre2 protein modeling output of the putative MVP1 TcdA toxin
Supplemental File 3: Phyre2 protein modeling output of the putative MVP1 TcdB toxin
Supplemental File 4: Phyre2 protein modeling output of the putative MVP1 TccC toxin
Supplemental File 5: InterProScan output of the NCBI-predicted MVP1 proteome.
Supplemental Table 1: NCBI BlastN output using the fuc genes of Vibrio parahaemolyticus MVP1 as the query to search against the Vibrio reference WGS database as of 21 Oct 2019.

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Faculty Opinions recommended

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25. Walker BJ, Abeel T, Shea T, et al.: Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One. 2014; 9(11): e112963. PubMed Abstract | Publisher Full Text | Free Full Text
26. Waterhouse RM, Seppey M, Simão FA, et al.: BUSCO Applications from Quality Assessments to Gene Prediction and Phylogenomics. Mol Biol Evol. 2017; 35(3): 543–8. PubMed Abstract | Publisher Full Text | Free Full Text
27. Hyatt D, Chen GL, Locascio PF, et al.: Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 2010; 11(1): 119. PubMed Abstract | Publisher Full Text | Free Full Text
28. Tatusova T, DiCuccio M, Badretdin A, et al.: NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res. 2016; 44(14): 6614–24. PubMed Abstract | Publisher Full Text | Free Full Text
29. Alikhan NF, Petty NK, Zakour NLB, et al.: BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genomics. 2011; 12(1): 402. PubMed Abstract | Publisher Full Text | Free Full Text
30. Kelley LA, Mezulis S, Yates CM, et al.: The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc. 2015; 10(6): 845–58. PubMed Abstract | Publisher Full Text | Free Full Text
31. UniProt Consortium: UniProt: a hub for protein information. Nucleic Acids Res. 2015; 43(Database issue): D204–D12. PubMed Abstract | Publisher Full Text | Free Full Text
32. Fu L, Niu B, Zhu Z, et al.: CD-HIT: accelerated for clustering the next-generation sequencing data. Bioinformatics. 2012; 28(23): 3150–2. PubMed Abstract | Publisher Full Text | Free Full Text
33. Katoh K, Standley DM: MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013; 30(4): 772–80. PubMed Abstract | Publisher Full Text | Free Full Text
34. Price MN, Dehal PS, Arkin AP: FastTree 2--approximately maximum-likelihood trees for large alignments. PLoS One. 2010; 5(3): e9490. PubMed Abstract | Publisher Full Text | Free Full Text
35. Muhire BM, Varsani A, Martin DP: SDT: a virus classification tool based on pairwise sequence alignment and identity calculation. PLoS One. 2014; 9(9): e108277. PubMed Abstract | Publisher Full Text | Free Full Text
36. Gan HM, Gan HY, Ahmad NH, et al.: Whole genome sequencing and analysis reveal insights into the genetic structure, diversity and evolutionary relatedness of luxI and luxR homologs in bacteria belonging to the Sphingomonadaceae family. Front Cell Infect Microbiol. 2015; 4: 188. PubMed Abstract | Publisher Full Text | Free Full Text
37. Krehenwinkel H, Wolf M, Lim JY, et al.: Estimating and mitigating amplification bias in qualitative and quantitative arthropod metabarcoding. Sci Rep. 2017; 7(1): 17668. PubMed Abstract | Publisher Full Text | Free Full Text
38. Gan HM, Grandjean F, Jenkins TL, et al.: Absence of evidence is not evidence of absence: Nanopore sequencing and complete assembly of the European lobster (Homarus gammarus) mitogenome uncovers the missing nad2 and a new major gene cluster duplication. BMC Genomics. 2019; 20(1): 335. PubMed Abstract | Publisher Full Text | Free Full Text
39. Gan HM, Linton SM, Austin CM: Two reads to rule them all: Nanopore long read-guided assembly of the iconic Christmas Island red crab, Gecarcoidea natalis (Pocock, 1888), mitochondrial genome and the challenges of AT-rich mitogenomes. Mar Genomics. 2019; 45: 64–71. PubMed Abstract | Publisher Full Text
40. Xiao J, Liu L, Ke Y, et al.: Shrimp AHPND-causing plasmids encoding the PirAB toxins as mediated by pirAB-Tn903 are prevalent in various Vibrio species. Sci Rep. 2017; 7: 42177. PubMed Abstract | Publisher Full Text | Free Full Text
41. Mahillon J, Chandler M: Insertion sequences. Microbiol Mol Biol Rev. 1998; 62(3): 725–74. PubMed Abstract | Free Full Text
42. Skipper KA, Andersen PR, Sharma N, et al.: DNA transposon-based gene vehicles - scenes from an evolutionary drive. J Biomed Sci. 2013; 20(1): 92. PubMed Abstract | Publisher Full Text | Free Full Text
43. Sheets JJ, Hey TD, Fencil KJ, et al.: Insecticidal toxin complex proteins from Xenorhabdus nematophilus: structure and pore formation. J Biol Chem. 2011; 286(26): 22742–9. PubMed Abstract | Publisher Full Text | Free Full Text
44. Phiwsaiya K, Charoensapsri W, Taengphu S, et al.: A Natural Vibrio parahaemolyticus ΔpirA^Vp pirB^Vp+ Mutant Kills Shrimp but Produces neither Pir ^Vp Toxins nor Acute Hepatopancreatic Necrosis Disease Lesions. Appl Environ Microbiol. 2017; 83(16): pii: e00680-17. PubMed Abstract | Publisher Full Text | Free Full Text
45. Williams SL, Jensen RV, Kuhn DD, et al.: Analyzing the metabolic capabilities of a Vibrio parahaemolyticus strain that causes early mortality syndrome in shrimp. Aquaculture. 2017; 476: 44–8. Publisher Full Text
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52. Gan HM, Austin C: Nanopore long reads enable the first complete genome assembly of a Malaysian Vibrio parahaemolyticus isolate bearing the pVa plasmid associated with acute hepatopancreatic necrosis disease [Data set]. Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3568485

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Author details Author details

¹ Centre for Integrative Ecology, School of Life and Environmental Sciences,, Deakin University, Geelong, Victoria, 3220, Australia
² Deakin Genomics Centre, Deakin University, Geelong, Victoria, 3220, Australia
³ School of Science, Monash University Malaysia, Petaling Jaya, Selangor, 47500, Malaysia

Han Ming Gan
Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Resources, Software, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Christopher M Austin
Roles: Funding Acquisition, Supervision, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

The author(s) declared that no grants were involved in supporting this work.

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Gan HM and Austin CM. Nanopore long reads enable the first complete genome assembly of a Malaysian Vibrio parahaemolyticus isolate bearing the pVa plasmid associated with acute hepatopancreatic necrosis disease [version 1; peer review: 1 approved with reservations, 1 not approved]. F1000Research 2019, 8:2108 (https://doi.org/10.12688/f1000research.21570.1)

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Reviewer Report 10 Mar 2020

Tomoo Sawabe, Laboratory of Microbiology, Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Japan

Not Approved

https://doi.org/10.5256/f1000research.23769.r60806

AHPND is one of the important threats for the shrimp aquaculture industry to be prevented worldwide. The authors succeeded to have the complete genome of a Vibrio parahaemolyticus strain MVP1 isolated from a Malaysian aquaculture farm. The study is so ... Continue reading

AHPND is one of the important threats for the shrimp aquaculture industry to be prevented worldwide. The authors succeeded to have the complete genome of a Vibrio parahaemolyticus strain MVP1 isolated from a Malaysian aquaculture farm. The study is so important but studies on AHPND should be moved to studies on population dynamics based on complete genomes.

Nanopore and Illumina hybrid assemblies are available commercially. The cost-effective methodology can achieve dozens of strains to be sequenced completely. The current manuscript is likely to trace such published known reports. In this aspect, the manuscript is unlikely to be technically sound.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

No source data required
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

CITE

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Reviewer Report 17 Feb 2020

Ballamoole Krishna Kumar, Nitte University Center for Science Education and Research (NUCSER), Mangalore, Karnataka, India

Approved with Reservations

https://doi.org/10.5256/f1000research.23769.r59964

V. para is not a member of Enterobacteriaceae, that needs correction. It is a Gram‐negative, halophilic bacterium belonging to the family of Vibrionaceae. Basically, this pathogen is the leading cause of seafood‐borne gastroenteritis due to the consumption of raw seafood, not the true pathogen of ... Continue reading

V. para is not a member of Enterobacteriaceae, that needs correction. It is a Gram‐negative, halophilic bacterium belonging to the family of Vibrionaceae. Basically, this pathogen is the leading cause of seafood‐borne gastroenteritis due to the consumption of raw seafood, not the true pathogen of cultured shrimps. However, this bacterium has recently gained importance following the identification of certain strains of Vibrio parahaemolyticus as the causative agent that caused large scale losses in farmed shrimp production in several parts of the world.

I am not clear about the true source of MVP1 used as no clear description is provided. Is it isolated from the surface of the infected animal or from haemolymph or hepatopancreas or from soil and water of the shrimp pond that was tested positive for AHPND?

What is the evidence that exists to show that authors have used unfragmented gDNA for library preparation?

The genome of Vibrio parahaemolyticus MVP1 is how much comparable to the Reference strain?

Additional insecticidal toxin gene generated in the chromosome of MVP1 - does it present in the strains used for the comparative analysis? Is it part of any VpaI identified previously? Do you think just mere presence of such genes would precipitate in the disease/pathogenesis? Could the presence of this gene be linked to demonstrable traits among the isolates?

What was the rationale behind the analysis of LuxM/OpaM/AinS family autoinducer?

The reference list is biased to the more recent articles published by members of the research group and few older references of others. An up to date literature review is clearly needed.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: I am a microbiologist working on vibrio pathogenesis.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

CITE

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Version 1 16 Dec 19	read	read

Ballamoole Krishna Kumar, Nitte University Center for Science Education and Research (NUCSER), Mangalore, India
Tomoo Sawabe, Hokkaido University, Hakodate, Japan

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Reviewer Report

12 Views

10 Mar 2020 | for Version 1

Tomoo Sawabe, Laboratory of Microbiology, Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Japan

12 Views Cite this report Responses(0)

Not Approved

AHPND is one of the important threats for the shrimp aquaculture industry to be prevented worldwide. The authors succeeded to have the complete genome of a Vibrio parahaemolyticus strain MVP1 isolated from a Malaysian aquaculture farm. The study is so important but studies on AHPND should be moved to studies on population dynamics based on complete genomes.

Nanopore and Illumina hybrid assemblies are available commercially. The cost-effective methodology can achieve dozens of strains to be sequenced completely. The current manuscript is likely to trace such published known reports. In this aspect, the manuscript is unlikely to be technically sound.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

No source data required
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

Respond to this report

Responses (0)

Back to all reports

Reviewer Report

15 Views

17 Feb 2020 | for Version 1

Ballamoole Krishna Kumar, Nitte University Center for Science Education and Research (NUCSER), Mangalore, Karnataka, India

15 Views Cite this report Responses(0)

Approved With Reservations

V. para is not a member of Enterobacteriaceae, that needs correction. It is a Gram‐negative, halophilic bacterium belonging to the family of Vibrionaceae. Basically, this pathogen is the leading cause of seafood‐borne gastroenteritis due to the consumption of raw seafood, not the true pathogen of cultured shrimps. However, this bacterium has recently gained importance following the identification of certain strains of Vibrio parahaemolyticus as the causative agent that caused large scale losses in farmed shrimp production in several parts of the world.

I am not clear about the true source of MVP1 used as no clear description is provided. Is it isolated from the surface of the infected animal or from haemolymph or hepatopancreas or from soil and water of the shrimp pond that was tested positive for AHPND?

What is the evidence that exists to show that authors have used unfragmented gDNA for library preparation?

The genome of Vibrio parahaemolyticus MVP1 is how much comparable to the Reference strain?

Additional insecticidal toxin gene generated in the chromosome of MVP1 - does it present in the strains used for the comparative analysis? Is it part of any VpaI identified previously? Do you think just mere presence of such genes would precipitate in the disease/pathogenesis? Could the presence of this gene be linked to demonstrable traits among the isolates?

What was the rationale behind the analysis of LuxM/OpaM/AinS family autoinducer?

The reference list is biased to the more recent articles published by members of the research group and few older references of others. An up to date literature review is clearly needed.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

I am a microbiologist working on vibrio pathogenesis.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Respond to this report

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[2] 2. Lee CT, Chen IT, Yang YT, et al.: The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin. Proc Natl Acad Sci U S A. 2015; 112(34): 10798–803. PubMed Abstract | Publisher Full Text | Free Full Text

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[13] 13. Gan HM, Rajasekaram G, Eng WWH, et al.: Whole-Genome Sequences of Two Carbapenem-Resistant Klebsiella quasipneumoniae Strains Isolated from a Tertiary Hospital in Johor, Malaysia. Genome Announc. 2017; 5(32): pii: e00768-17. PubMed Abstract | Publisher Full Text | Free Full Text

[14] 14. Gan HM, Eng WWH, Dhanoa A: Data on whole-genome sequencing of extended-spectrum beta-lactamases producing Enterobacteriaceae isolates from Malaysia. Data Brief. 2019; 25: 104257. PubMed Abstract | Publisher Full Text | Free Full Text

[15] 15. Gan HM, Eng WWH, Dhanoa A: First genomic insights into carbapenem-resistant Klebsiella pneumoniae from Malaysia. J Glob Antimicrob Resist. 2019; pii: S2213-7165(19)30174-2. PubMed Abstract | Publisher Full Text

[16] 16. Yap KP, Ho WS, Gan HM, et al.: Global MLST of Salmonella Typhi revisited in post-genomic era: genetic conservation, population structure, and comparative genomics of rare sequence types. Front Microbiol. 2016; 7: 270. PubMed Abstract | Publisher Full Text | Free Full Text

[17] 17. Yan CZY, Austin CM, Ayub Q, et al.: Genomic characterization of Vibrio parahaemolyticus from Pacific white shrimp and rearing water in Malaysia reveals novel sequence types and structural variation in genomic regions containing the Photorhabdus insect-related (Pir) toxin-like genes. FEMS Microbiol Lett. 2019; 366(17): pii: fnz211. PubMed Abstract | Publisher Full Text

[18] 18. Theethakaew C, Nakamura S, Motooka D, et al.: Plasmid dynamics in Vibrio parahaemolyticus strains related to shrimp Acute Hepatopancreatic Necrosis Syndrome (AHPNS). Infect Genet Evol. 2017; 51: 211–8. PubMed Abstract | Publisher Full Text

[19] 19. Yang YT, Chen IT, Lee CT, et al.: Draft genome sequences of four strains of Vibrio parahaemolyticus, three of which cause early mortality syndrome/acute hepatopancreatic necrosis disease in shrimp in China and Thailand. Genome Announc. 2014; 2(5): pii: e00816-14. PubMed Abstract | Publisher Full Text | Free Full Text

[20] 20. Watson M, Warr A: Errors in long-read assemblies can critically affect protein prediction. Nat Biotechnol. 2019; 37(2): 124–6. PubMed Abstract | Publisher Full Text

[21] 21. Gan HM, Lee YP, Austin CM: Nanopore Long-Read Guided Complete Genome Assembly of Hydrogenophaga intermedia, and Genomic Insights into 4-Aminobenzenesulfonate, p-Aminobenzoic Acid and Hydrogen Metabolism in the Genus Hydrogenophaga. Front Microbiol. 2017; 8: 1880. PubMed Abstract | Publisher Full Text | Free Full Text

[22] 22. Austin CM, Tan MH, Harrisson KA, et al.: De novo genome assembly and annotation of Australia's largest freshwater fish, the Murray cod (Maccullochella peelii), from Illumina and Nanopore sequencing read. GigaScience. 2017; 6(8): 1–6. PubMed Abstract | Publisher Full Text | Free Full Text

[23] 23. Wick RR, Judd LM, Gorrie CL, et al.: Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol. 2017; 13(6): e1005595. PubMed Abstract | Publisher Full Text | Free Full Text

[24] 24. Kolmogorov M, Yuan J, Lin Y, et al.: Assembly of long, error-prone reads using repeat graphs. Nat Biotechnol. 2019; 37(5): 540–546. PubMed Abstract | Publisher Full Text

[25] 25. Walker BJ, Abeel T, Shea T, et al.: Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One. 2014; 9(11): e112963. PubMed Abstract | Publisher Full Text | Free Full Text

[26] 26. Waterhouse RM, Seppey M, Simão FA, et al.: BUSCO Applications from Quality Assessments to Gene Prediction and Phylogenomics. Mol Biol Evol. 2017; 35(3): 543–8. PubMed Abstract | Publisher Full Text | Free Full Text

[27] 27. Hyatt D, Chen GL, Locascio PF, et al.: Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 2010; 11(1): 119. PubMed Abstract | Publisher Full Text | Free Full Text

[28] 28. Tatusova T, DiCuccio M, Badretdin A, et al.: NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res. 2016; 44(14): 6614–24. PubMed Abstract | Publisher Full Text | Free Full Text

[29] 29. Alikhan NF, Petty NK, Zakour NLB, et al.: BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genomics. 2011; 12(1): 402. PubMed Abstract | Publisher Full Text | Free Full Text

[30] 30. Kelley LA, Mezulis S, Yates CM, et al.: The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc. 2015; 10(6): 845–58. PubMed Abstract | Publisher Full Text | Free Full Text

[31] 31. UniProt Consortium: UniProt: a hub for protein information. Nucleic Acids Res. 2015; 43(Database issue): D204–D12. PubMed Abstract | Publisher Full Text | Free Full Text

[32] 32. Fu L, Niu B, Zhu Z, et al.: CD-HIT: accelerated for clustering the next-generation sequencing data. Bioinformatics. 2012; 28(23): 3150–2. PubMed Abstract | Publisher Full Text | Free Full Text

[33] 33. Katoh K, Standley DM: MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013; 30(4): 772–80. PubMed Abstract | Publisher Full Text | Free Full Text

[34] 34. Price MN, Dehal PS, Arkin AP: FastTree 2--approximately maximum-likelihood trees for large alignments. PLoS One. 2010; 5(3): e9490. PubMed Abstract | Publisher Full Text | Free Full Text

[35] 35. Muhire BM, Varsani A, Martin DP: SDT: a virus classification tool based on pairwise sequence alignment and identity calculation. PLoS One. 2014; 9(9): e108277. PubMed Abstract | Publisher Full Text | Free Full Text

[36] 36. Gan HM, Gan HY, Ahmad NH, et al.: Whole genome sequencing and analysis reveal insights into the genetic structure, diversity and evolutionary relatedness of luxI and luxR homologs in bacteria belonging to the Sphingomonadaceae family. Front Cell Infect Microbiol. 2015; 4: 188. PubMed Abstract | Publisher Full Text | Free Full Text

[37] 37. Krehenwinkel H, Wolf M, Lim JY, et al.: Estimating and mitigating amplification bias in qualitative and quantitative arthropod metabarcoding. Sci Rep. 2017; 7(1): 17668. PubMed Abstract | Publisher Full Text | Free Full Text

[38] 38. Gan HM, Grandjean F, Jenkins TL, et al.: Absence of evidence is not evidence of absence: Nanopore sequencing and complete assembly of the European lobster (Homarus gammarus) mitogenome uncovers the missing nad2 and a new major gene cluster duplication. BMC Genomics. 2019; 20(1): 335. PubMed Abstract | Publisher Full Text | Free Full Text

[39] 39. Gan HM, Linton SM, Austin CM: Two reads to rule them all: Nanopore long read-guided assembly of the iconic Christmas Island red crab, Gecarcoidea natalis (Pocock, 1888), mitochondrial genome and the challenges of AT-rich mitogenomes. Mar Genomics. 2019; 45: 64–71. PubMed Abstract | Publisher Full Text

[40] 40. Xiao J, Liu L, Ke Y, et al.: Shrimp AHPND-causing plasmids encoding the PirAB toxins as mediated by pirAB-Tn903 are prevalent in various Vibrio species. Sci Rep. 2017; 7: 42177. PubMed Abstract | Publisher Full Text | Free Full Text

[41] 41. Mahillon J, Chandler M: Insertion sequences. Microbiol Mol Biol Rev. 1998; 62(3): 725–74. PubMed Abstract | Free Full Text

[42] 42. Skipper KA, Andersen PR, Sharma N, et al.: DNA transposon-based gene vehicles - scenes from an evolutionary drive. J Biomed Sci. 2013; 20(1): 92. PubMed Abstract | Publisher Full Text | Free Full Text

[43] 43. Sheets JJ, Hey TD, Fencil KJ, et al.: Insecticidal toxin complex proteins from Xenorhabdus nematophilus: structure and pore formation. J Biol Chem. 2011; 286(26): 22742–9. PubMed Abstract | Publisher Full Text | Free Full Text

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[52] 52. Gan HM, Austin C: Nanopore long reads enable the first complete genome assembly of a Malaysian Vibrio parahaemolyticus isolate bearing the pVa plasmid associated with acute hepatopancreatic necrosis disease [Data set]. Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3568485

Nanopore long reads enable the first complete genome assembly of a Malaysian Vibrio parahaemolyticus isolate bearing the pVa plasmid associated with acute hepatopancreatic necrosis disease

Abstract

Keywords

Introduction

Methods

Nanopore sequencing

De novo genome assembly and genome polishing

Comparative Genomic analysis and protein homology modeling

Identification and phylogenetic analysis of LuxM, OpaM and AinS proteins

Results and discussion

Chromosomal assembly enabled by Nanopore long reads

Table 1. Statistics and BUSCO completeness assessment of the Vibrio parahaemolyticus MVP1 assembly.

Reduced Illumina sequencing depth in several low-GC genomic regions

Figure 1. Genomic comparisons of pVa plasmid-harboring Vibrio parahaemolyticus.

Multiple localization of IS5 family transposase genes in the complete genomes of pVa-carrying V. parahaemolyticus

Figure 2. Alignment of Illumina-assembled contigs, Illumina and Nanopore reads to the annotated Vibrio parahaemolyticus MVP1 pVa plasmid sub-region containing the pirABVp genes.

Structural variations in the MVP1 genome

Figure 3. InterProScan identification of conserved protein domains in the putative toxin complex family proteins coded by three linked genes located on Chromosome 2 of Vibrio parahaemolyticus MVP1.

High prevalence and divergence of the LuxM/OpaM/AinS family autoinducer synthesis proteins among Vibrio spp

Figure 4. Phylogenetic analysis and sequence divergence of the AinS-like family proteins in Vibrio spp.

Conclusions

Data availability

Underlying data

Extended data

References

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Figure 2. Alignment of Illumina-assembled contigs, Illumina and Nanopore reads to the annotated Vibrio parahaemolyticus MVP1 pVa plasmid sub-region containing the pirAB^Vp genes.