The plant virus transmissions database.

Plant viruses are transmitted mechanically or by vegetative propagation, and by vectors such as arthropods, fungi, nematodes, or parasitic plants. Sources to access available information regarding plant virus transmissions are scattered and require extensive literature searches. Here, a recently created plant virus transmission database is described. This was developed to provide access to the modes of transmission and vectors of over 1600 plant viruses. The database was compiled using over 3500 publication records spanning the last 100 years. The information is publicly accessible via https://library.wur.nl/WebQuery/virus and fully searchable by virus name, taxonomic position, mode of transmission or vector.

Detection of single nucleotide polymorphisms in virus genomes assembled from high-throughput sequencing data: large-scale performance testing of sequence analysis strategies.

Recent developments in high-throughput sequencing (HTS) technologies and bioinformatics have drastically changed research in virology, especially for virus discovery. Indeed, proper monitoring of the viral population requires information on the different isolates circulating in the studied area. For this purpose, HTS has greatly facilitated the sequencing of new genomes of detected viruses and their comparison. However, bioinformatics analyses allowing reconstruction of genome sequences and detection of single nucleotide polymorphisms (SNPs) can potentially create bias and has not been widely addressed so far. Therefore, more knowledge is required on the limitations of predicting SNPs based on HTS-generated sequence samples. To address this issue, we compared the ability of 14 plant virology laboratories, each employing a different bioinformatics pipeline, to detect 21 variants of pepino mosaic virus (PepMV) in three samples through large-scale performance testing (PT) using three artificially designed datasets. To evaluate the impact of bioinformatics analyses, they were divided into three key steps: reads pre-processing, virus-isolate identification, and variant calling. Each step was evaluated independently through an original, PT design including discussion and validation between participants at each step. Overall, this work underlines key parameters influencing SNPs detection and proposes recommendations for reliable variant calling for plant viruses. The identification of the closest reference, mapping parameters and manual validation of the detection were recognized as the most impactful analysis steps for the success of the SNPs detections. Strategies to improve the prediction of SNPs are also discussed.

Evaluation of sequencing and PCR-based methods for the quantification of the viral genome formula.

Viruses show great diversity in their genome organization. Multipartite viruses package their genome segments into separate particles, most or all of which are required to initiate infection in the host cell. The benefits of such seemingly inefficient genome organization are not well understood. One hypothesised benefit of multipartition is that it allows for flexible changes in gene expression by altering the frequency of each genome segment in different environments, such as encountering different host species. The ratio of the frequency of segments is termed the genome formula (GF). Thus far, formal studies quantifying the GF have been performed for well-characterised virus-host systems in experimental settings using RT-qPCR. However, to understand GF variation in natural populations or novel virus-host systems, a comparison of several methods for GF estimation including high-throughput sequencing (HTS) based methods is needed. Currently, it is unclear how HTS-methods compare a golden standard, such as RT-qPCR. Here we show a comparison of multiple GF quantification methods (RT-qPCR, RT-digital PCR, Illumina RNAseq and Nanopore direct RNA sequencing) using three host plants (Nicotiana tabacum, Nicotiana benthamiana, and Chenopodium quinoa) infected with cucumber mosaic virus (CMV), a tripartite RNA virus. Our results show that all methods give roughly similar results, though there is a significant method effect on genome formula estimates. While the RT-qPCR and RT-dPCR GF estimates are congruent, the GF estimates from HTS methods deviate from those found with PCR. Our findings emphasize the need to tailor the GF quantification method to the experimental aim, and highlight that it may not be possible to compare HTS and PCR-based methods directly. The difference in results between PCR-based methods and HTS highlights that the choice of quantification technique is not trivial.

Arabidopsis latent virus 1, a comovirus widely spread in Arabidopsis thaliana collections.

Transcriptome studies of Illumina RNA-Seq datasets of different Arabidopsis thaliana natural accessions and T-DNA mutants revealed the presence of two virus-like RNA sequences which showed the typical two-segmented genome characteristics of a comovirus. This comovirus did not induce any visible symptoms in infected A. thaliana plants cultivated under standard laboratory conditions. Hence it was named Arabidopsis latent virus 1 (ArLV1). Virus infectivity in A. thaliana plants was confirmed by quantitative reverse transcription polymerase chain reaction, transmission electron microscopy and mechanical inoculation. Arabidopsis latent virus 1 can also mechanically infect Nicotiana benthamiana, causing distinct mosaic symptoms. A bioinformatics investigation of A. thaliana RNA-Seq repositories, including nearly 6500 Sequence Read Archives (SRAs) in the NCBI SRA database, revealed the presence of ArLV1 in 25% of all archived natural A. thaliana accessions and in 8.5% of all analyzed SRAs. Arabidopsis latent virus 1 could also be detected in A. thaliana plants collected from the wild. Arabidopsis latent virus 1 is highly seed-transmissible with up to 40% incidence on the progeny derived from infected A. thaliana plants. This has probably led to a worldwide distribution in the model plant A. thaliana with as yet unknown effects on plant performance in a substantial number of studies.

ICTV Virus Taxonomy Profile: Secoviridae 2022.

Members of the family Secoviridae are non-enveloped plant viruses with mono- or bipartite linear positive-sense ssRNA genomes with a combined genome of 9 to 13.7 kb and icosahedral particles 25-30 nm in diameter. They are related to picornaviruses and are members of the order Picornavirales. Genera in the family are distinguished by the host range, vector, genomic features and phylogeny of the member viruses. Most members infect dicotyledonous plants, and many cause serious disease epidemics. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) report on the family Secoviridae, which is available at ictv.global/report/secoviridae.

Characterization and Tissue Tropism of Newly Identified Iflavirus and Negeviruses in Glossina morsitans morsitans Tsetse Flies.

Tsetse flies cause major health and economic problems as they transmit trypanosomes causing sleeping sickness in humans (Human African Trypanosomosis, HAT) and nagana in animals (African Animal Trypanosomosis, AAT). A solution to control the spread of these flies and their associated diseases is the implementation of the Sterile Insect Technique (SIT). For successful application of SIT, it is important to establish and maintain healthy insect colonies and produce flies with competitive fitness. However, mass production of tsetse is threatened by covert virus infections, such as the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV). This virus infection can switch from a covert asymptomatic to an overt symptomatic state and cause the collapse of an entire fly colony. Although the effects of GpSGHV infections can be mitigated, the presence of other covert viruses threaten tsetse mass production. Here we demonstrated the presence of two single-stranded RNA viruses isolated from Glossina morsitans morsitans originating from a colony at the Seibersdorf rearing facility. The genome organization and the phylogenetic analysis based on the RNA-dependent RNA polymerase (RdRp) revealed that the two viruses belong to the genera Iflavirus and Negevirus, respectively. The names proposed for the two viruses are Glossina morsitans morsitans iflavirus (GmmIV) and Glossina morsitans morsitans negevirus (GmmNegeV). The GmmIV genome is 9685 nucleotides long with a poly(A) tail and encodes a single polyprotein processed into structural and non-structural viral proteins. The GmmNegeV genome consists of 8140 nucleotides and contains two major overlapping open reading frames (ORF1 and ORF2). ORF1 encodes the largest protein which includes a methyltransferase domain, a ribosomal RNA methyltransferase domain, a helicase domain and a RdRp domain. In this study, a selective RT-qPCR assay to detect the presence of the negative RNA strand for both GmmIV and GmmNegeV viruses proved that both viruses replicate in G. m. morsitans. We analyzed the tissue tropism of these viruses in G. m. morsitans by RNA-FISH to decipher their mode of transmission. Our results demonstrate that both viruses can be found not only in the host's brain and fat bodies but also in their reproductive organs, and in milk and salivary glands. These findings suggest a potential horizontal viral transmission during feeding and/or a vertically viral transmission from parent to offspring. Although the impact of GmmIV and GmmNegeV in tsetse rearing facilities is still unknown, none of the currently infected tsetse species show any signs of disease from these viruses.

First report of Soybean Mosaic Virus in commercially grown soybean in the Netherlands.

In July 2020, plants with crinkled, chlorotic, occasionally necrotic leaves, typical for Soybean Mosaic Virus (SMV), were observed in eight soybean fields (Glycine max L.) in Flevoland, The Netherlands (Supp. Fig. 1). Disease incidence varied from 5-50% and the plants affected often occurred in small or extensive patches. Leaves from several symptomatic plants were sampled from each of two fields planted with soybean variety Green Shell or Summer Shell. Total RNA was extracted from one plant leaf sample per field using InviTrap Spin Plant RNA Mini Kit (Invitek, Germany). One-tube RT-PCRs employing potyvirus generic primers P9502 and CPUP (Van der Vlugt et al, 1999) and SMV-specific primers SMV-dT (5'-TTTTTTTTTTTTTTTAGGACAAC-3') and SMV-Nib-Fw (5'-CAAGGATGARTTTAAGGAG-3') combined with Sanger sequencing confirmed the presence of SMV in all leaf samples. To exclude the presence of other agents in the samples, total RNA from each cultivar was used in standard Illumina library preparation with ribosomal RNA depletion followed by sequencing on an Illumina NovaSeq6000 (paired-end, 150 bp) which yielded 66,579,158 reads (Summer Shell) and 223,953,206 reads (Green Shell). After quality trimming in CLC Genomics Workbench 20.0.4 (CLC-GWB; Qiagen, Hilden), four million reads were randomly sampled for de novo assembly. Contigs over 500 nucleotides (nts) in length with a minimum of 500 reads were annotated by BLASTn against NCBI GenBank. This identified one contig of 9,883 nts (6,233,397 reads) in Summer Shell and one contig of 9,727 nts (3,139,927 reads) in Green Shell with clear homology to SMV (E-value = 0.0). No other viruses were identified in the datasets. Reference assemblies against the SMV reference sequence (NC_002634) mapped 24,090,763 reads (36.2%) for Summer Shell and 175,459,637 reads (78.3%) for Green Shell. Extracted consensus sequences for SMV in both soybean cultivars were 9,584 nts long (excluding the poly-A tail). Sequence data from the de novo and reference assemblies were combined into consensus sequences which showed over 98% overall nt sequence identity to NC_002634 and 99.6% to each other. Both consensus sequences were deposited in GenBank under accession numbers MW822167 (SMV-Summer Shell) and MW822168 (SMV-Green Shell). In addition, the presence of SMV in the field samples was confirmed with an inoculation assay. Leaf samples from both fields were ground in phosphate buffer (0.1M, pH 7.2) and inoculated on cotyledons and first expanded leaves of soybean plants (unknown cv.) 12 days post-germination. Plants showed veinal chlorosis in systemic leaves from 12 days post-inoculation, which developed into veinal necrosis. SMV infections were confirmed by RT-PCR in systemic, chlorotic leaf samples of all symptomatic plants using the SMV-specific primers described above. To our knowledge, this is the first report of SMV in The Netherlands. As soybean is a relatively new but expanding crop in this country, information about emerging diseases is highly relevant. SMV can be transmitted via seeds and aphids, where seeds can serve as primary source of virus inoculum (Cui et al., 2011; Hartman et al., 2016; Hajimorad et al., 2018). Weeds and non-commercial plants can also serve as origin of SMV, particularly in subsequent growing seasons, although this virus infects a limited host range of six plant families (Cui et al., 2011; Hill & Whitham, 2014). Special monitoring would be advised for the recurrence and possible damage by SMV in Dutch soybean fields.

[From boots on the ground to nucleotides in the sequencer: a century of advances in the study of the plant virus ecology]. / De la paire de bottes à la paire de bases : de l'intérêt d'étudier l'écologie des viromes de plantes.

Plant virus ecology began to be explored at the end of the 19th century. Since then, major advances have revealed complex virus-host-vector interactions in a variety of environments. These advances have been accelerated by development of new technologies for virus detection and characterization, the latest of which being high-throughput sequencing (HTS). HTS technologies have proved to be effective for non-targeted characterization of all or nearly all viruses present in a sample without requiring prior information about virus identity, as would be needed for virus-targeted tests. Phytoviromic studies have thus made important advances, including characterization of the complex interactions between phytovirus dynamics and the structure of multi-species host communities, and documentation of the effects of anthropogenic ecosystem simplification on plant virus emergence and diversity. However, such studies must overcome challenges at every stage, from plant sampling to bioinformatics analysis. This review summarizes major advances in plant virus ecology, in association with technological developments, and presents key considerations for use of HTS in the study of the ecology of phytovirus communities.

Illuminating an Ecological Blackbox: Using High Throughput Sequencing to Characterize the Plant Virome Across Scales.

The ecology of plant viruses began to be explored at the end of the 19th century. Since then, major advances have revealed mechanisms of virus-host-vector interactions in various environments. These advances have been accelerated by new technlogies for virus detection and characterization, most recently including high throughput sequencing (HTS). HTS allows investigators, for the first time, to characterize all or nearly all viruses in a sample without a priori information about which viruses might be present. This powerful approach has spurred new investigation of the viral metagenome (virome). The rich virome datasets accumulated illuminate important ecological phenomena such as virus spread among host reservoirs (wild and domestic), effects of ecosystem simplification caused by human activities (and agriculture) on the biodiversity and the emergence of new viruses in crops. To be effective, however, HTS-based virome studies must successfully navigate challenges and pitfalls at each procedural step, from plant sampling to library preparation and bioinformatic analyses. This review summarizes major advances in plant virus ecology associated with technological developments, and then presents important considerations and best practices for HTS use in virome studies.

Proposed revision of the family Secoviridae taxonomy to create three subgenera, "Satsumavirus", "Stramovirus" and "Cholivirus", in the genus Sadwavirus.

We present a taxonomic proposal for revision of the family Secoviridae, a taxon of plant viruses in the order Picornavirales. We propose the reorganization of the genus Sadwavirus to create three new subgenera and to update the classification of five existing species. The proposed subgenera are "Satsumavirus" (one species: Satsuma dwarf virus), "Stramovirus" (two species: Strawberry mottle virus and Black raspberry necrosis virus) and "Cholivirus" (two species: Chocolate lily virus A and Dioscorea mosaic associated virus).

ICTV Virus Taxonomy Profile: Secoviridae.

Members of the family Secoviridae are non-enveloped viruses with mono- or bipartite (RNA-1 and RNA-2) linear positive-sense ssRNA genomes with the size of the RNAs combined ranging from 9 to 13.7 kb. They are related to picornaviruses and are classified in the order Picornavirales. The majority of known members infect dicotyledonous plants and many are important plant pathogens (e.g. grapevine fanleaf virus and rice tungro spherical virus). This is a summary of the current International Committee on Taxonomy of Viruses (ICTV) report on the taxonomy of the family Secoviridae available at www.ictv.global/report/secoviridae.

Aphid transmission of Lettuce necrotic leaf curl virus, a member of a tentative new subgroup within the genus Torradovirus.

Lettuce necrotic leaf curl virus (LNLCV) was described as the first non-tomato-infecting member of the genus Torradovirus. Until today, the virus was found only in The Netherlands in two different areas in open field crops of lettuce. In 2015, LNLCV was accepted by the ICTV as a new member of the genus Torradovirus. The tomato-infecting (TI) torradoviruses Tomato torrado virus (ToTV), Tomato marchitez virus (ToMarV) and Tomato chocolàte virus (ToChV) are transmitted by at least three whitefly species in a semi-persistent and stylet-borne manner. As LNLCV was transmitted in open fields in The Netherlands, where whiteflies are present only in low incidence, transmission studies were set up to identify the natural vector of LNLCV. Whitefly species which survive Dutch open field conditions during summer, as well as lettuce colonizing aphid species, were tested for their ability to transmit LNLCV. Lengths of acquisition and inoculation periods were chosen in accordance with the conditions for TI torradoviruses. Transmission experiments involving whiteflies were never successful. Transmission with aphids was only successful in case of the lettuce-currant aphid, Nasonovia ribisnigri. Localization of LNLCV virions in N. ribisnigri with a nested RT-PCR indicated the stylets as possible retention sites. The willow-carrot aphid Cavariella aegopodii did not transmit LNLCV in our transmission experiment but the virus could be detected in the stylets of this aphid, leaving C. aegopodii as a possible vector for LNLCV.

Consensus statement: Virus taxonomy in the age of metagenomics.

The number and diversity of viral sequences that are identified in metagenomic data far exceeds that of experimentally characterized virus isolates. In a recent workshop, a panel of experts discussed the proposal that, with appropriate quality control, viruses that are known only from metagenomic data can, and should be, incorporated into the official classification scheme of the International Committee on Taxonomy of Viruses (ICTV). Although a taxonomy that is based on metagenomic sequence data alone represents a substantial departure from the traditional reliance on phenotypic properties, the development of a robust framework for sequence-based virus taxonomy is indispensable for the comprehensive characterization of the global virome. In this Consensus Statement article, we consider the rationale for why metagenomic sequence data should, and how it can, be incorporated into the ICTV taxonomy, and present proposals that have been endorsed by the Executive Committee of the ICTV.

Down-regulation of Arabidopsis DND1 orthologs in potato and tomato leads to broad-spectrum resistance to late blight and powdery mildew.

Multiple susceptibility genes (S), identified in Arabidopsis, have been shown to be functionally conserved in crop plants. Mutations in these S genes result in resistance to different pathogens, opening a new way to achieve plant disease resistance. The aim of this study was to investigate the role of Defense No Death 1 (DND1) in susceptibility of tomato and potato to late blight (Phytophthora infestans). In Arabidopsis, the dnd1 mutant has broad-spectrum resistance against several fungal, bacterial, and viral pathogens. However this mutation is also associated with a dwarfed phenotype. Using an RNAi approach, we silenced AtDND1 orthologs in potato and tomato. Our results showed that silencing of the DND1 ortholog in both crops resulted in resistance to the pathogenic oomycete P. infestans and to two powdery mildew species, Oidium neolycopersici and Golovinomyces orontii. The resistance to P. infestans in potato was effective to four different isolates although the level of resistance (complete or partial) was dependent on the aggressiveness of the isolate. In tomato, DND1-silenced plants showed a severe dwarf phenotype and autonecrosis, whereas DND1-silenced potato plants were not dwarfed and showed a less pronounced autonecrosis. Our results indicate that S gene function of DND1 is conserved in tomato and potato. We discuss the possibilities of using RNAi silencing or loss-of-function mutations of DND1 orthologs, as well as additional S gene orthologs from Arabidopsis, to breed for resistance to pathogens in crop plants.

Torradoviruses.

Torradoviruses are an example of a group of recently discovered plant viruses. The first description of Tomato torrado virus, now the type member of the newly established genus Torradovirus within the family Secoviridae, was published in 2007 and was quickly followed by findings of other torradoviruses, initially all on tomato. Their characterization led to the development of tools that allowed recognition of still other torradoviruses, only very recently found on non-tomato crops, which indicates these viruses have a much wider host range and diversity than previously believed. This review describes the characteristics of this newly emerged group of plant viruses. It looks in detail at taxonomic relationships and specific characteristics in their genomes and encoded proteins. Furthermore, it discusses their epidemiology, including host range, semipersistent transmission by whitefly vectors, and impact on diverse cropping systems.

Multiplex detection of plant pathogens through the Luminex MagPlex bead system.

Here we describe a versatile multiplex method for both the serological and molecular detection of plant pathogens. The Luminex MagPlex bead system uses small paramagnetic microspheres ("beads"), either coated with specific antibodies or oligonucleotides, which capture respectively viruses and/or bacteria or PCR products obtained from their genetic material. The Luminex MagPlex bead system allows true multiplex detection of up to 500 targets in a single sample on a routine basis. The liquid suspension nature of the method significantly improves (1) assay speed, (2) detection limits and (3) dynamic range. It can also considerably reduce labor and consumables costs.

Development of a multiplexed bead-based suspension array for the detection and discrimination of pospiviroid plant pathogens.

Efficient and reliable diagnostic tools for the routine indexing and certification of clean propagating material are essential for the management of pospiviroid diseases in horticultural crops. This study describes the development of a true multiplexed diagnostic method for the detection and identification of all nine currently recognized pospiviroid species in one assay using Luminex bead-based suspension array technology. In addition, a new data-driven, statistical method is presented for establishing thresholds for positivity for individual assays within multiplexed arrays. When applied to the multiplexed array data generated in this study, the new method was shown to have better control of false positives and false negative results than two other commonly used approaches for setting thresholds. The 11-plex Luminex MagPlex-TAG pospiviroid array described here has a unique hierarchical assay design, incorporating a near-universal assay in addition to nine species-specific assays, and a co-amplified plant internal control assay for quality assurance purposes. All assays of the multiplexed array were shown to be 100% specific, sensitive and reproducible. The multiplexed array described herein is robust, easy to use, displays unambiguous results and has strong potential for use in routine pospiviroid indexing to improve disease management strategies.

Torradoviruses are transmitted in a semi-persistent and stylet-borne manner by three whitefly vectors.

Members of the genus Torradovirus (family Secoviridae, type species Tomato torrado virus, ToTV) are spherical plant viruses transmitted by the whitefly species Trialeurodes vaporariorum and Bemisia tabaci. Knowledge on the mode of vector transmission is lacking for torradoviruses. Here, the mode of transmission was determined for Tomato marchitez virus (ToMarV). A minimal acquisition access period (AAP) and inoculation access period (IAP) of approximately 2h each was required for its transmission by T. vaporariorum, while optimal transmission required an AAP and IAP of at least 16h and 8h, respectively. Whiteflies could retain the virus under non-feeding conditions for at least 8h without loss of transmission efficiency, but upon feeding on a non-host plant in between the AAP and IAP they retained the virus for no more than 8h. Similar conditions supported transmission of isolates of ToTV and Tomato chocolàte virus (ToChV) by T. vaporariorum and B. tabaci. Additionally, similar experiments revealed the banded-winged whitefly (Trialeurodes abutilonea) as a vector for all three virus species. The results are congruent with acquisition and retention periods for semi-persistent virus transmission. RT-PCR detection analysis of ToTV and ToMarV in the vector's body revealed their presence in the stylet, but not in the head where the pharynx of the foregut is located. The results altogether indicate a semi-persistent stylet-borne mode of vector transmission for torradoviruses. Additionally, this is the first group of spherical viruses transmitted by at least three different species of whiteflies.

Lettuce necrotic leaf curl virus, a new plant virus infecting lettuce and a proposed member of the genus Torradovirus.

A new virus was isolated from a lettuce plant grown in an open field in the Netherlands in 2011. This plant was showing conspicuous symptoms that consisted of necrosis and moderate leaf curling. The virus was mechanically transferred to indicator plants, and a total RNA extract of one of these indicator plants was used for next-generation sequencing. Analysis of the sequences that were obtained and further biological studies showed that the virus was related to, but clearly distinct from, viruses belonging to the genus Torradovirus. The name "lettuce necrotic leaf curl virus" (LNLCV) is proposed for this new torradovirus.

Methods in virus diagnostics: from ELISA to next generation sequencing.

Despite the seemingly continuous development of newer and ever more elaborate methods for detecting and identifying viruses, very few of these new methods get adopted for routine use in testing laboratories, often despite the many and varied claimed advantages they possess. To understand why the rate of uptake of new technologies is so low, requires a strong understanding of what makes a good routine diagnostic tool to begin. This can be done by looking at the two most successfully established plant virus detection methods: enzyme-linked immunosorbant assay (ELISA) and more recently introduced real-time polymerase chain reaction (PCR). By examining the characteristics of this pair of technologies, it becomes clear that they share many benefits, such as an industry standard format and high levels of repeatability and reproducibility. These combine to make methods that are accessible to testing labs, which are easy to establish and robust in their use, even with new and inexperienced users. Hence, to ensure the establishment of new techniques it is necessary to not only provide benefits not found with ELISA or real-time PCR, but also to provide a platform that is easy to establish and use. In plant virus diagnostics, recent developments can be clustered into three core areas: (1) techniques that can be performed in the field or resource poor locations (e.g., loop-mediated isothermal amplification LAMP); (2) multiplex methods that are able to detect many viruses in a single test (e.g., Luminex bead arrays); and (3) methods suited to virus discovery (e.g., next generation sequencing, NGS). Field based methods are not new, with Lateral Flow Devices (LFDs) for the detection being available for a number of years now. However, the widespread uptake of this technology remains poor. LAMP does offer significant advantages over LFDs, in terms of sensitivity and generic application, but still faces challenges in terms of establishment. It is likely that the main barrier to the uptake of field-based technologies is behavioural influences, rather than specific concerns about the performance of the technologies themselves. To overcome this, a new relationship will need to develop between centralised testing laboratories offering services and those requiring tests; a relationship which is currently in its infancy. Looking further into the future, virus discovery and multiplex methods seem to converge as NGS becomes ever cheaper, easier to perform and can provide high levels of multiplexing without the use of virus specific reagents. So ultimately the key challenge from a routine testing lab perspective will not be one of investment in platforms-which could even be outsourced to commercial sequencing services-but one of having the skills and expertise to analyse the large datasets generated and their subsequent interpretation. In conclusion, only time will tell which of the next-generation of methods currently in development will become the routine diagnostics of the future. This will be determined through a combination of factors. And while the technology itself will have to offer performance advantages over existing methods in order to supplant them, it is likely to be human factors e.g., the behaviours of end users, laboratories and policy makers, the availability of appropriate expertise, that ultimately determine which ones become established. Hence factors cannot be ignored and early engagement with diagnostic stakeholders is essential.