Historical Perspective, Development and Applications of Next-Generation Sequencing in Plant Virology

1Next-generation (high throughput, deep) sequencing (NGS) has been developed in recent years. These technologies have lowered the costs of DNA sequencing beyond what is possible with standard dye-terminator methods. NGS describes platforms that produce large amounts (typically millions to billions) of DNA reads, with lengths between 25 and 400 bp. These reads are shorter than the traditional Sanger sequence reads (300 to 750 bp). Recently, however, NGS technologies have advanced to produce DNA reads longer than 750 bp. The roots of NGS development go back to the discovery of the structure of the DNA double helix in 1953 by James Watson and Francis Crick, an American geneticist and a British physicist, respectively. Robert Holley, an American biochemist, was the first to sequence a nucleic acid when he and colleagues developed sequencing methods for tRNA in 1964 and 1965. Sequencing methods for long DNA were successfully developed independently in 1977 by Frederick Sanger, a British biochemist, and by Walter Gilbert, an American biochemist/physicist. The Nobel Prize was received by Watson and Crick in 1962, by Holley in 1968, and by Sanger and Gilbert in 1980.

2Several years before any attempt was made to sequence a DNA molecule, in 1964 Robert Holley (1922–1993) was the first to sequence an RNA molecule. He determined the complete sequence and structure of the 77 ribonucleotides of alanine tRNA, the molecule that incorporates the amino acid alanine into protein. Holley’s pioneer work opened the door for others to determine the sequence of other RNAs as well as DNA.

3In 1972, Paul Berg (1926–present) developed DNA technology, which permits isolation of defined fragments of DNA. Prior to this, the only accessible samples for sequencing were from phages or virus DNA. Also, his discovery led to the development of the modern genetic engineering.

4In 1973, the first nucleotide sequence of 24 bp out of 27 bp of the lac operator DNA was published. In 1977, Frederick Sanger (1918–2013) was the first to sequence the complete DNA genome of bacteriophage ΦX 174. He also developed “DNA sequencing with chain-terminating inhibitor”. Moreover, in 1977 Walter Gilbert (1932–present) developed “DNA sequencing by chemical degradation”.

5Paul Berg, Frederick Sanger and Walter Gilbert received the Nobel Prize in Chemistry in 1980. Frederick Sanger received the Noble Prize twice, the first one in 1958 for his work on the structure of proteins, especially that of insulin. Sanger is one of only four people to win two Nobel Prizes and the only Nobel Laureate to win two chemistry prizes.

61984: Medical Research Council scientists completed the DNA sequence of the Epstein-Barr virus (EBV) 172,282 bp using the dideoxynucleotide/M13 sequencing procedure. EBV causes infectious mononucleosis.

71986: LeRoy Hood at the California Institute of Technology (Cal Tech., Pasadena, CA, USA) announced the invention of the first-semi-automated DNA sequencing machine. The machine automated the enzymatic chain termination procedure for DNA sequence analysis developed by Sanger and became a key instrument in mapping and sequencing genetic material.

81987: Applied Biosystems (USA) marketed the first automated sequencing machine, the model ABI 370. Constant improvements in the technology resulted in faster sequencing capacity, which was significant for advanced scientific research in projects such as mapping the human genome.

91990: The International Project on Human Genome was formally started and it was expected to take 15 years. It involved mainly the United States, the United Kingdom, France, Germany, Japan, China, and India.

101990: The US National Institutes of Health (NIH) began large-scale sequencing trials on Mycoplasma capricolumn, Escherichia coli, Caenorhabditis elegans, and Saccharomyces cerevisiae.

111995: Craig Venter, Hamilton Smith and colleagues (USA) completed the first complete genome of a free-living organism, the bacterium Haemophilus influenza. The circular chromosome contains 1,830,137 bp. This is the bacterium from which Smith had first isolated a restriction enzyme for which he shared the Nobel Prize in 1978.

121996: Applied Biosystems markets the capillary sequencer, ABI 310. It is an automatic single-capillary genetic analyzer designed for a wide range of sequencing and fragment analysis approaches.

131996: Pal Nyren and Mostafa Ronaghi (Sweden) developed the method of DNA pyrosequencing without the need for electrophoresis. It relies on the detection of DNA polymerase activity by an enzymatic luminometric inorganic pyrophosphate detection assay developed by P. Nyren in 1987.

141998: Eric Kawashima, Laurent Farinelli and Pascal Mayer developed “Method of nucleic acid amplification” and obtained WO 98/44151 patent for its development while working at the Glaxo Wellcome’s, Geneva Biomedical Research Institute. This method described DNA colony sequencing which is one of the milestones in developing the massive parallel sequencing technologies such as Illumina (San Diego, CA, USA) and Life Sciences 545 (a Roche Company, Branford, CT, USA).

151998: Phil Green and Brent Ewing (USA) developed the base calling program “Phred” for sequencer data analysis. It has the ability to estimate a probability of error for each base-call, as a function of certain parameters computed from the tracer data.

161998: Genome of the nematode Caenorhabditis elegans sequenced. The 97-megabase pair genomic sequence revealed over 19,000 genes. The complete genome sequence, the first from a multicellular organism and from an animal, was a joint sequencing project between the Welcome Trust Sanger Institute (Hinxton, UK) and the Genome Institute, Washington University, St. Louis, MO, USA.

172000: Human Genome Project: due to widespread international cooperation and advances in the field of genomics (especially in sequence analysis), as well as major advances in computing technology, a “rough draft” of the genome was finished in 2000 (announced jointly by U.S. President Bill Clinton and the British Prime Minister Tony Blair on June 26, 2000).

182001-2003: Human Genome Project: Key findings of the draft (2001) and essentially complete genome were announced in April 2003, 2 years earlier than planned (3.3 billion-base pairs; approximately 23,000 genes).

19The automated Sanger method had led to a number of major accomplishments, including the completion of the human genome and other selected animal and plant genomes. However, the method limitations showed a need for new and improved technologies for sequencing large numbers of human and other genomes. In the late 20th and early 21st century, efforts have been made towards the development of new methods to replace the automated Sanger method, which is considered as a “first-generation” technology. The newer methods are referred to as next-generation sequencing (NGS) and their use has changed the scientific approaches in both basic and applied research in many of scientific disciplines, especially in many branches of the biological field, including plant pathology and plant virology.

20The major advance offered by NGS is the ability to produce an enormous volume of data, in several cases in excess of one billion short reads per instrument run, as well as its ability to deliver fast, inexpensive and accurate genome information.

21In 2000, Massively Parallel Signature Sequencing (MPSS) Lynx Therapeutics (USA) Company launched the first of the NGS technologies. The company was later purchased by Illumina.

22In 2004, 454 Life Sciences (Branford, CT, USA) marketed a paralleled version of pyrosequencing. The first version of their machine reduced sequencing costs 6-fold compared to automated Sanger sequencing and was the second of a new generation of sequencing technologies, after MPSS. Life Sciences acquired by Roche Company (Headquartered in Basel, Switzerland). Pyrosequencing provides intermediate read lengths and price per base compared to Sanger sequencing on the one hand and Illumina and SOLiD on the other. In 2005–2006, the 454 GS 20 Roche sequencing platform was introduced which revolutionized DNA sequencing as it could produce 20 million bases (20 Mbp). This was replaced by the GS FLX model in 2007 which is capable of producing over 100 Mbp of sequence in just four hours, which increased in 2008 to 400 Mbp. This model was then upgraded to the 454 GS−FLX+ Titanium sequencing platform which is capable of producing over 600 Mbp of sequence data in a single run with Sanger-like read lengths of up to 1,000 bp. Another sequencing platform system produced by Roche is the GS Junior which is small in size and is placed on a laboratory bench top. It provides long 400 bp sequencing reads with a fast sequencing run.

23In 2005, Solexa released the Genome Analyzer (GA). Its sequencing technology is based on sequencing by synthesis (SBS) using reversible dye-terminators chemistry. Solexa was purchased by Illumina in 2007. The GAIIx platform generates up to 50 billion bases (50 Bbp) of usable data per run and the latest model can attain 85 Bbp per run.

24During the last 3–4 years, Illumina has developed the HiSeq platform series which include HiSeq® 2500, HiSeq 2000, HiSeq 1500 and HiSeq 1000 sequencing platforms. They vary in their outputs, run times, cluster generations, paired end reads, and maximum reads length with HiSeq 2500 the longest length of 200 bp and HiSeq 1000 the shortest one. HiSeq 2500 platform has the capacity to sequence a human genome in about 24 hours, “Genome in a day”. It may also sequence 20 exomes in a day, or 30 RNA sequencing samples in as little as five hours. It generates 120 billion b (120 Bbp) of data in 27 hours. Standard HiSeq 2000 generates 600 billion bases (600 Bbp) per run. The output of HiSeq 2500 can reach 600 Gbp. Illumina also released MiSeq in 2011, a bench top platform which shared most technologies with HiSeq series. It generates 1.5 Gbp per run in about 10 h.

25SOLiD technology employs sequencing by oligo ligation detection. The result is sequences of quantities and length comparable to Illumina sequencing. SOLiD was purchased by Applied Biosystems (AB, founded in 2006 in Foster City, CA, USA and became a division of Life Technologies in 2008 Headquartered in Carlsbad, CA, USA). Owing to a two-base sequencing method, SOLiD accuracy may reach 99.99%.

26In 2007, the first SOLiD sequencing system was released by AB, followed by the SOLiD 5500 w and 5500 xlw sequencing systems in 2010. The SOLiD 5500 xlw has read lengths of 85 bp, with 99.99% accuracy and 30 Gbp per run. A complete run could be finished within a week. Applications of SOLiD include analysis of whole genome clusters. The SOLiD website, see, may be helpful.

27Other recently developed methods of NGS technologies include: Helicos sequencer released in 2009, Life Technologies Ion Torrent sequencer released in 2011, Pacific Biosciences (Menlo Park, CA, USA) single molecule real-time (smrt) sequencer which also became available in 2011, and Oxford Technologies Nanopore (Oxford, UK) single molecule sequencer with ultra long single molecule reads that became available in 2012–2013. In November 2012, Helicos Biosciences (Cambridge, MA, USA) filed for bankruptcy in Cambridge, MA, USA, and it is currently reorganizing its operation. For this reason its website is no longer available.

28Additional methods of NGS were also developed. These methods may include Polonator sequencing, Polony sequencing, DNA Nanoball sequencing, and VisiGen Biotechnologies sequencing.

29Table 1 shows current major methods of NGS technologies and Table 2 shows current models of major sequencing platform systems and their applications in NGS technologies.

30Important factors in selecting a sequencing platform may include the size or expected size of the genome being studied, its complexity (including G+C content), and the depth of coverage and accuracy needed. Thus, it is important and advisable to contact the providers of the next-generation sequencing services for guidance. For de novo genome sequencing, longer read length may be appropriate. For fast turnover times and limited throughput, smaller laboratory bench top platforms may offer greater flexibility. For amplicon sequencing, the Roche 454 platform is suitable because of its longer reads, however, it is currently expensive. Recently, the laboratory bench tops Roche 454 GS Junior, Illumina MiSeq, and Ion PGM have claimed that their sequencing platforms are suitable for sequencing amplicons. For RNA-seq and those projects that require high depths of coverage, Illumina and SOLiD platforms may offer the best all-round value for money, accuracy and throughput. Roche 454 has the longest read length, Illumina HiSeq 2500 features the biggest output and lowest sequencing cost, and SOLiD 5500 xlw has the highest accuracy. Table 3, Table 4, Table 5, Table 6, Table 7 and Table 8 show that during the last five years, Illumina sequencing platforms were used more frequently than those of Roche’s in different plant virology projects in several countries.

31The software functions for next-generation sequencing data analysis may be classified into four general categories which include alignment of sequence reads, base-calling and/or polymorphism detection, de novo, and genome browsing and annotation. Several software packages have been developed for each category. For example, a variety of bioinformatics’ software tools for short-read sequencing used for de novo assembly of genomes and transcriptomes are available. A practical application of de novo genome assembly software tools has been also recently reported. New categories and new software tools for short-read sequencing analysis are continuously being developed commercially world-wide, especially in the U.S., Europe and Australia. Many commercial sequencing companies, i.e., Roche, Illumina, SOLiD, etc., offer their software services to customers for different types of DNA or RNA sequencing analysis. It is worth mentioning that alignment software such as BLAST or BLAT are not suitable for short-read sequencing analysis of next generation sequencing as they were developed specifically for long reads generated by conventional first generation sequencing.

32A full review of software tools available for quality control assembly and quantitative analysis of next-generation sequencing is beyond the scope of this article, but have been the subject of several review articles, i.e., and a book. Moreover, the journal Bioinformatics devoted one of its volumes published in 2009 (volume 25, No.4) to bioinformatics tools and algorithms that have been developed for next-generation sequencing data analysis. These bioinformatics software tools and algorithms are continuously being developed and improved to keep abreast and pace with the latest advances of next-generation sequencing technologies.

33According to the latest information released by the National Human Genome Research Institute, U.S. National Institues of Health, the cost per raw megabase of DNA sequence and the cost per genome were reduced dramatically from July 2001 to July 2013. During this time period, first generation sequencing methods were used from 2001 through 2007, then, second generation NGS platforms were used from 2008 to present, 2013. The cost per raw megabase of DNA sequence was reduced from about $8,000 in 2001 to $700 in 2007 and then to less than $0.1 in 2013 (Figure 1, top). Similarly, the cost per genome DNA sequence went from $100, 000,000 in 2001 to $10,000,000 in 2007 then to about $8,000 in 2013 (Figure 1, bottom).

34In the next few years, it is expected that the third generation of DNA sequencing platforms will increase sequencing capacity and speed while reducing cost. A genome may be sequenced for about $1,000 or less in about 15 minutes. That is 85% less than the current cost.

35To date the biological applications of next-generation sequencing focused primarily on:

36Full or complete genome sequencing (de novo sequencing and/or resequencing genomics): Its aim is to sequence the entire genome of an organism such as humans, primates, dogs, cats, mice, nematodes, fungi, bacteria, viruses, etc. It is worth noting that unlike full genome sequencing, DNA profiling only determines the likelihood that genetic material came from a particular individual or group; single nucleotide polymorphism (SNP) genotyping covers less than 0.1% of the genome.

37Metagenomics: The term “metagenomics” was first used in publications in 1998. Consequently in 2005 Kevin Chen and Lior Pachter at University of California (Berkeley, CA, USA), defined metagenomics as “the application of modern genomics techniques to the study of communities of microbial organisms directly in their natural environments, by passing the need for isolation and lab cultivation of individual species.” The goals of this approach are to characterize organisms present in a sample and identify what roles each organism has within a specific natural environment. Metagenomics microbial samples are found almost everywhere such as the human body, soil samples, plant samples, water samples, etc.

38Transcriptome sequencing: It includes sequencing and analysis of full-length mRNA and small RNA (microRNA profiling and discovery); mRNA transcript expression analysis (full-length mRNA, expressed sequence tags [ESTs] and ditags, and allele-specific expression); quantification of gene expression and alternative splicing, transcript annotation, discovery of transcribed SNPs or somatic mutation.

39Sequencing of the cDNAs of diverse RNAs (genomic, mRNA, micro RNA, ribosomal RNA, etc.) has been coined RNA-seq..

40Amplicon sequencing: This method allows the detection of mutations at extremely low frequency levels in PCR amplified specific targeted regions of DNA. It has been used to identify low frequency somatic mutations in cancer samples, discovery of rare variants in HIV-infected patients, etc. It also has been used recently for sequencing phytoplasmas from grapevines.

41Other applications of NGS in specific categories may include reduced representation sequencing, (i.e., large-scale polymorphism discovery), targeted genomic resequencing, (i.e., targeted polymorphism and mutation discovery), paired end sequencing, (i.e., discovery of inherited and acquired structural variation), sequencing of bisulfite-treated DNA, (i.e., for determining patterns of cytosine methylation in genomic DNA), chromatin immunoprecipitation sequencing (ChIP-Seq), (i.e., genome-wide mapping of protein-DNA interactions), nuclease fragmentation and sequencing, (i.e., nucleosome positioning). In addition, other applications include: pharmogenomics sequenceing to determine suitable drugs for individuals; diagnosis and discovery of new human diseases; cancer therapy; humans origin, migration of humans, evolution, and others.

42The first applications of next-generation sequencing in the field of medical virology were to monitor population diversity in HIV. In September 2008, an outbreak of unexplained hemorrhagic fever in people was reported in South Africa which resulted in death of four out of five patients. Such outbreaks required a rapid response to control both infection and public anxiety. NGS of infected blood serum samples revealed the discovery of a novel arenavirus. Rapid pathogen discovery using NGS is now available to monitor all outbreaks, regardless of the disease.

43Currently, next-generation sequence technologies applications in medical virology may include, but are not limited to, determination of: full-length viral genome sequence; study of viral genome variability; characterization of viral quasi-species; viral evolution. Metagenomics-based strategies have been used for detection of unknown viral pathogens, discovery of novel viruses, detection and identification of tumor viruses; characterization of the human “viral metagenome” or “virome” in healthy and disease conditions and epidemiology of viral infection. These strategies have also been used for analysis of resistance profiles to drugs and host immunity; quality control of viral vaccines; transcriptomics studies for measurements of mRNA to achieve new insights into genome expression and how this may be modified in health and disease as well as for exploring the role of microRNAs in virus replication and pathogenesis (for review, see).

44NGS technologies combined with sophisticated bioinformatics have been recently changing the field of plant virology, particularly in the areas of genome sequencing, ecology, discovery, epidemiology, transcriptomics, replication, detection and identification. Currently a small number of plant RNA viruses and viroids have been identified from infected tissues and sequenced by RNA-seq. Either total nucleic acid or total double-stranded RNA (dsRNA) from pathogen infected tissue was isolated and the virus or viroid was identified by NGS; alternatively, in some cases, host nucleic acid was partially eliminated by hybridization to nucleic acid isolated from healthy plants to enrich for virus sequences in the infected plant material prior to sequencing. All the viruses and/or viroids detected and identified in infected plants or viruses in viruliferous vectors using NGS are termed “virome”.

45Plant viruses or viroids can also be detected indirectly. In response to infection by RNA/DNA viruses or viroids, the host plant generates specific RNA molecules, 21 to 24 nt in length, called short interfering RNAs (siRNA). RNA silencing (RNAi) is a cytoplasmic cell surveillance system to recognize dsRNA and specifically destroy single and double stranded RNA molecules homologous to the inducer, using small interfering RNAs as a guide (for review see). NGS of siRNAs offers good opportunities to identify viruses or viroids infecting plants, even at extremely low titers, in symptomless infections, and including previously unknown viruses or viroids. Next-generation sequencing can provide thousands to millions of siRNA sequences from virus or viroid infected plant materials. When virus or viroid-derived siRNAs are abundant enough, virus or viroid genome fragments can be assembled. Since the virus or viroid siRNAs are 21–24 nt in length, their sequences can be employed directly as primer sequences to amplify viral or viroid fragments by PCR or RT-PCR.

46Table 3, Table 4, Table 5 and Table 6 summarize the use of next generation technologies in analyzing virus or viroid virome in infected plant species. NGS technologies have also been used for detection and identification of virus virome in viruliferous vectors (Table 7) and insect viruses, and the bacterial “virome” of the bacterium ‘Candidatus Liberibacter asiaticus’ in its insect vector (Table 7) and infected host plant (Table 8). In addition, these technologies have been used for analyzing phytoplasma “virome” in infected plants (Table 8).

47The recent use of NGS technologies in plant virology revealed that some diseases of unknown etiology that affect herbaceous and grass hosts or latent agent infections in wild hosts of different species are caused by new or known viruses (Table 3). This table shows that the following novel viruses were discovered: (a) in sweet potato, two dsDNA viruses (Badnavirus) and one ssDNA virus (Mastrevirus); (b) in Gomphrena globosa mechanically inoculated with sap from Liatris spicata, a Cucumovirus provisionally named Gayfeather mild mottle virus; (c) in wild plant species from 15 families, unclassified virus families and numerous novel virus species; (d) in wild cocksfoot grass, Cereal dwarf virus (Luteovirus) discovered infecting this plant species; (e) in pepper and eggplant, Pepper yellow leaf curl virus (Polerovirus) and Eggplant mild leaf mottle (Ipomovirus), respectively; (f) watercress, a virus provisionally named Watercress white vein virus; (g) in tomato, a Potyvirus species with the proposed name Tomato necrotic stunt virus, also in tomato in a different study, an unspecified Tospovirus and a squash-infecting geminivirus; (h) in 17 wild plant species among them 14 Australian indigineous species, four novel viruses (Potyvirus, Sadwavirus, Trichovirus); (i) in black pepper, two DNA viruses (Caulimoviridae) with proposed names DNA virus 1 and DNA virus 2.

48Most, if not all, diseases of unknown etiology that affect stone, pome and small fruit crops, as well as citrus, and grapevine are graft transmissible. That may imply that systemic pathogens are most likely involved in the etiology of these diseases. Currently there are over 35 diseases of unknown etiology that affect stone fruits and a similar number that affect pome fruits. As revealed recently by next generation sequencing, the disease apple green crinkle is a disease complex and at least 6 known viruses—the 3 apple latent viruses and 3 stone fruit viruses—are associated with it (Table 4). Also, it was established that Shirofugen stunt disease syndrome could be caused by Little cherry virus 1 (Table 4). Among major diseases of unknown etiology that affect small fruits are blueberry fruit drop, blueberry bronze leaf curl, blackberry yellow vein, raspberry leaf curl, raspberry yellow spot and strawberry latent C. Quito-Avila et al. discovered a novel reovirus in the family Reoviridae, subfamily Spinareovirinae infecting raspberry; Thekke-Veetil et al. identified a novel Ampelovirus as one of the viruses associated with blackberry yellow vein disease (Table 4).

49In citrus, a novel DNA geminivirus was discovered and provisionally named Citrus chlorotic dwarf-associated virus; two novel RNA viruses were also discovered, one was provisionally named Citrus leprosies virus cytoplasmic type 2 (Cilevirus) and the other was named Citrus vein enation virus (Enamovirus) (Table 4).

50NGS has also been used for detection and identification of virus virome in viruliferous vectors as well as of the bacterium, “Candidatus Liberibacter asiaticus” in its vector (Table 7). It has also been utilized for the detection and discovery of insect viruses.

51In addition to viruses and viroids, NGS technologies have been used for analyzing phytoplama “virome” in infected grapevine, periwinkle or citrus (Table 8). Total DNA and/or RNA or siRNAs from infected host were used in these studies. Moreover, it was also demonstrated that, upon citrus infection by the hard to grow bacterium, “Candidatus Liberibacter asiaticus”, the causal agent of citrus Huanglongbing (greening) disease, several miRNAs and siRNAs were highly induced and can be potentially developed into early diagnosis markers of the disease (Table 8).

52Next generation sequencing has been shown to be a good option for investigating diseases of unknown etiology in grapevine. A novel Marafivirus (Grapevine Syrah 1 virus) was identified associated with grapevine syrah decline (Table 5). In addition, the following novel RNA viruses were discovered infecting grapevine and given the provisional names: Grapevine Pinto gris virus, Grapevine virus F. (Vitivirus). Two DNA viruses were also discovered, the first belongs to the genus Badnovirus, family Caulimoviridae, provisionally named Grapevine vein clearing virus; the second is a DNA geminivirus and given the provisional name Grapevine red-blotch-associated virus or Grapevine red-leaf-associated virus (Table 5). A viroid-like circular RNA with a hammerhead structure was discovered in infected grapevine (Table 6). Currently, infectivity studies showed that the RNA is not infectious which may suggest it is a viral satellite.

53NGS technologies have become available during the last several years and have been widely used in whole genome sequencing, metagenomics, RNA sequencing, etc. The technologies are in continuous development and improvement. Sequencing chemistries are becoming established, maturing and evolving, read lengths and fidelity are increasing, allowing us to investigate thoroughly viral genome diversity, virus-host interaction, virus epidemiology, virus diagnosis and elimination, etc. The major sequencing platforms are being developed to be easier to use and more cost effective. Moreover, very recently Roche, Illumina, Life Technologies Ion Torrent and Oxford Technologies Nanopore sequencing companies have developed and released in the market reasonably priced bench top platforms with the aim of making next-generation sequencing technologies more readily available to more research laboratories as well as to diagnostic laboratories for viral, bacterial and fungal human, animal, and plant diseases. In 2012–2013 single-molecule DNA sequencing in a miniaturized disposable device for single use has been developed by Oxford Technologies Nanopore that allows simple and cheap high throughput sequencing. It is worth mentioning that Nanopore sequencing platform models have the potential to rapidly generate ultra long single molecule reads.

54The most recent report of the International Committee for the Taxonomy of Viruses lists about 900 plant viruses. The utilization of NGS in plant virology will definitely increase this number very significantly as new viruses are being discovered and characterized in different plant host species, including wild ones, as well as in different insect vectors. Conventional serological or molecular detection and identification methods of plant viruses or viroids depend on prior knowledge of antibody or sequence of the virus or viroid of interest. The NGS technologies have provided a very powerful alternative for detection and identification of these pathogens without a priori knowledge. Metagenomics developed by NGS technologies has been proven to be sensitive, accurate and fast in detection and identification of known and unknown viral and viroid viromes without bias in infected plant species, including woody perennial crops which have low titers of these pathogens. Thus, NGS technologies have the potential to be used in quarantine and certification programs of grapevine, pome and stone fruits, small fruits as well as citrus. The potential use of these technologies in diagnostics has been recently suggested in temperate fruit crops, citrus, grapevine and other crops. Data generated by these technologies can be used effectively to improve efficiency and reliability of these programs as well as in programs aimed at virus and viroid elimination from vegetatively propagative material. Accordingly, NGS technologies will be a significant and powerful tool in controlling virus and viroid diseases.

55The next-generation high throughput sequencing technologies have been available for several years. These technologies and bioinformatics provide rapid and low cost DNA sequencing for biological material, including plant viruses and viroids. NGS platforms, which have different underlying biochemistries and differ in sequencing protocol, produce large amounts (typically millions to billions) of generally short DNA sequence reads, length typically between 25 bp and 400 bp. These reads are shorter than the 750 bp first-generation Sanger sequences. The beginning of the era of NGS technologies was in 2000 and the first marketed sequencing platform was in 2004. Since then the NGS industry has expanded with several companies marketing different models of sequencing platforms, most recently the bench top platforms. The sequencing platforms are continually improved to become faster, more efficient and cheaper in order to bring next-sequencing technologies into many more laboratories to expand their use in biological research and diagnostics.

56NGS technologies combined with sophisticated bioinformatics were successfully utilized in plant virology since 2009, particularly in the areas of genome sequencing, detection and identification, discovery, transcriptomics, replication, ecology, and epidemiology. Known and novel plant RNA and DNA viruses and their satellites as well as viroids and phytoplasmas from different infected plant species were successfully studied using these technologies. The number of published studies, however, are still small but significant (Table 3, Table 4, Table 5, Table 6, Table 7 and Table 8). The utilization of NGS in plant virology in the near future will definitely increase whether in research or diagnostics. The potential use of these technologies in plant certification and quarantine programs can effectively improve the efficiency and reliability of these programs and in controlling virus and viroid diseases at both the national and international levels.