Role of Next Generation Sequencing (NGS) in Plant Disease Management: A Review

: A high throughput technique used to determine a part of the nucleotide sequence of an organism’s genome is called next generation sequencing (NGS). NGS has been Proven revolutionary in genomics. Clinical diagnostics, Plant diseases diagnostic and other aspects of medical are now made possible by sequencing. Techniques of NGS: there are different techniques of NGS which are being used in real life sciences i.e., Illumina sequencing, Pyrosequencing, Roche 454 sequencing and Ion torrent sequencing. All vintage methods like culturing in bacterial, fungal, and viral samples are being suppressed by next generation sequencing. The potential for random metagenomic sequencing of sick samples to find potential pathogens has surfaced with the development of next-generation high-throughput parallel sequencing technology. NGS enables highly efficient, rapid, low-cost DNA or RNA high-throughput sequencing of plant virus and viroids genomes, as well as specific small RNAs generated during infection. Although this technique is not so much familiar in the field of plant diseases. However, its widespread application in agronomic sciences will make it possible to create solutions to future food-related challenges that involve biotic stress.


Introduction
A high throughput technique used to determine a part of the nucleotide sequence of an organism's genome is called next generation sequencing (NGS).This methodology is also known as second generation sequencing.This Method uses the sequencing technologies of DNA which are good capable of processing numbers of DNA sequences in parallel.Next generation sequencing also called massively parallel sequencing.Simply an advanced DNA, RNA sequencing technique to sequence big or large genome sample at accurately and quickly as compared to Sanger technique.Next-generation sequencing techniques utilizing methylation, RNA, or DNA sequencing left a great impact on life science.As compared to Sanger traditional method which is also called first generation sequencing technique, NGS cost lower and time saving techniques with gigabase range of base pairs.The utilization of molecular biology and genetic information by different species to reproduce and live with or without disease, mutations, and diversity within their population networks and changing surroundings is now more or much more studied by NGS than ever before.
The history of DNA sequencing starts from 1965 when Robert Holley was awarded the Nobel Prize as he sequenced first tRNA, in 1986.In his Nobel Prize speech, he said, "without minimizing the pleasure of receiving awards and prizes, I think it is true that the greatest satisfaction for a scientist comes from carrying a major piece of research to a successful conclusion" (Holley, 1968).From that different technologies are being used to sequence whole genome.In 1977, a scientist named Sanger sequenced the first-time whole genome of a virus (Berg, 2014).Human genome was sequenced in 20 years with 3 billion dollars expenditures.Different techniques got evolutional to reach next generation sequencing.In a many of previous reviews (Lam et al., 2012;Mardis, 2008;Margulies et al., 2005) history of next generation was published in detailed.Techniques of NGS: there are different techniques of NGS which are being used in real life sciences.Illumina sequencing: Clonal array building and its unique reversible terminator technology are used in Illumina's sequencing approach for efficient and accurate large-scale sequencing.The process identifies DNA bases while also including them in a chain of nucleic acids.The specific fluorescent signal that each base produces as it is integrated into the growing strand establishes the DNA sequence's order (Quail et al., 2008).Sample-specific barcodes must be incorporated into sequencing libraries in order to parallelize target capture and sequencing for numerous samples while maintaining the ability to identify the origin of each sequence.This technique outlines a quick and dependable process for creating barcoded ("indexed") sequencing libraries for the Genome Analyzer platform from Illumina (Meyer & Kircher, 2010).
A significant number of sequence data may now be produced quickly and for a lot less money than with capillary sequencing because to improvements in DNA sequencing technology.These new technologies have unique traits and constraints that must be taken into account either during project design or throughout data analysis (Figure 1).In both the laboratory and statistical stages of project planning and analysis, specific knowledge is required to produce high quality data from these new platforms.The Illumina sequencers that allow parallel reading of several hundred million immobilized sequences using fluorescent-dye reversible-terminator chemistry include the new HI Scan, Genome Analyzers, and Hi Seq.The effectiveness of the sequencing library, sample handling, instrument settings, and sequencing chemistry all have a big impact on the sequencing process (Kircher, Heyn, & Kelso, 2011).Pyrosequencing: Pyrosequencing is a method of genetic material sequencing that is based on the synthesis principle.It works by locating nucleotides that a DNA polymerase has incorporated into the sequence.A chain reaction-based light detection system is also used in pyrosequencing.This system is also based on an enzymatic system.There are many present and future applications of this method (Ahmadian, Ehn, & Hober, 2006).
Roche 454 sequencing: The scientific community now conducts far more sequencing thanks to the 454 Sequencer, and there is a wider range of issues that can be solved using direct readouts of DNA sequence.Higher throughput streamlined in vitro sample preparation procedures, and the miniaturization of sequencing chemistries all of which contributed to the development of the 454-sequencing platform made massively parallel sequencing reactions feasible at a scale and price that were previously unthinkable (Figure 2).The 454 platform has begun to democratize sequencing along with other recently announced nextgeneration technologies, giving independent laboratories access to capacities that were previously only available at a small number of major sequencing centers (Rothberg & Leamon, 2008).Figure 3.An overview of whole-genome sequencing of a pure organism from cultured growth (Simner, Miller, & Carroll, 2018).

Ion torrent sequencing:
The detection of hydrogen ions generated during DNA polymerization is the foundation of the DNA sequencing technique known as ion semiconductor sequencing or Ion torrent sequencing.A complementary strand is constructed using this "sequencing by synthesis" technique, which bases it on the sequence of a template strand (Merriman et al., 2012).3).

NGS as disease
A study conducted by Lecuit and Eloit (2015), proved that next generation sequencing can easily sequence the infectious illness whole genome.This uncommon disease has a huge impact on 30 million Europeans 25 to 50 million americans10 million Australians.Rare diseases provide a significant challenge to clinicians because they can be difficult to precisely diagnose and because they significantly raise the cost of healthcare globally.This article will understand us the way that how next generation sequencing methodologies left a great impact on discoveries of rare infectious diseases and also the impact of NGS on research and technology.Next generation sequencing facilitates the development of potential novel medications that are easy to use and reasonably priced.Genetic abnormalities can now be detected with this technique, the importance of mosaic and de novo mutations have been highlighted, cases of digenic inheritance have been discovered, the vast phenotypic range of the majority of genes has been exposed, and patients with numerous uncommon diseases have been identified.NGS has made all these things feasible.Although there are some back draws of this technique too.But this is really an amazing technique in modern medicine.NGS role in plant diseases: Crop disease resistance has been improved by using next generation sequencing because these genomic based approaches have the ability to sequence the whole genome at once.Gratitude must be paid to NGS technology, due to which it become simple to sequence the whole genome of plants and their pathogens.By using this technology, metagenomic studies of different microbes and their hosts are now conceivable.Thanks to NGS techniques, as it is now routinely and affordably possible to generate plant and pathogen genome or their transcriptome marker sequences, that are related to virulence phenotypes in the pathogen or resistance phenotypes in the plant.This might lead to better management of plant diseases.Now a days next generation sequencing tools are being used to detect plant parasitic pathogens i.e., fungi, bacteria, nematodes, and viruses.The potential for random metagenomic sequencing of sick samples to find potential pathogens has surfaced with the development of next-generation high-throughput parallel sequencing technology (Cox-Foster et al., 2007).From tissue samples acquired from three transplant recipients who had passed away after getting transplants from the same donor, Palacios et al. (2008) sequenced the cDNA.
NGS technology has a good potential for detecting diseases in plants.It is used for certain diagnoses because it provides sequencing of every nucleic acid found in a sample taken from diseased tissue or cell, regardless of standard culture protocols.Because the disease agent-specific barcodes are not used, the presequencing information of the illness-causing organism is not necessary in this technique.However, it is advised to favor a database having information on the fungal pathogens' genomes when examining the outcomes.
Because plant pathogenic fungus genetic data databases are uncommon.One of the primary studies in this regard is that Yang 2022 and colleagues used metagenomic analyses for the detection and identification of Calonectria pseudonaviculata, which causes boxwood blight in plants, which is one of the key works on defining fungal pathogenesis in plants (Yang et al., 2022).A fungus called boxwood blight results in severe financial losses for attractive plants.Due to this, readings are performed using a mixture of barcodes that are currently utilized in research to detect bacterial and fungal infections.NGS has created new opportunities for the study of the microbial diversity associated with either specific plants or the environment in which they thrive, in addition to enabling us to comprehend the range of bacterial plant illnesses.Plant microbiome or phytobiome research is a young area that has great potential for unravelling the complex connections between plants and their phytobiomes.The growing knowledge might potentially be used to construct phytobiomes to lessen the prevalence and severity of disease or by employing components of the phytobiome as biopesticides to control particular infections.A systems framework is suggested by Poudel et al. (2016) (Angly et al., 2006;Breitbart et al., 2003;Edwards et al., 2006;Fierer et al., 2007;Sogin et al., 2006;Venter et al., 2004;Williamson et al., 2008;Zhang et al., 2006)

Conclusion
Next generation sequencing (NGS) is fast, up to date, time lapsing and precise data recording technique, which is being used in all the fields of life sciences.Although many Phyto doctors are using NGS techniques but, not much work was found as to use NGS techniques in detecting plant pathogens and to address them.There are several reasons behind this, i.e., it is very costly technique still now.However, its widespread application in agronomic sciences will make it possible to create solutions to future food-related challenges that involve biotic stress.To improve agricultural and crop yields, infectious agents must be found and identified in the phyllo-sphere of plants.

Figure 1 .
Figure 1.Factors contribution platform accuracy in Illumina sequencing.
torrent sequencing: The detection of hydrogen ions generated during DNA polymerization is the foundation of the DNA sequencing technique known as ion semiconductor sequencing or Ion torrent sequencing.A complementary strand is constructed using this "sequencing by synthesis" technique, which bases it on the sequence of a template strand(Merriman, D Team, & Rothberg, 2012).

Figure 2 .
Figure 2. Genetic diversity and abundance of genes in Roche 454 and Illumina (Luo, Tsementzi, Kyrpides, Read, & Konstantinidis, 2012).NGS as disease diagnostic tool: The new molecular diagnostic tools in the medical field have changed the scenario in detection of different diseases.All vintage methods like culturing in bacterial, fungal, and viral samples are being suppressed by next generation sequencing.Culturing technique is not perfect reliable diagnostic tool: The new molecular diagnostic tools in the medical field have changed the scenario in detection of different diseases.All vintage methods like culturing in bacterial, fungal, and viral samples are being suppressed by next generation sequencing.Culturing technique is not perfect reliable method to diagnostic, because we can't study the whole genome but only the specific targeted area.
Besnard et al. (2014)have used the next generation sequencing technology in plant nematology too.Different species of root-knot nematodes are being separated easily by this technique.According toBesnard et al. (2014)different genomic rearrangements were detected profound as when compared with other species of the Hoplolaimoidea subfamily.In this study repeat elements of different base pairs were discovered.This study illustrate the capability of next-generation sequencing to produce whole mitochondrial genomes, even in the absence of a reference sequence, and suggests new directions for nematode species/race identification, phylogenetics, and population genetics.