2. Pioneer Landmarks in DNA Sequencing
3. Major Landmarks in DNA Sequencing during the Last Three Decades
4. The Need for Fast, Inexpensive and Accurate DNA Sequencing Technologies
5. Development of NGS Platforms (2000–present)
6. Platform Selection
|Sequencing platform||Amplification method||Sequencing chemistry||Read length (bp)||Sequencing Speed/h||Maximum Output Per run||Accuracy (%)||M1 I2 D3|
|454 (Roche)||Emulsion PCR||Pyrosequencing||400–700||13 Mbp||700 Mbp||99.9||0.10, 0.3, 0.02 |
|Illumina (Illumina)||Bridge PCR||Reversible terminators||100–300||25 Mbp||600 Gbp||99.9||0.12, 0.004, 0.006 |
|SOLiD (Life Technologies)||Emulsion PCR||Ligation||75–85||21–28 Mbp||80–360 Gbp||99.9||Error is higher than Illumina |
|PacBio (Pacific Biosciences)||No amplification Single molecule real-time (or SMRT)||Fluorescently labeled nucleotides||4, 000–5,000||50–115 Mbp||200 Mb–1 Gbp||95||1, 2, 12 |
|Helicos (Helicos Biosciences)||No amplification Single molecule||Reversible terminators||25–55||83 Mbp||35 Gbp||97||Error is in the range of few percent but higher than 454 and Illumina and biased toward InDels |
|Ion Torrent (Life Technologies)||Emulsion PCR||Detection of released H||100–400||25 Mb–16 Gbp||100 Mb–64 Gbp||99||M, 0.06, I + D 1.38 |
|Nanopore (Oxford Technologies)||No amplification Single molecule||Very long reads up to 50 kbp||150 Mbp||Tens of Gbp||96|
|454 GS FLX + Systems (GS FLX Titanium XL+/GS FLX Titanium XLR70)|
* GS Junior System (bench top)
|DNA sequencing: whole genome sequencing, de novo and resequencing of large genes in a single run with read length up to 1 kbp. Amplicon sequencing; RNA sequencing: transcriptome sequencing, sequencing capture metagenomics.|
Run time: 10–23 h; * Fast sequencing run with read length of 400 bp. Similar applications as the above system.
HiSeq Systems (2500/2000/1500/1000), Genome Analyzer IIx, HiScan SQ, * MiSeq (bench top)
|DNA sequencing including candidate region targeted sequencing; Epigenetic sequencing: chromatin immunoprecipitation sequencing (ChIP-Seq), methylation analysis by sequencing; RNA sequencing: transcriptome analysis, small RNA and mRNA sequencing, gene expression profiling by sequencing; Run time: 8–14 days; * Up to 15 Gbp of output with 25 M sequencing reads and 2 × 300 bp read length; Access more sequencing applications such as exome, metagenomics, human leukocyte antigen (HLA) gene typing, mRNA sequencing, targeted gene expression (proteins and non-protein coding genes) such as rRNA, tRNA, or smRNA genes; Run time: 20–35 h.|
5500 W Series Genetic Analysis Systems (5500 W, 5500 xlw)
|DNA sequencing: whole genome and exome; Epigenetic Sequencing; RNA Sequencing.|
Run time: 7–12 days.
|PacBio PACBIO RSII||DNA sequencing using single molecule real-time (SMRT) system with the longest read lengths of any sequencing technology.|
Characterization of genetic variation, methylation, targeted sequencing such as SNP detection and validation, indels, structural variants, haplotypes and phasing, base modification detection to understand gene expression, host-pathogen interactions, DNA damage and DNA repair.
Run time: 30 min.
|Helicos Genetic Analyzer System||DNA sequencing and RNA sequencing.|
Run time: 8 days.
|Ion Torrent Ion PGM System (bench top)|
*Ion Proton System (bench top)
|Semiconductor sequencing with 400-bp length- Ideal for sequencing small genes and genomes.|
DNA sequencing for microbial: genes and genomes, amplicons, exomes (unreveal disease-causing variants), targeted sequencing, viral typing and other microbial typing.
Run time: 4.5 h
* Semiconductor sequencing: Sequencing microbial genomes, exomes, transcriptomes.
Run time: 4.5 h
|Nanopore GridION System (bench top)|
*MinION System (a miniaturized disposable device for single use)
|DNA sequencing; Epigenetic sequencing; Characterization of genetic variation.|
RNA sequencing: the system is designed to analyze the original sample RNA directly, without undergoing conversion to cDNA.
Run time possibly under 60 min.
* For DNA sequencing only, i.e., blood DNA.
|Host||Study finding/virome||Sample preparation/target||Sequencing platform||Ref.|
|Sweet potato||Detected: Sweet potato feathery mottle virus, Sweet potato chlorotic stunt virus; Discovered: two Badnavirus species (dsDNA), one Mastrevirus species (ssDNA)||siRNAs||Illumina|||
|Gomphrena globosa||Plants were inoculated with unknown virus-a new Cucumovirus was identified. Proposed name: Gayfeather mild mottle virus||Total RNA + subtractive hybridization||Roche 454 GS-FLX|||
|Arabidopsis thaliana||Tobacco mosaic virus siRNAs mediate virus-host interactions which may contribute to viral pathogenicity and host specificity||siRNAs||Illumina Genome Analyzer|||
|Nicotiana benthamiana, Arabidopsis thaliana, Cucumis milo and tomato||Nine different viruses including Cucumber mosaic virus, Tobacco rattle virus, Pepper mild mosaic virus, Potato virus X were studied. The study extended the knowledge of distribution and composition of siRNAs in virus-infected plants and contributed to a better understanding of siRNAs biogenesis.||siRNAs of nine different viruses||Roche 454|||
|Cassava||The complete sequence of the Tanzanian strain of Cassava brown streak virus was determined and compared with that of the Ugandan strain. The virus is highly heterogeneous at both the isolate and strain levels with nucleotide identity at the isolate level of 76%||Total RNA + subtractive hybridization||Roche 454 GS− FLX|||
|Nicotiana benthamiana||Profiled Cymbidium ringspot virus-derived siRNAs. These RNAs were primarily produced from the positive strand of the virus, produced with different frequency, and had 5' monophosphate and were not perfect duplexes.||siRNAs||Roche 454 and Solexa (Illumina)|||
|Wild plant species from 15 families naturally infected with viruses were utilized. The families are: Acanthaceae, Bignoniaceae, Caesalpinaceae Commelinaceae, Cyperaceae, Cucurbitaceae, Euphorbiaceae, Gesneriaceae, Lamiaceae, Mimosaceae, Myrtaceae, Papilionaceae, Poaceae, Rubiaceae and Solanaceae||Identification of 11 virus families in infected plants which include: Bromoviridae, Caulimoviridae, Chrysoviridae, Closteroviridae, Endornaviridae, Luteoviridae, Narnaviridae, Partitiviridae, Potyviridae, Totiviridae, and Tymoviridae. Unclassified virus families were also identified in some samples of infected plants. Discovered several thousand novel viruses, all linked to their specific plant hosts||dsRNAs||Roche 454-GS-FLX|||
|Wild cocksfoot grass||Cereal yellow dwarf virus (Luteovirus) discovered infecting this grass (not previously reported infecting this host). Cocksfoot streak virus (Potyvirus) was detected||siRNAs||Roche 454|||
|Nicotiana benthamiana, Arabidopsis thaliana||Profiling siRNAs of Bamboo mosaic virus and its interfering and non-interfering associated satellite RNAs. The overall composition of virus siRNAs and satellite RNAs in the infected plants reflect the combined action of virus, satellite RNA and different DCLs in host plants.||siRNAs (virus and satellites)||Solexa (Illumina)|||
|Rice||Characterization of siRNAs of Rice stripe virus four genome RNAs in infected rice plants||siRNAs||Illumina Solexa|||
|Cotton||Characterization of siRNAs of Cotton leafroll dwarf virus (genus Polerovirus, family Luteoviridae) in infected cotton plants||siRNAs||Illumina Genome Analyzer|||
|Wild Passiflora caerula (Blue-crown passion flower) (a vine)||The complete nucleotide sequence of the Passion fruit woodiness virus (Potyvirus) was determined||Polyadenylated RNA||Illumina Solexa GAIIx|||
|Pepper, eggplant||The complete nucleotide sequences of two new viruses Pepper yellow leaf curl virus (Polerovirus) and Eggplant mild leaf mottle virus (Ipomovirus) were determined||Purified virons viral RNA||SOLiD|||
|Different hosts||A novel virus infecting watercress was identified (proposed name: Watercress white vein virus). Viruses such as Piper yellow mottle virus, Arachacha virus B, and Potato black ring virus were detected and sequenced||Partial virus and RNA purification then fragmented||Roche 454 GS−FLX|||
|Tomato||siRNAs of Tomato spotted wilt virus were used to detect the virus in infected tomato before symptoms appeared at levels too low for conventional detection methods. Also, used for analysis of the virus quasi species and for identification of an unspecified Tospovirus and a squash-infecting geminivirus||siRNAs||Illumina|||
|Arabidopsis thaliana||Characterization of the siRNAs and transcriptome profiles of Oilseed rape mosaic virus (Tobamovirus)-infected Arabidopsis plants.||Total RNAs and siRNAs||Illumina Genome Analyzer|||
|Tomato, Nicotiana benthamiana||Characterization of the siRNAs for the monopartite begomovirus Tomato yellow leaf curl China virus and its associated betasatellite in infected tomato and Nicotiana benthamiana plants . Also, it was found that the betasatellite affected the amount of virus siRNAs detected in both plant species.||siRNAs||Solexa-Illumina|||
|Tomato||Identification of Potato spindle tuber virod, Pepino mosaic virus, differentiation of two strains of the virus, and a novel Potyvirus from infected tomato plants by obtaining the complete genome sequence of the mixed infected pathogens without prior knowledge of their existence. Based on the severity of symptoms on tomato, the novel virus is provisionally named “Tomato necrotic stunt virus”||siRNAs||Illumina Genome Analyzer IIx|||
|Tobacco cv Xanthi nc||Identification of gene expression changes associated with disease development in tobacco plants induced by infection with the M strain of Cucumber mosaic virus Sequencing analysis of tobacco transcriptome identified 95,916 unigenes, 34,408 of which were new transcripts by database searches.||Total RNA then treated with DNase I||Illumina Hi Seq 2000|||
|Corn (maize)||Detection and identification of Maize chlorotic mottle virus and Sugarcane mosaic virus by obtaining over 90% of both viral genomes sequencing which allowed also strain characterization. Next generation sequencing may be used for rapidly identifying potential disease causing agents, in this case viruses||Total RNA was purified from diseased tissue with virus symptoms||Roche 454 GS−FLX+|||
|Seventeen plant species, among them 14 Australian indigenous species||Detection and identification of 12 viruses described previously members of the genera Potyvirus, Nepovirus, Allexivirus and Carlavirus. Four novel viruses were identified and proposed as members of the genera Potyvirus, Sadwavirus and Trichovirus. Moreover, in 3 cases, 2–3 distinct isolates of a virus species co-infected the same plant.||Polyadenylated RNA||Illumina Genome Analyzer IIx|||
|Nicotiana benthamiana, Laodelphgax striatellus (small brown leafhopper), rice||The presence Rice stripe virus (RSV) siRNAs was demonstrated in infected rice as well as in infected N. benthamiana and viruliferous L. striatellus. Also, results indicate the potential existence of RNAi-mediated immunity against RSV infection in L. striatellus, a member of Hemipteran that transmits about 55% of the known plant viruses. Moreover, demonstrated that siRNA are generated differentially in different hosts.||siRNAs||Illumina|||
|Sweet potato||Detected Sweet potato feathery mottle virus strain RC, Sweet potato virus C (Potyvirus), Sweet potato chlorotic stunt virus strain WA (Crinivirus), Sweet potato leaf curl Georgia virus (Begomovirus), and Sweet potato pakakuy virus strain B (synonym: Sweet potato badnovirus B) infecting sweet potato crops in Central America. Also, 4 viruses were detected in a sweet potato sample from the Galapagos Islands. Results suggest that siRNAs deep sequencing analysis is suitable for use as a reliable method for detection of plant viruses in infected crops.||si RNA||Illumina Genome Analyzer|||
|Pepper (genotype Yolo Wonder)||Developing a mathematical model that estimates genetic drift and selection intensities using next generation sequencing data of 4 variants of Potato virus Y that differ by 1–2 substitutions involved in pathogenicity.||Total RNA||Roche 454|||
|Arabidopsis thaliana||Analysis of viral siRNAs from DNA virus-infected cells showed that the entire circular genomes of Cauliflower mosaic virus (genus: Caulimovirus, family: Caulimoviridae) and cabbage leaf curl virus (genus: Begomovirus, family: Geminiviridae) are densely covered with siRNAs in both sense and antisense polarities without gaps. This would enable de novo reconstruction of the complete DNA virus genomes from siRNAs.||siRNAs||Illumina Genome Analyzer||[51,52,53]|
|Black pepper||The complete genome sequence of Piper yellow mosaic virus was determined. It was also established that the virus is a member of the genus Badnovirus and the family Caulimoviridae. Fragments of two additional novel viruses belong to Caulimoviridae were sequenced and the viruses were tentatively named Piper DNA virus 1 and 2.||Viral and plant DNA were isolated from virus-enriched fraction||Roche 454 GS−FLX Titanium|||
|Host||Study finding/virome||Sample preparation/target||Sequencing platform||Ref.|
|Raspberry||A novel virus isolated from infected raspberry plants was completely sequenced and characterized. It was designated as Raspberry latent virus. The virus is a novel dicot-infecting reovirus in the family Reoviridae, subfamily Spinareovirinae.||dsRNA||Illumina|||
|Citrus||In Citrus tristeza virus (CTV) infected citrus plants It was shown that the citrus homologues of Dicer-like ribonucleases mediate the genesis of the 21 and 22 nt CTV siRNAs and that the ribonucleases act not only on the genomic RNA but also on the 30 co-terminal subgenomic RNAs and, particularly, on their dsRNA forms. A novel citrus miRNAs was also indentified and how CTV influences their accumulation was determined.||CTV sRNAs, gRNA sgRNAs||Illumina|||
|Citrus||Genomic organization and other molecular characterizations were determined for Citrus yellow vein clearing virus. Analyses suggested that the virus is the causal agent of yellow vein clearing disease of lemon trees and represent a new species in the genus Mandarivirus.||siRNAs||Illumina|||
|Citrus||A novel DNA virus species, member of the family Geminiviridae, was identified and associated with citrus chlorotic dwarf disease. A provisional name of Citrus chlorotic dwarf-associated virus was proposed.||siRNAs and total DNA||Illumina HiSeq2000|||
|Apple, Citrus, Grapevine||Detected ASPV, ACLSV and an unknown mycovirus. Detected two variants of CTV and ASGV. Detected variants of GLRaV-3, GVA and an unknown mycovirus.||siRNAs||Illumina|||
|Apple||Identified agents associated with green crinkle disease of apple trees. The disease is a complex one as the following viruses were identified associated with it: ASGV, ASPV, ACLSV, ApLV, ApPCLSV and PCMV.||siRNAs||Illumina HiSeq2000|||
|Prunus||Detected and identified known Prunus viruses such as PPV, PNRS, etc. and novel virus agents.||dsRNA||Roche 454|||
|Fig||Detected Fig mosaic virus and Fig latent virus-1 for their elimination from infected clones. It is the first application of next-generation sequencing technology to detect and identify known and new species of viruses infecting fig trees.||dsRNAs||Illumina|||
|Blackberry||A novel Ampelovirus in the family Closteroviridae was identified as one of the viruses associated with blackberry yellow vein disease complex.||dsRNAs||Illumina|||
|Cherry||Characterization of the genome of the divergent Little cherry virus 1 (LChV1) isolate and establishing that LChV1 isolates could be responsible for Shirofugen stunt disease syndrome.||dsRNAs||Roche 454 Pyrosequencing multiplex approach|||
|Citrus||The complete nucleotide sequence and structure of a novel virus of the genus Cilevirus was determined. The novel virus causes symptoms similar to citrus leprosies and it is suggested to be called Citrus leprosis virus cytoplasmic type 2.||siRNAs||Illumina|||
|Citrus||A novel virus was discovered by analysis of the contigs assembled from the virus siRNAs sequences which showed similarity with luteovirus sequence, particularly with Pea enation mosaic virus, the type member of the genus Enarnovirus. The complete genome of the virus was determined and the new virus was provisionally named Citrus vein enation virus.||siRNAs||Solexa-Illumina|||
|Host||Study finding/virome||Sample preparation/target||Sequencing platform||Ref.|
|Grapevine||A novel Marafivirus (Grapevine Syrah 1virus) was identified associated with grapevine syrah decline. The virus was also identified in leafhopper vector. Also detected in plant tissue GRSPaV, GRVFV, GLRaV-9, and viroids.||Total RNA or dsRNA||Roche 454|||
|Grapevine||Grapevine virus E, not previously in South Africa. A mycovirus similar to Penicillium chrysogenum virus, two other mycoviruses, GLRaV-3, GRSPaV, GVA.||dsRNA||Illumina|||
|Grapevine||Viruses of the genera Foveavirus, Maculavirus, Marafivirus, and Nepovirus were detected. siRNAs originate from both genomic and antigenomic strands with the exception of tymoviruses, the majority are derived from antigenic virus strand.||siRNAs||Illumina|||
|Grapevine||A novel DNA virus was discovered associated with the grapevine vein-clearing and vine decline syndrome. The virus belongs to genus Badnavirus in the family Caulimoviridae. It is the first DNA virus discovered in grapevine. It has been provisionally named Grapevine vein clearing virus.||siRNAs||Illumina Genome Analyzer|||
|Grapevine||Twenty six fungal groups were identified in a single plant source. Three of the mycoviruses were associated with Botrytis cinerea. Most of the rest were undescribed.||dsRNA||Roche 454|||
|Grapevine||A novel species of virus was discovered for which the provisional name Grapevine Pinot gris virus is proposed. Also, detected and identified GRSPaV, GRVFV, GSy 1V, and viroids.||siRNAs||Illumina|||
|Grapevine||A novel circular DNA virus was identified associated with red blotch disease in grapevine in California. A provisional name of Grapevine red blotch-associated virus is proposed for the novel virus.||dsRNA extracted without DNase treatment||Illumina Genome Analyzer IIx|||
|Grapevine||A novel Vitivirus was identified. The virus is provisionally named Grapevine virus F.||dsRNA||Illumina Genome Analyzer IIx|||
|Grapevine||Characterization of siRNAs associated with grapevine leafroll disease.||siRNAs||Illumina|||
|Grapevine||Complete sequence of a novel single-stranded DNA virus associated with grapevine red leaf disease (GRD). The virus is tentatively named Grapevine red leaf-associated virus (GRLaV). The virus represents an evolutionary distinct lineage in the family Geminiviridae. Also detected in plant tissue GRSPaV, GFV, and viroids.||Total RNA treated with DNase||Illumina Genome Analyzer IIx|||
|Host||Study finding/virome||Sample preparation/target||Sequencing platform||Ref.|
|Grapevine||Different Dicer-like enzymes target RNAs of Hop stunt viroid, Grapevine yellow speckle viroid 1. Also, study suggested that the viroid RNAs may interact with host enzymes involved in the RNA-directed DNA methylation pathway||siRNAs||Solexa, Illumina|||
|Grapevine||Detection and identification of Australian grapevine viroid, Hop stunt viroid and Grapevine yellow speckle viroid||Total RNA or dsRNA||Roche 454|||
|Peach||To study the genesis of Peach latent mosaic viroid siRNA and viroid pathogenesis||siRNAs||Illumina|||
|Nicotiana benthamiana||RNA-dependent RNA polymerase 6 restricts accumulation and precludes meristem invasion of Potato spindle tuber viroid which replicates in nuclei||Plant and viroid siRNAs||Illumina EAS269 GAII|||
|Cucumber||To study Hop stunt viroid pathway involved in the biogenesis of the viroid siRNAs||siRNAs||Illumina|||
|Tomato||Detection and identification of Potato spindle tuber viroid||siRNAs||Illumina Genome Analyzer IIx|||
|Grapevine||Detection and identification of Grapevine yellow speckle viroid 1 and Hop stunt viroid||siRNAs||Illumina|||
|Grapevine||Detection and identification of Grapevine yellow speckle viroid 1 and Hop stunt viroid||siRNAs and dsRNAs||Illumina|||
|Grapevine||Discovery of viroid-like circular RNA 375 nt long with hammerhead ribozymes. Currently, infectivity studies showed that the RNA is not infectious which may suggest that it is viral satellite||siRNAs||Illumina|||
|Grapevine||Characterization of siRNAs of Hop stunt viroid, Grapevine yellow speckle viroid 1, and Grapevine yellow speckle viroid 2||siRNAs||Illumina|||
|Grapevine||Detection and identification of Grapevine yellow speckle viroid 1, Hop stunt viroid, Citrus exocortis Yucatan viroid and Citrus exocortis viroid from both symptomatic and non-symptomatic samples of grapevine read leaf disease||Total RNA treated with Dnase||Illumina Genome Analyzer IIx|||
|Vector||Study finding/virome||Sample preparation/target||Sequencing platform||Ref.|
|Grapevine leafhopper||A novel Merafivirus associated with grapevine syrah decline was detected in the vector||Total nucleic acids of the viruliferous vector||Roche 454|||
|Citrus psyllid||A complete genomic sequence of the bacterium, “Candidatus Liberibacter asiaticus” was obtained. The genome is circular and its size is about 1.23 Mb, The bacterium is the causal agent of citrus Huanglongbing (greening) disease||DNA extracted from a single “Ca. L. asiaticus”-infected Asian citrus psyllid (Diaphorina citri)||Roche 454 GS−FLX|||
|Bemesia tabaci||Four novel Begomovirus species were discovered in their viruliferous vectors||Purified viral DNA||Metagenomic reads 100–700 nt|||
|Aodelphgax striatellus (small brown leafhopper)||The presence of Rice stripe virus siRNAs was demonstrated in the viruliferous vector L. striatellus||siRNAs||Illumina|||
|Host||Study finding/pathogen||Sample preparation/target||Sequencing platform||Ref.|
|Grapevine||It was demonstrated that sequences of infected phytoplasmas belonged to 16 SrV and 16 SrXII groups, as well as to Candidatus Phytoplasma prunorum (16SrX-B) whereas some sequences could not be assigned to a single phytoplasma group. Also a high number of single nucleotide polymorphisms (SNPs) were found. Suggested NGS may be used for future phytoplasma detection in quarantine.||Total DNA from mid-vein leaf tissue||Amplicon sequencing by Roche 454 GS FLX||[85,86]|
|Grapevine||Demonstrated significant changes in the transcriptome of Aster yellows phytoplama-infected Grapevine cv. Chardonnay. The study could contribute to understanding the unknown mechanisms of phytoplasma pathogenicity.||Total RNA and DNA||Illumina HiSeq 2000|||
|Periwinkle||Genomic analysis of four phytoplasma strains of 16SrIII group and two strains of the 16SrI-B subgroup revealed the significant role of horizontal gene transfer among different “Ca. Phytoplasma” species in shaping phytoplasma genomes and promoting their diversity.||Standard DNA preparation from infected periwinkle||Illumina|||
|Citrus (Mexican lime)||Identified miRNA families that are expressed differentially upon infection of Mexican lime trees with Candidatus Phytoplasma aurantifolia. The study increases our understanding of the molecular basis of witches’ broom disease which may lead to development of new strategies for its control.||miRNAs were isolated from infected and from healthy tissues||Illumina HiSeq 2000|||
|Citrus||Demonstrated that several miRNAs and siRNAs were highly induced by Ca. L. Asiaticus (Las) infection, which can be potentially developed into early diagnosis markers of huanglongbing (HLB) disease (citrus greening disease). MiR399 was induced specifically by infection of Las. MiR399 is induced by phosphorous starvation in other plant species. Applying phosphorous significantly reduced HLB symptoms in citrus.||RNA fragments 18–28 nt in length obtained after fractionation of total RNA on denaturing polyacrylamide gel electrophoresis. The total RNA was extracted from Las-infected tissue with HLB symptoms||Illumina|||
7. Bioinformatics’ Software Tools for Data Analysis
8. Cost of DNA Sequencing
9. Biological Applications of Next-Generation Sequencing
10. Applications of Next-Generation Sequencing in Medical Virology
11. Current Applications of Next-Generation Sequencing in Plant Virology
12. Next-Generation Sequencing and Revealing the Etiology of Unknown Diseases and Latent Infections
Conflicts of Interest
References and Notes
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