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Next Generation Sequencing (NGS)
Next Generation Sequencing (NGS) is a deep sequencing technique that uses massively paralleled DNA or RNA sequences to piece together genomes. Millions of small (or long) DNA sequences are analyzed in unison, and these fragments are bioinformatically mapped in relation to a known genome, highlighting potential variants. NGS can pinpoint even the smallest changes in base pairs, insertions, deletions and substitutions, or can be used to identify larger genomic changes, including exon or whole gene deletions, inversions, rearrangements and translocations. NGS has been used heavily in research to understand differentiating subtypes of cancer genomes to potentially provide a more accurate diagnosis or means of therapy. NGS also gives the potential for rare-disease identification, as well as understanding pathogen morphology which can lead to the understanding of drug sensitivity or used to trace infection and outbreak sources.
Even though NGS is a growing field, improving daily, optimized NGS techniques still have a long way to go. Systems must be low cost while still performing at a high rate; they must accurately lower possibilities of error and minimize artifacts while requiring as little DNA or RNA samples as possible.
Even though NGS is a growing field, improving daily, optimized NGS techniques still have a long way to go. Systems must be low cost while still performing at a high rate; they must accurately lower possibilities of error and minimize artifacts while requiring as little DNA or RNA samples as possible.
History of DNA Sequencing
In 1977 Frederick Sanger developed a chain-termination method of DNA sequencing, where dideoxynucleosides that lacked a 3' -OH group prevented phosphodiester bond formation by DNA polymerase, thereby halting DNA production. The ddNTPs can either be radically tagged in sequencing gels or fluorescently labeled through automated machines, allowing for clear visibility of the DNA sequence. Innovations to Sanger sequencing have revolutionized NGS techniques, keeping them alive and well in commercial laboratories, the first being the construction of fluorescent dyes that attach to the 5' end of the oligonucleotide.
Another advancement is the use of thermal-cycle sequencing and thermostable polymerases, systems like PCR, that drastically reduce the amount of DNA needed for a test, precisely incorporate dyes at target points in a DNA, and increase high-throughput potential. Lastly, ever-growing technological advancements in bioinformatic software have given researchers the ability to better interpret, analyze and understand data.
Previous Advancements in NGS
By Synthesis (SBS)
Most SBS technology uses wells or microchannels in a solid substrate for individual molecular DNA adherence, generally does not use dideoxy terminators, and typically use short templates, around 300-500 bases. SBS relies heavily on massively parallel sequencing of short DNA sequences, 50-300 nucleotides, to accurately eliminate error rates, however can produce enormous loads of genetic information in a short time span. Because the DNA is amplified isothermally, this process provides incredibly high-throughput repeatable results. Some notable SBS systems are as follows: Roche 454 pyrosequencing (though discontinued) utilized the detection of pyrophosphate to identify base incorporation in a growing DNA chain. Ion Torrent releases a H+ ion when correct base pairs are released on a growing DNA chain, changing the pH gradient that is detected by an ion sensor. Illumina technology uses bridge amplification to create identifiable clonal clusters of DNA after repeated rounds of amplification.
By Ligation
NGS by ligation uses DNA ligase, versus polymerase, to identify nucleotides at precise points in a DNA sequence, and requires the use of clonally amplified DNA, instead of a single molecule. A previously used method was SOLiD, which used a two-base sequencing method with a library including a ligation site, cleavage site, and 4 different fluorescent dyes attached to the last base. After 5 rounds of sequencing, the fragment can be fully deduced using complementing ladder primer sets. Due to not requiring DNA polymerase, fragments can also be read in the 5'-3' direction, however for this reason palindromic sequences may be difficult to discern. Due to high associated costs, long turnaround times (~1 week), and low-throughput potential, SOLiD, and other ligation methods like Polonator, are no longer used often commercially.
Table 1. Building Blocks for Preparing Fluorescent Conjugates in NGS
By Hybridization
NGS through hybridization is a microarray technique that uses specially, synthetically crafted probes to semiquantitative profile gene expression. Repeat cycles of hybridization and washing steps provide DNA fragments that can be overlapped, where information will decipher an underlying genetic code. Sensitivity and specificity are limited to probe types, so typically this form of NGS is only used in diagnostic measures, in identification of disease-related single-nucleotide polymorphisms and chromosome malformations.
Current and Upcoming NGS
Long DNA Segments Reading Systems
Traditional NGS methods use short-read DNA sequences from 35-300, and the data output availability paired with decreased cost in base sequences makes these ideal options for many applications. Short-read sequencing has effectively been used to target information about structural variants, however this NGS method comes with its own set of complications, namely in ability to characterize de novo sequences, or novel DNA sequences that have no reference arrangement.
Some long-read technologies have been developed to sequence over 30-50 kb, and though these methods have a relatively high error rate the errors are stochastic in nature and random at best, so when run in parallel or in consensus sequencing this technology remains highly accurate. Not only do long-read processes have the potential to significantly reduce the cost of NGS, with notably quicker turn-around times, they can bring to light information from gray areas in known genomes that typically fall short in short-read sequencing alone.
Nanopore Systems
Nanopore-based NGS is the newest development in the field and offers ultra long length base pair reads, from 104-106 units, which require no fluorescent labeling and little starting material. The essence of the nanopore system is that single DNA molecules pass through nanometer size openings in either a membrane or film by means of an ion current. Cis and trans elements are sieved accordingly, targeting the physical and chemical properties of DNA molecules.
Biological, synthetic, or hybrid nanopore systems exist, all with upsides and drawbacks. Biological nanopores have impressive applications in ssDNA sequencing, single-molecule detection, and disease diagnosis, but systems lack structural stability and have uniform pore size and conformation. Synthetic, solid-state pore systems are stronger and chemically stable, have mass-production potential, and are used typically in DNA and protein translocation applications but lack the resolution to obtain structural information of molecules on a single-nucleotide level.
NGS Applications
On top of rapidly sequencing whole genomes and target regions, NGS can also be applied to give information on RNA variants, splice locations, and mRNA quantification. NGS can be used to analyze gene expression and epigenetics on an elemental scale, like DNA-protein relationships. NGS continues to assist microbiome understanding across all areas of biology, and the dependence genes, proteins and transcriptomes have on each other. New NGS systems are compact, use little energy and require little reagent use or maintenance.
Today's NGS systems have lower error rates, high-throughput capabilities, and speedier processing times than ever before, and advances in bioinformatics too have made data analysis a simpler process. Long-read techniques and nanotechnologies have also expanded the horizon to what NGS can do, clarifying complexities behind exome and intron architecture and allowing for advancements in clinical applications.
Product Ordering Information
Table 2. DNA Sequencing Building Blocks
| Cat# ▲ ▼ | Product Name ▲ ▼ | Unit Size ▲ ▼ |
| 300 | 5-dR6G [5-Carboxy-4,7-dichlororhodamine 6G] | 25 mg |
| 301 | 6-dR6G [6-Carboxy-4,7-dichlororhodamine 6G] | 25 mg |
| 302 | 5-dR6G, succinimidyl ester | 5 mg |
| 305 | 5-dTMR [5-Carboxy-4,7-dichlorortetramethylrhodamine] | 25 mg |
| 306 | 6-dTMR [6-Carboxy-4,7-dichlorortetramethylrhodamine] | 25 mg |
| 307 | 5-dTMR, succinimidyl ester | 5 mg |
| 310 | 5-dROX [5-Carboxy-4,7-dichloror-X-hodamine] | 25 mg |
| 311 | 6-dROX [6-Carboxy-4,7-dichloror-X-hodamine] | 25 mg |
| 312 | 5-dROX, succinimidyl ester | 5 mg |
| 315 | 5-dR110 [5-Carboxy-4,7-dichlororhodamine 110] | 25 mg |
Table 3. Available MagaDye™ fluorescent ddNTPs for Sanger sequencing.
| Product Name ▲ ▼ | Nucleotide ▲ ▼ | Ex (nm) ▲ ▼ | Em (nm) ▲ ▼ | Abs(nm) ▲ ▼ | Unit Size ▲ ▼ | Cat No. ▲ ▼ |
| MagaDye™ 535-ddGTP | Guanine | 503 nm | 536 nm | 503 nm | 5 nmoles | 17063 |
| MagaDye™ 535-ddGTP | Guanine | 503 nm | 536 nm | 503 nm | 50 nmoles | 17067 |
| MagaDye™ 561-ddATP | Adenine | 498 nm | 561 nm | 498 nm | 5 nmoles | 17062 |
| MagaDye™ 561-ddATP | Adenine | 498 nm | 561 nm | 498 nm | 50 nmoles | 17066 |
| MagaDye™ 588-ddTTP | Thymine | 498 nm | 588 nm | 498 nm | 5 nmoles | 17061 |
| MagaDye™ 588-ddTTP | Thymine | 498 nm | 588 nm | 498 nm | 50 nmoles | 17065 |
| MagaDye™ 613-ddCTP | Cytosine | 498 nm | 614 nm | 498 nm | 5 nmoles | 17060 |
| MagaDye™ 613-ddCTP | Cytosine | 498 nm | 614 nm | 498 nm | 50 nmoles | 17064 |
References
Behjati, Sam, and Patrick S Tarpey. “What Is Next Generation Sequencing?” Archives of Disease in Childhood - Education & Practice Edition, vol. 98, no. 6, 2013, pp. 236-238., https://doi.org/10.1136/archdischild-2013-304340.
Slatko, Barton E., et al. “Overview of next-Generation Sequencing Technologies.” Current Protocols in Molecular Biology, vol. 122, no. 1, 2018, https://doi.org/10.1002/cpmb.59.
Levy, Shawn E., and Richard M. Myers. “Advancements in next-Generation Sequencing.” Annual Review of Genomics and Human Genetics, vol. 17, no. 1, 2016, pp. 95-115., https://doi.org/10.1146/annurev-genom-083115-022413.
Liu, Lin, et al. “Comparison of next-Generation Sequencing Systems.” Journal of Biomedicine and Biotechnology, vol. 2012, 2 Apr. 2012, pp. 1-11., https://doi.org/10.1155/2012/251364.
Feng, Yanxiao, et al. “Nanopore-Based Fourth-Generation DNA Sequencing Technology.” Genomics, Proteomics & Bioinformatics, vol. 13, no. 1, 2015, pp. 4-16., https://doi.org/10.1016/j.gpb.2015.01.009.