In the context of DNA and RNA sequencing, sequence terminators play a crucial role as specific molecules that indicate the end of a of oligonucleotide sequence. For instance, in Sanger sequencing, terminator bases are instrumental in halting the DNA polymerization process at specific points, thereby enabling the determination of the DNA sequence. However, next-generation sequence terminators are chemical or biological molecules used in modern next-generation sequencing (NGS) technologies. NGS, also known as high-throughput sequencing, allows for the rapid sequencing of genomes or targeted genomic regions of interest.
Chemistry of Terminators in NGS
The first-generation sequencing (FGS) which included Maxam Gilbert methods and Sanger sequencing opened a new door into diagnostics of genetic diseases since 1977. In the traditional Sanger sequencing, chain terminators, known as dideoxynucleotides (ddNTPs), were the norm. These terminators, lacking a hydroxyl group on the 3'-carbon of the sugar molecule, when incorporated into oligonucleotides during DNA synthesis, terminate polymerization. The advent of next-generation sequencing technologies has seen a significant evolution of this concept in several ways. General structures of deoxynucleotides (dNTPs) and dideoxynucleotides (ddNTPs) are shown below as well as the DNA elongation and termination reactions catalyzed by DNA polymerase.
| Deoxynucleic acid triphosphate | Dideoxynucleic acid triphosphate |
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| | 1977 Sanger: Irreversible terminator dideoxynucleotide. |
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| DNA polymerase reaction to elongate the DNA oligonucleotide strand. DNA polymerase-catalyzed esterification of the normal dNTP with the 3’-terminal nucleotide of DNA. The reaction extents the length of a primer by a single nucleotide. |
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| DNA polymerase reaction with a terminator to terminate the DNA oligonucleotide strand. DNA polymerase-catalyzed esterification of the a ddNTP, which does not have a 3’-hydroxyl residue, with the 3-terminal nucleotide of DNA. The ddNTP when incorporated into the growing chain by DNA polymerase acts as a terminator because it does not have the 3’-hydroxyl group reqired for formation of another 5’->3’-phosphodiester bond. |
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| DNA polymerase beta (E.C.2.7.7.7)-DNA complex. PDB ID 7ICG: Pelletier et al. 1996. https://www.ncbi.nlm.nih.gov/Structure/pdb/7ICG Pelletier H, Sawaya MR, Wolfle W, Wilson SH, Kraut J. A structural basis for metal ion mutagenicity and nucleotide selectivity in human DNA polymerase beta. Biochemistry. 1996 Oct 1;35(39):12762-77. https://pubs.acs.org/doi/10.1021/bi9529566 When crystals of human DNA polymerase β (pol β) complexed with DNA are soaked in the presence of dATP and Mn2+, X-ray structural analysis revealed that nucleotidyl transfer to the primer 3‘-OH takes place directly in the crystals, even though the DNA is blunt-ended at the active site. [Pelletier, H., Sawaya, M. R., Wolfle, W., Wilson, S. H., & Kraut, J. (1996) Biochemistry 35, 12742−12761] |
Reversible Terminators
Next generation sequencing (NGS) utilizes sequencing by synthesis (SGS). Older versions called second sequencing (SGS) are [1] Roche/454 sequencing, [2] Ion torrent; proton/PGM sequencing; [3] Illumina (Solexa) sequencing, and [4] SOLID sequencing. Modern NGS platforms often use reversible terminators. Reversible terminators are nucleotide analogs that, like ddNTPs, stop DNA synthesis at each step but can be chemically removed to allow the addition of the next nucleotide. Since these analogs allow reversion of the terminating reactions, they are called "reversible terminators." Illumina's sequencing-by-synthesis (SBS) platform uses this approach.
Third-generation sequencing (TGS) methods utilize single molecular and real-time sequencing technologies. For example, the Pacific Biosciences platform uses a single-molecule real-time technology. Since a closed and circular ssDNA template can be replicated automatically during DNA library preparation, no PCR is required. During sequencing, fluorescence signals are activated by a laser as soon as a labeled dNTPs is incorporated into DNA. The color and duration of the emitted light is recorded by a camera system in real time in the flow cell equipped with zero mode waveguides. Because the time of the base incorporation is longer as the base is modified, the time called “interpulse duration” indicates the DNA modification event. However, nanopore sequencing utilizes a nanopore inserted in an electrical resistant membrane. The potential applied across the membrane results in a current flowing only through nanopore. During sequencing, a characteristic disruption in the electrical current is measured as a nucleotide passes through the nanopore, identifying the nucleotide.
Specifics of NGS Technologies
Illumina Sequencing as an Example
The Illumina sequencing platform uses reversible terminator chemistry. In this approach, multiple single template DNA fragments are attached to a flat surface, and nucleotides are added one by one during sequencing by synthesis. Each incorporated nucleotide is tagged with a fluorescent reversible terminator deoxyribonucleotide. After the incorporation of a nucleotide, the dye is read or imaged to determine the base, and chemical removal of the terminator allows the next sequencing cycle to proceed.
(For more information see https://www.biocompare.com/Editorial-Articles/590720-Guide-to-NGS-Platforms/)
Advantages of Reversible Terminators
Higher Accuracy
Reversible terminators help achieve high accuracy in sequencing by allowing precise control over the sequencing process and reducing errors.
Longer Reads
Although shorter than PacBio or Oxford Nanopore technologies, NGS technologies using reversible terminators can achieve reasonably long read lengths compared to older methods, making next-generation sequence terminators crucial components of modern sequencing technologies, allowing for rapid and accurate sequencing of large amounts of DNA. [Read length information: https://support.illumina.com/ko-kr/bulletins/2020/04/maximum-read-length-for-illumina-sequencing-platforms.html]
The reversible termination sequencing-by-synthesis approach amplifies the sequence of a template by stepwise primer elongation. It is known as a second-generation-sequencing technology on the Illumina platform. The general reversible termination sequencing process involves
(i) immobilizing the sequencing templates and primers on a solid support;
(ii) primer extension by one base plus termination;
(iii) recognizing the color of the fluorophore carried by the extended base to identify the incorporated nucleotide after washing away the unincorporated nucleotides;
(iv) removal of the fluorescent tag and the 3′-O blocking group;
(v) washing again and repeating the former steps
(ii–iv). The whole process can be summarized as extension–termination–cleavage–extension cycle.
Structure schematics of irreversible and reversible terminators utilized in sequencing technologies.
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| Sanger type dye linked irreversible terminator. |
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| 3’-O-blocked reversible terminator. The blocking group -OR, a reversible terminating or capping group, is linked to the oxygen atom of the 3’-OH of the pentose. The fluorescence label linked to the base can be cleaved. |
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| 3’-unblocked reversible terminator. |
Reversible terminators have their advantages and disadvantages.
The 3’-reversible blocking group of the 3’-O-blocked reversible terminator should result in a better termination effect. However, the 3-unblocked reversible terminator is more easily accepted by DNA polymerases due to the missing modified moiety on the 3’-OH. Polymerases discriminate between ribonucleoside triphosphates and 2’-deoxyribonucleotide triphosphates by inspecting the 2’- and 3—positions more closely. The missing oxygen atom at the 3’-position will prevent DNA polymerases from further catalytic elongation of additional nucleosides.
Commercially available reversible terminators with a blocking group at 3’-OH.
All three reversible terminators shown below showed a good performance in their reversible termination function, nearly achieving 100% of 3’-O blocking efficiency and fluorescent label group cleavage after primer extension termination.
| 3’-O-NH2 reversible terminator | 3’-O-allyl reversible terminator | 3’-O-azidomethyl reversible terminator |
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| Chen et al. 2010; Hutter et al. 2010. | Guo et al. 2010. | Bently et al. 2008; Li et al. 2003; Wu et al. 2007; Guo et al. 2008; Ju et al. 2008; Chen et al. 2010; Hutter et al. 2010. |
Phosphoramidites of chain terminators allows their use in automated solid phase oligonucleotide synthesis.
Unblocked reversible terminators
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| Bowers et al. (2009) synthesized reversible terminators with tethered inhibitors useful for next generation sequencing and called these “Virtual Terminator” nucleotides. These nucleotides are efficiently incorporated but block incorporation of a second nucleotide on a homopolymer template. The terminators were tested by resequencing mammalian DNA. |
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| Another recently developed 3′-OH unblocked reversible terminator called a “Lightning terminator” is also a 3′-OH unblocked reversible terminator. Using UV light allows the fluorescent group to be cleaved off for this terminator. (Gardener et al. 2012; Litosh et al. 2011; Stupi et al. 2012; Wu et al. 2007). |
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