DNA Sequencing Methods

DNA sequencing is a basic technique used to understand genetic information and solve biological processes. It has a wide range of uses from genetic research to clinical diagnosis. In this article, we will discuss DNA sequencing methods in detail.

1. Sanger Sequencing

Sanger sequencing is a method developed by Frederick Sanger in 1977 and considered the gold standard for many years. This method is based on the chain termination technique. Sanger method requires template DNA (single or double-stranded DNA), primer DNA, DNA polymerase enzyme, deoxynucleotide triphosphates (dNTPs) and di-deoxynucleotide triphosphates (ddNTPs). First, DNA sample is multiplied to reach a sufficient amount for sequencing. This process is usually carried out using the polymerase chain reaction (PCR). The PCR process is a technique used to multiply specific DNA regions, and during the process, the DNA is denatured (opened into two strands) with heat changes, the primers are annealed and new chains are synthesized by DNA polymerase. For sequencing, a primer DNA is added to bind to a specific region of the DNA. The primer is necessary for DNA synthesis to begin and determines the starting point of the target sequence. After the primer is attached, the DNA polymerase enzyme is activated and synthesis begins. dNTPs are deoxynucleotide molecules (dATP, dGTP, dTTP, dCTP) that form the basic building blocks of DNA. The DNA polymerase enzyme uses dNTPs to create a new DNA strand during DNA synthesis. dNTPs have a 3' hydroxyl (-OH) group, and this group allows the formation of a phosphodiester bond with the new nucleotide. Thus, DNA synthesis can continue. Specially modified ddNTPs are added during the sequencing process. ddNTPs do not have a 3' hydroxyl (-OH) group and therefore DNA synthesis stops when they are added. This mechanism allows DNA to be broken into fragments of different lengths. Each ddNTP pairs with a specific base, causing random terminations. Once the reaction is complete, the DNA fragments formed are separated according to their size by gel electrophoresis or capillary electrophoresis. The fluorescently labeled ddNTPs determine which base each chain ends with and the DNA sequence is analyzed using a laser reader (1).

Advantages of Sanger Sequencing

• Provides high accuracy thanks to the reliable synthesis mechanism of DNA polymerase.

• Provides long read lengths (~800-1000 base pairs).

• Can detect specific sequences with high sensitivity.

• Data is easy to process and interpret.

Disadvantages of Sanger Sequencing

• Can be more expensive than other sequencing methods due to the use of chemical reagents and special equipment.

• Only a limited number of DNA sequences can be sequenced at a time, which creates a disadvantage in high-volume genome studies.

• Sequencing organisms with large genomes, such as humans, takes a long time.

Figure 1. Sanger Method (2).

2. Maxam-Gilbert Sequencing

Maxam-Gilbert sequencing was developed in 1977 as an alternative to Sanger sequencing, but over time it has become less preferred due to its complexity and the use of dangerous chemicals. Before the Maxam-Gilbert sequencing process begins, a radioactively labeled phosphate group is added to the 5' or 3' end of the DNA molecule. This labeling allows the fragments to be identified during the sequencing process. DNA is then cut using chemical reagents specific to certain bases. These chemicals target specific nucleotides to separate the DNA into fragments of different lengths. The cut DNA fragments are separated according to their sizes using polyacrylamide gel electrophoresis (PAGE). After the DNA fragments are separated in the gel, the DNA sequence is determined using the autography method thanks to the radioactive labeling (3).

Figure 2. Principle of Maxam-Gilbert sequencing. Single-stranded DNA is split into its bases by four different chemical reactions (A+G, G, C+T, C). This process causes the DNA to be broken into fragments of different lengths and loaded into four separate wells of a polyacrylamide gel. The sequence is read from the bottom of the gel to the top (for example, GTATGC). If a G is found opposite a gap, this indicates the presence of 5-methylcytosine (4).

Advantages of Maxam-Gilbert Sequencing

• Since it uses a direct chemical-based cutting method, it provides high sensitivity in short DNA sequences.

• It can be applied to sequence single or double-stranded DNA.

• Sensitive mutation analyses can be performed in certain regions thanks to the base-specific cutting mechanism.

Disadvantages of Maxam-Gilbert Sequencing

• The process is very laborious and requires careful chemical treatments in a laboratory environment.

• It contains hazardous chemicals such as hydrazine and radioactive markers, so it poses a high security risk.

• It is a much slower and more costly method compared to Sanger sequencing and next-generation sequencing (NGS) methods.

• It is not suitable for large-scale sequencing projects.

3. Next-Generation Sequencing (NGS)

Next-Generation Sequencing (NGS) is the general name of modern sequencing technologies that allow rapid, high-accuracy, and low-cost determination of DNA and RNA sequences. Compared to traditional Sanger sequencing, NGS can sequence millions of DNA fragments simultaneously in parallel. Thus, it has become a revolutionary method for large-scale genome projects, cancer research, genetic disease analysis, and metagenomic studies. NGS is divided into two main categories: short-read and long-read technologies. While methods such as Illumina, SOLiD, and Ion Torrent are based on short-read technology, methods such as PacBio and Oxford Nanopore provide long-read. While short-read methods are generally more widely used with their high accuracy and low-cost advantages, long-read technologies offer significant advantages in the analysis of complex genome regions and structural variations. This technology is used in many different biotechnological and medical applications such as sensitive mutation detection, analysis of epigenetic modifications, cancer biology studies, single-cell sequencing and transcriptome analyses (5).

3.1. Illumina Sequencing

Illumina sequencing is one of the next-generation sequencing (NGS) methods and provides fast, low-cost and high-accuracy sequencing by reading millions of DNA fragments simultaneously. Reversibly terminated fluorescently labeled nucleotides are added one by one during DNA synthesis, read, and then the blockage is removed and synthesis continues. DNA fragments are attached to a special glass surface called a flow cell. There are adapters on the flow cell that allow DNA to bind. After the DNA fragments are attached to these adapters, a process called bridge amplification is performed. In this process, DNA molecules remain attached to the surface, form a curved structure and replicate themselves. Thus, thousands of copies are produced from each DNA fragment. This amplification process increases the signal strength during sequencing, allowing for high-sensitivity reading, and the DNA becomes ready for sequencing. During the sequencing phase, the DNA polymerase enzyme begins to synthesize the opposite strand of DNA. During this process, fluorescently labeled and reversibly terminated (A, T, G, C) are added one by one. Each nucleotide is read by scanning with a laser, and which base is added is determined. Thanks to the "reversibly terminated fluorescent nucleotides" principle, DNA synthesis is temporarily stopped at each nucleotide addition stage, thus the base sequence is determined with precision. After the nucleotide is read, the blockage is removed, allowing the next nucleotide to be added, and DNA synthesis continues. This method provides high accuracy, allowing the DNA sequence to be determined step by step. Since it is performed on millions of DNA fragments at the same time, Illumina sequencing is a very fast and efficient sequencing method (6).

Figure 3. (a) DNA fragments are randomly cut and combined with oligonucleotide adapters to make them double-stranded. (b) DNA is made single-stranded and attached to oligonucleotides on the surface of the flow cell. DNA clusters are formed by bridging amplification. (c) One of the DNA strands in the cluster is cut to obtain a single-stranded template for sequencing. Bridging is repeated for bidirectional sequencing. (d) The ends of long DNA fragments are marked with biotin, made circular, and randomly fragmented, and the matching ends are used for sequencing (6).

Advantages of Illumina Sequencing

• Fast and high accuracy

• Low cost

• Capacity to produce large data

Disadvantages of Illumina Sequencing

• Short read lengths

• High data processing requirements

3.2. Ion Torrent Sequencing

In Ion Torrent sequencing, DNA fragments are bound to small magnetic beads and amplified using the emulsion PCR (emPCR) method. This process allows multiple copies of each DNA fragment to be produced and helps to obtain a stronger signal during the sequencing process. During the sequencing phase, the DNA polymerase enzyme works on the template DNA strand, adding the appropriate nucleotide for each base. Hydrogen ions are released during the addition of each nucleotide, and these ions cause a pH change. Sensitive semiconductor sensors in the Ion Torrent chip measure pH changes and determine which nucleotide has been added. With this method, DNA sequence is determined by reading electrical signals (7).

Figure 4. Ion Torrent sequencing workflow (8).

Advantages of Ion Torrent Sequencing

• Since it does not require optical sensors, the sequencing process is faster and more cost-effective than fluorescent-based technologies.

• The sequencing process is simpler since it does not require additional chemical processes such as fluorescent labeling.

• It can be used with compact and portable devices, which makes it suitable for clinical and field research.

Disadvantages of Ion Torrent Sequencing

• The error rate is high in long homopolymer sequences (sequences where the same base is repeated consecutively) because sensors can sometimes incorrectly detect how many bases are added.

• It offers lower throughput compared to methods such as Illumina or PacBio for large genome projects.

• It has limited scalability and cannot produce much data in large-scale genome analyses.

3.3. SOLiD Sequencing

SOLiD (Sequencing by Oligonucleotide Ligation and Detection) sequencing is a ligation-based next-generation sequencing (NGS) method that is particularly notable for its high accuracy rate. Developed by Life Technologies (now Thermo Fisher Scientific), this technology falls into the short-read sequencing category and is used particularly for mutation detection and sensitive genome analyses. In the SOLiD sequencing process, DNA fragments are attached to magnetic beads and amplified by the emulsion PCR (emPCR) method. In this way, thousands of copies are created from each DNA fragment, increasing the signal strength. During the sequencing phase, DNA fragments are matched with fluorescently labeled probe sequences with the help of ligation enzymes and which bases are sequenced are determined. This method uses the "two-base encoding" system, which allows two bases to be read simultaneously instead of one, thus significantly reducing the error rate (9).

Figure 5. SOLiD sequencing ligation steps. A) Sequencing occurs on the surface where the DNA primer is annealed to one of the library adapters. Di-base probes compete for binding; once annealed, a portion of the octamethad is excised and a fluorescent signal is emitted. B) Different bases are identified after ligation cycles, which are usually repeated 10 times. This process involves resetting and re-adding the primer, ensuring that each base is interrogated twice in two different reactions. C) This two-base coding system uses a special matrix that allows the DNA sequence to be decoded accurately (10).

Advantages of SOLiD Sequencing

• It has a very high accuracy rate (up to 99.99%), so it is suitable for mutation detection and genome analysis.

• Thanks to the special algorithm (two-base encoding) that allows two bases to be read simultaneously, the error rate is kept low.

• It is very effective for cancer research, genetic variation analysis and small genome sequencing.

Disadvantages of SOLiD Sequencing

• Difficulty may be experienced in complex genome analyses due to short read lengths (50-75 bp).

• Data analysis is more complex compared to other NGS methods.

• Its use has decreased with the development of faster and more efficient methods such as Illumina.

3.4. PacBio (SMRT) Sequencing

PacBio sequencing is one of the long-read sequencing methods based on the technology called Single Molecule Real-Time (SMRT) sequencing. This method is used in sequencing large genomes, detecting structural variations, and epigenetic studies by sequencing DNA molecules in real time at the single molecule level. In PacBio sequencing, DNA fragments are loaded into special wells called SMRT cells. In these wells, the DNA polymerase enzyme initiates the synthesis process and sequencing is performed by adding fluorescently labeled nucleotides one by one. As each nucleotide is added, it emits a fluorescent signal and this signal is recorded in real time by a special detector. In this way, the DNA synthesis process is directly observed and both the base order is determined and epigenetic modifications of DNA can be detected during sequencing (11).

Figure 6. PacBio DNA Preparation Workflow (12).

Advantages of PacBio (SMRT) Sequencing

• Provides very long read lengths (can vary between 10-100 kb), which is ideal for large genome projects.

• Since it is based on the principle of single molecule sequencing, it does not require PCR, thus avoiding amplification errors.

• It can detect epigenetic modifications.

  
  

Disadvantages of PacBio (SMRT) Sequencing

• The raw data error rate is slightly higher than other sequencing methods, but this error rate can be significantly reduced with special error correction algorithms (HiFi reads).

• It is more costly and has a longer data generation time than short read methods such as Illumina.

3.5. Nanopore Sequencing

Nanopore sequencing is one of the next generation sequencing (NGS) technologies and provides direct sequencing of DNA and RNA molecules. This method, developed by Oxford Nanopore Technologies (ONT), is based on the principle of reading electrical signals instead of optical detection, unlike other sequencing techniques. In this method, DNA molecules are passed through the nanopores one by one using a membrane consisting of biological nanopores with the help of an electric field. In this process, DNA is made single-stranded by enzymes and passed through the nanopore at a certain speed with the help of a motor protein. During the passage of DNA through the nanopore, changes occur in the ion current and each five-base-long (5-mer) sequence creates a unique electrical signal. These signals are processed by bioinformatics software such as Metrichor (13) and converted into DNA sequence (14).

Figure 7. Principle of nanopore sequencing (15).

Advantages of Nanopore Sequencing

• It can perform real-time sequencing and provide results instantly.

• It can read very long DNA or RNA sequences (50 kb or longer).

• It can be used in the field with compact and portable devices (MinION).

Disadvantages of Nanopore Sequencing

• Its error rate is higher than methods such as Illumina.

• It requires powerful bioinformatics tools for data analysis.

• There is a risk of some bases being read incorrectly due to signal changes.

DNA sequencing technologies have a wide range of uses from genetic research to clinical diagnosis. Although old-generation sequencing methods are used in the analysis of certain genome regions with high accuracy rates, large-scale genome projects are completed more quickly and economically thanks to Next Generation Sequencing (NGS) methods. While short-read methods allow sensitive mutation detection with lower error rates, long-read technologies play an important role in large genome analyses and structural variation detection. As a result, DNA sequencing technologies are rapidly evolving and continue to revolutionize many fields, including medical genetics, personalized medicine, agricultural biotechnology, and forensic science.

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