Genomic sequencing has revolutionized biology and medicine and is now one of the most important tools in biomedical research. It has led to major advances in our understanding of genetics, disease and evolution. Genomic sequencing has also brought about innovations in drug development. It encompasses DNA and RNA sequencing approaches to unlock answers to questions rooted in the genome.

What is DNA Sequencing?

DNA sequencing determines the linear order of nucleotides in a DNA molecule. DNA sequences can be derived from short segments of 10 nucleotide bases to long reads over thousands of bases.

In the 1970s, Fredrick Sanger developed the dideoxy sequencing method that researchers used for the Human Genome Project. Focused on speed and accuracy, newer sequencing platforms facilitate many DNA reads simultaneously, allowing the human genome to be sequenced in a day.

Common Types of DNA Sequencing Methods

There are different DNA sequencing methods available including Sanger dideoxy sequencing and next-generation sequencing (NGS). Newer techniques, such as Single-Molecule Real-Time (SMRT) and nanopore sequencing allow for long sequence determinations.

Sanger DNA Sequencing

In Sanger DNA sequencing, fluorescent chain-terminating inhibitors (also known as fluorescent ddNTPs) are used to label the DNA fragments generated during the sequencing reaction. These fluorescent ddNTPs are like standard ddNTPs but lack the 3’ hydroxyl group necessary for the next nucleotide to be added to the growing strand. This causes the DNA synthesis to terminate at the point where a fluorescent chain-terminating inhibitor is incorporated, creating a series of DNA fragments of different lengths.

Each of the four different fluorescent chain-terminating inhibitors (A, C, G, T) has a different color or wavelength of fluorescence. Once the DNA synthesis is terminated and the fragment is separated by gel electrophoresis, the fluorescence can be detected. The order of nucleotides in the original DNA molecule can be read by detecting the color of the fluorescent label at the end of each fragment.

Next-Generation Sequencing (NGS)

NGS, also known as short-read sequencing, can sequence large amounts of DNA approximately 100-300 nucleotides in length.

NGS platforms generate enormous amounts of data, which must be processed and analyzed using specialized software tools. The data generated by NGS can be used for a variety of applications, such as whole genome sequencing, gene expression profiling and epigenetic studies.

For whole genome sequencing, NGS platforms typically produce millions to billions of short sequence reads that are then computationally reassembled into a complete genome. However, depending on the accuracy of the reads and software algorithms, the assemblies can range from highly accurate to very fragmented. Once an assembly is generated, it can be annotated with gene predictions and other functional information.

Third-Generation Sequencing

Third-generation sequencing, commonly known as long-read sequencing, is a method of determining the sequence of a DNA molecule typically >1,000 bases. Long-read sequencing allows for the detection of structural variations (e.g., insertions, deletions and inversions), and is preferred for de novo assemblies and haplotyping.

Single-Molecule Real-Time (SMRT) sequencing measures fluorescent-labeled nucleotide incorporation in real-time. Nanopore sequencing allows for long sequence reads without labels or PCR.

What is RNA Sequencing?

RNA sequencing (RNA-seq) is a technique used to determine the order of nucleotides in an RNA molecule. RNA-seq can be used to study the transcriptome and quantify gene expression in cells and tissues. It can be used to detect differentially expressed genes between two groups of samples, such as cancerous and healthy tissue. It can also be used to identify gene fusions, point mutations, and other types of sequence variations.

The process of RNA-seq begins with the isolation and purification of RNA, followed by reverse transcription to convert the RNA into its complementary DNA (cDNA) strand. The cDNA is sequenced using NGS then analyzed to infer information about gene expression of the sample. However, third-generation sequencing platforms can facilitate native RNA sequencing. This is desirable because modifications are retained, and cDNA conversion errors are avoided.

NGS Applications

NGS is revolutionizing the field of genomics by providing new insights into disease etiology and treatments. NGS is used for a variety of applications, including:

Genome sequencing

• NGS has enabled the sequencing of entire genomes with unprecedented speed and accuracy. This has greatly facilitated the study of genetic variation, evolution and disease.

Transcriptome analysis

• NGS has made it possible to sequence and quantify the expression of all the genes in a tissue or organism at once. This has greatly improved our understanding of gene regulation and the complexity of cellular processes.

Epigenetics

• NGS has enabled the analysis of epigenetic modifications, such as DNA methylation and histone modifications, at a genome-wide scale. This has greatly improved our understanding of gene regulation and the role of epigenetics in disease.

Metagenomics

• NGS has made it possible to sequence the DNA of entire microbial communities. This has greatly improved our understanding of the diversity and function of microbial ecosystems.

NGS in Clinical Practice

NGS has impacted human health significantly by providing new insights and solutions, including:

Diagnosis of Genetic Disorders

• NGS can be used to identify genetic mutations or variations that are responsible for inherited diseases. This can greatly improve the accuracy of diagnosis and enable personalized treatment.

Infectious Disease Diagnosis

• NGS can be used to identify the specific pathogen causing an infection, enabling targeted treatment and better management of outbreaks.

Cancer Diagnosis and Treatment

• NGS can be used to identify genetic mutations or alterations in cancer cells, which can inform treatment decisions and help identify targeted therapies.

Pharmacogenomics

• NGS can be used to identify genetic variants that affect a patient's response to specific drugs. This can help optimize drug selection and dosing for individual patients.

Prenatal Diagnosis

• NGS can be used to analyze fetal DNA from maternal blood samples, enabling non-invasive prenatal diagnosis of genetic disorders.

Future trends in sequencing

Genomic sequencing has advanced rapidly in recent years, and there are several trends that are expected to shape the field soon:

Increased Speed and Affordability

• Next-generation sequencing (NGS) technologies are becoming faster and more cost-effective, making it possible to sequence entire genomes quickly and at a lower cost. This trend is expected to continue, with the goal of making genomic data more widely accessible to researchers and clinicians.

Improved Accuracy and Resolution

• DNA sequencing technologies are constantly being refined to increase accuracy, resolution and sensitivity. This includes developing new algorithms to improve error correction and increasing the read length of sequenced DNA fragments.

Increasing use of Cloud Computing

• As DNA sequencing data becomes larger and more complex, cloud computing is becoming an increasingly important tool for data management and analysis. This trend is expected to continue, with cloud-based platforms becoming more widely used for storing, analyzing and sharing genomic data.

Overall, these trends are expected to contribute to the continued growth and advancement of genomic sequencing, leading to new discoveries and innovations in many areas of biomedical research.

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