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What is next-generation sequencing?
Next-Generation Sequencing (NGS) is a DNA and RNA sequencing method that uses massively parallel processing to quickly and accurately determine genetic sequences. It can sequence entire genomes or targeted regions, identifying genetic variations such as single nucleotide changes, structural variants, and RNA fusions. Compared to Sanger sequencing, NGS offers faster results, higher throughput, and lower costs, making it essential for research, clinical diagnostics, and genetic analysis.
Applications of Next-Generation Sequencing (NGS)
Next-Generation Sequencing (NGS) is used in many areas of genetic research. Some of the main applications are:
1. Whole Genome Sequencing (WGS): NGS can sequence entire genomes, identifying genetic variations like mutations and structural changes. It is useful for studying diseases, evolution, and genetic diversity.
2. Targeted Sequencing: This method focuses on specific parts of the genome to study mutations related to certain diseases, reducing sequencing costs and time.
3. RNA Sequencing (RNA-Seq): NGS is used to measure gene expression, detect new RNA changes, and study gene regulation. It helps understand how genes are turned on or off.
4. Cancer Research: NGS helps find mutations in cancer cells, track tumor changes, and monitor how tumors grow. Liquid biopsies, which use blood samples to analyze tumor DNA, are becoming more common for tracking cancer.
6. Rare Disease Research: NGS can quickly find genetic mutations linked to rare and inherited diseases, helping with diagnosis and research.
5. Microbiology and Infectious Diseases: NGS is used to identify pathogens, track disease outbreaks, and study antimicrobial resistance by sequencing the genomes of bacteria, viruses, and other microbes.
7. Multiomics: NGS can be combined with other techniques to study DNA, RNA, proteins, and other molecules at once. This approach gives a fuller picture of how biological systems work and is useful in personalized medicine.
Steps in the NGS Process :
The NGS workflow consists of several key steps, from extracting nucleic acids to analyzing sequencing data. Here’s an overview of each step in the process:
Step 1: Nucleic Acid Extraction
The first step is isolating genetic material (DNA or RNA) from samples such as tissue, individual cells, or biofluids. After extraction, a quality control (QC) step is needed. Common methods for QC include UV spectrophotometry for purity and fluorometric methods for quantifying nucleic acids.
Step 2: Library Preparation
The extracted DNA or cDNA is fragmented into smaller pieces to create a sequencing library. This library is necessary for the sequencing process, as it allows the NGS instrument to read and analyze the genetic material.
Step 3: Sequencing
Libraries are loaded onto a sequencing platform, such as Illumina, where the DNA is read. Sequencing involves determining the order of nucleotides in the sample. The read length (the size of each DNA fragment read) and depth (the number of reads per sample) depend on the specific research needs. Illumina uses sequencing by synthesis (SBS) to read bases as they are incorporated into DNA strands. Different sequencing platforms are available to meet the requirements of various applications and throughput needs.
Step 4: Data Analysis and Interpretation
Once sequencing is complete, bioinformatics tools process the sequence data. The sequence reads (As, Ts, Gs, Cs) are analyzed using algorithms to make sense of the genetic information. Some sequencing systems offer built-in analysis tools, making data interpretation accessible to users with minimal bioinformatics experience. Advanced tools can perform tasks like read quality control, alignment, variant calling, and gene expression analysis.
