Next-Generation Sequencing

The first venture into DNA sequencing was The Human Genome Project, a 13 year long, 3 billion dollar endeavor. The Human Genome Project was accomplished with the help of the first-generation Sanger sequencing. Since then, the need for accessible, faster, and efficient DNA sequencing methods has increased dramatically. This has resulted in the birth of Next-Generation Sequencing (NGS). NGS performs a process known as parallel sequencing. Millions of DNA sequences from a single sample are simultaneously sequenced, allowing the entire human genome to be sequenced within a day.

The methodology behind NGS involves three main steps:
Library Preparation
Sequencing and Imaging
Data Analysis

Library Preparation
This is the first step involved in NGS and includes building a library of nucleic acids and then amplifying it. DNA extracted from the cell or tissue is fragmented; these fragments are then converted into the library by ligating them with sequencing adapters containing specific sequences designed to interact with the NGS platform. Once built, these DNA libraries are clonally amplified and prepared for sequencing.

Sequencing and Imaging
DNA sequencing of whole chromosomes or whole genomes as well as targeted regions, such as an exome, can be carried out using NGS. The library's DNA fragments act as a template, off of which a new DNA fragment is created. The fragments formed are subjected to cycles of washing and flooding with known nucleotides in a sequential order. As these nucleotides get incorporated into the fragment, they are digitally recorded as a sequence.

Data Analysis
This includes both variant identification and annotation followed by visualization. Variant identification is a vital part of NGS data analysis. In this, sequence coverage is the main parameter, as identified mutations should be supported by several reads. Tools of variant identification are divided into four categories:

1. Germline callers,
2. Somatic callers,
3. Copy Number Variants identification, and
4. Structural Variants identification. Annotation of variants provides biological significance by identifying disease-causing variants. After annotating the variants, they are visualized using visualization tools and genome browsers. By visualizing the variants, we can obtain information about variants, such as mapping quality, aligned reads, and annotation information.

Next-generation sequencing has a plethora of clinical applications. Any given tumor can have multiple mutations; in order to identify all of them, numerous assays need to be performed. In contrast, by using NGS, all targets can be interrogated using a single test. NGS can also be used to identify rare genetic diseases when there are no specific mutations; NGS can sequence the individual's whole genome to provide information on the disease-associated mutations. One of the main aims of NGS is to replace the conventional characterization of pathogens with genomic identification; it is also extensively employed in analyzing the human microbiome. Next-generation sequencing provides essential information on disease diagnostic classification, selection of therapeutic agents, and prognostic evaluation, thus becoming an important tool in personalized precision medicine.