Next-Generation Sequencing

Human Genome Project

International effort to sequence the entire human genome and identify all genes

Bermuda Principles

Guidelines requiring genomic data to be publicly released within 24 hours to promote collaboration

Sanger Sequencing

First-generation DNA sequencing method using chain termination to determine nucleotide order

Human Genome Project Outcomes

Led to precision medicine, disease gene identification, biotechnology advances, and global data sharing

Precision Medicine

Approach that tailors treatment based on an individual’s genetic profile

Next Generation Sequencing (NGS)

Massively parallel sequencing that allows millions of DNA fragments to be sequenced simultaneously

Massively Parallel Sequencing

Technique where many DNA fragments are sequenced at the same time to increase speed and throughput

Library Preparation

Process of fragmenting DNA and adding adapters and barcodes for sequencing

DNA Library

Collection of DNA fragments with adapters ready for sequencing

Adapters

Short DNA sequences added to fragments to allow binding, amplification, and sequencing

Barcoding/Indexing

Unique sequences added to samples so multiple samples can be sequenced together and later separated

Clonal Amplification

PCR-based process that creates many copies of a DNA fragment to increase detection signal

Flow Cell

Solid surface where DNA fragments attach and are amplified during sequencing

Sequencing by Synthesis

Method where nucleotides are added one at a time and detected as they are incorporated

Fluorescent Detection

Sequencing method where labeled nucleotides emit light to identify bases

Semiconductor Sequencing

Sequencing method that detects pH changes from hydrogen ion release during nucleotide incorporation

Ion Torrent Sequencing

NGS platform that detects voltage changes from pH shifts caused by hydrogen ion release

Emulsion PCR (emPCR)

Amplification method used in Ion Torrent where DNA is amplified on beads in droplets

Sequence Read

Short DNA sequence generated from a single fragment during sequencing

FASTQ Format

File format that stores sequence reads and their quality scores

Data Analysis in NGS

Computational process of aligning reads and identifying genetic variants

Single Nucleotide Polymorphism (SNP)

Variation of a single nucleotide in the DNA sequence

Insertion

Addition of one or more nucleotides into DNA

Deletion

Loss of one or more nucleotides from DNA

Indel

Combination of insertion and deletion mutations

Targeted Sequencing

Sequencing of specific genes or regions of interest

Amplicon-Based PCR

Target enrichment method using primers to amplify specific regions

Hybridization Capture

Target enrichment method using probes to isolate DNA regions of interest

Cancer Mutation Detection

NGS application that identifies mutations driving tumor growth and guides therapy

EGFR/KRAS/BRAF Mutations

Common cancer-related gene mutations detected by NGS

Immunotherapy Biomarkers

Genetic markers used to determine response to immune-based cancer treatments

Microsatellite Instability (MSI)

Genetic condition indicating defective DNA mismatch repair

Tumor Mutational Burden (TMB)

Measure of total mutations in a tumor genome مرتبط with immunotherapy response

Inherited Disease Testing

Use of NGS to detect mutations causing hereditary disorders

Infectious Disease Detection

Use of NGS to identify pathogens and resistance genes

Progressive Multifocal Leukoencephalopathy (PML)

Demyelinating disease caused by JC virus reactivation in immunocompromised patients

JC Virus (JCV)

Virus that infects oligodendrocytes and causes PML

Oligodendrocytes

Cells responsible for myelin production in the central nervous system

Demyelination

Loss of myelin sheath leading to impaired nerve conduction

NGS in PML

Detects JC virus DNA and helps analyze viral genetic characteristics