What is NGS?
NGS involves massively parallel sequencing of millions of DNA fragments at once, producing billions of nucleotides of data in a single run.
Main Steps in NGS Workflow
- Sample preparation
- Library preparation
- The fragments with adapters form a “library.”
- May include barcoding (indexing) for multiplexing samples.
- Amplification
- Clonal amplification of DNA fragments (e.g., bridge PCR on Illumina, emulsion PCR on Ion Torrent).
- Sequencing
- Sequencing-by-synthesis (Illumina)
- Semiconductor sequencing (Ion Torrent)
- Single-molecule real-time sequencing (PacBio, Nanopore)
- Data analysis (bioinformatics)
- Base calling, quality control
- Alignment to reference genome
- Variant calling (SNPs, indels, structural variants)
- Annotation and interpretation
Applications of NGS
- Clinical/Medical
- Cancer genomics (tumor profiling, minimal residual disease)
- Pharmacogenomics
- Rare genetic disorder diagnosis
- Infectious disease detection (e.g., COVID-19 variants, HIV resistance testing)
- Research
- Whole-genome sequencing (WGS)
- Whole-exome sequencing (WES)
- Transcriptome sequencing (RNA-seq)
- Epigenomics (ChIP-seq, methylation studies)
- Metagenomics (microbiome studies)
Advantages
- High throughput (millions of reads per run)
- Broad applications (DNA, RNA, epigenetics, microbiome)
- Rapid turnaround time
- Detects rare/low-frequency variants
Limitations
- Expensive equipment and analysis infrastructure
- Requires advanced bioinformatics
- Errors in repetitive or GC-rich regions
- Interpretation challenges (variants of uncertain significance)
Common NGS Platforms
- Illumina (MiSeq, NextSeq, NovaSeq) → sequencing by synthesis, short reads, high accuracy
- Ion Torrent (Thermo Fisher) → semiconductor sequencing
- PacBio SMRT → long reads, useful for structural variants
- Oxford Nanopore → portable, real-time sequencing, long reads