What is Next-Generation Sequencing (NGS)?

What is Next-Generation Sequencing (NGS)?

NGS is a term used to represent different technologies that enable massively parallel sequencing of clonally amplified or single DNA molecules, allowing high-throughput and comprehensive sequencing of DNA or RNA in a short time frame at an affordable cost per patient. 1

Core Technology Principles

NGS technologies differ from traditional sequencing by enabling the simultaneous sequencing of millions of DNA fragments, dramatically reducing both cost and turnaround time while increasing throughput. 1, 2 The various commercially available platforms differ in:

• Sequencing chemistries used 1
• Read lengths produced (ranging from hundreds of bases to tens of kilobases) 1
• Throughput capabilities 1
• Cost per base sequenced 1

Platform Categories

NGS platforms can be divided into two main categories:

• Short-read technologies: Produce reads in the hundreds of bases, offer lower per-base cost, and are sufficient for microbial genomic analyses, strain typing, outbreak tracing, and pangenome surveys 1
• Long-read technologies: Produce reads tens of kilobases in length, allow for finishing of microbial genomes for under $1,000 per genome, and are essential for resolving structural variants, genome rearrangements, duplications, deletions, and interspersed repeats 1

Standard NGS Workflow Components

Wet-Lab Steps (Sample Processing)

The laboratory processing involves several sequential steps:

• DNA extraction and quantification (or RNA-to-cDNA conversion by reverse transcription for RNA viral sequencing or transcriptome profiling) 1
• Library preparation: DNA is fragmented, adaptors are added to each fragment, and fragments are amplified prior to sequence generation 1
• Sequencing: The prepared library undergoes the actual sequencing reaction on the NGS platform 1

Dry-Lab Steps (Bioinformatics Pipeline)

The computational analysis is divided into three distinct phases:

Primary Analysis: Converting images or signals from the instrument into base calls with assigned quality scores that describe the probability of correct base assignment 1

Secondary Analysis: Processing and quality assessment of primary sequence data, including trimming and filtering based on laboratory-established quality thresholds, followed by either alignment to a reference sequence or de novo assembly to create full-length sequences 1

Tertiary Analysis: Interpretation of results, identification of clinically significant findings, and generation of final reports, which may include pathogen identification, variant calling, functional annotation, and taxonomic classification 1

Clinical Applications

NGS has transformed multiple areas of clinical practice:

• Cancer genomics: Simultaneous examination of point mutations, amplifications, deletions, fusions, microsatellite-instability status, and tumor mutation burden 1
• Inherited disorders: Whole-exome sequencing has become a frontline diagnostic tool for Mendelian conditions 1
• Precision oncology: Targeted panels interrogate medically relevant subsets of genes to guide treatment decisions 1
• Infectious disease diagnostics: Three distinct approaches include whole-genome sequencing, targeted NGS, and metagenomic NGS 3
• Microbial genomics: Strain typing, outbreak tracing, and tracking emergence of drug resistance 1

Sample Types for NGS

Both DNA and RNA can be isolated from various sources:

• Tissue biopsies or cytological specimens: Historically the standard sample type 1
• Circulating tumor DNA (ctDNA): Can be used as an alternative source in liquid biopsy 1
• DNA-based assays: More stable than RNA, facilitating convenient extraction, but less sensitive for gene fusions and alterations involving intronic regions 1
• RNA-based assays: More sensitive for gene fusions and intronic alterations, though less stable than DNA 1

Key Advantages Over Traditional Methods

• Massively parallel processing: Enables sequencing of multiple genes simultaneously rather than sequential single-gene testing 1
• Cost-effectiveness: Inexpensive production of large volumes of sequence data compared to conventional methods 2
• Comprehensive analysis: Can identify novel disease-associated genes and variants, aiding in development of targeted therapies 4
• Speed: Faster identification of patients with therapeutically targetable molecular alterations 1
• Tissue preservation: Reduces need for multiple biopsies by testing multiple targets from a single sample 1

Critical Infrastructure Requirements

The implementation of NGS requires substantial infrastructure that differs significantly from traditional laboratory operations:

• Large computational servers and databases necessary for NGS applications 1
• Professional IT support: Due to increasingly demanding technical requirements, e-infrastructure development and maintenance should be handled by professional service units 1
• Automated informatics components: Substantial automated systems for primary, secondary, and tertiary analysis 1
• Collaboration between researchers and IT professionals is essential for successful implementation 1

Important Limitations and Considerations

Turnaround time: Results generally take 2-4 weeks for comprehensive NGS panels, which can be an important consideration for patients with significant tumor burden 1

Quality requirements: All NGS testing requires high-quality DNA or RNA, and reliability is highly dependent on sample quality 1

Cost considerations: NGS is associated with higher cost compared with single-gene testing methods like immunohistochemistry and FISH, though this is offset by comprehensive analysis 1

Data analysis challenges: The sheer amount and variety of data generated require sophisticated computational methods and bioinformatics expertise 5