Next Generation Sequencing (NGS) vs. PCR

What is PCR?

PCR, short for Polymerase Chain Reaction, serves as a pivotal tool for detecting known mutations. It operates by identifying specific DNA fragments, thereby discerning whether a gene has undergone mutation. This technique mimics the natural process of DNA replication, albeit in a controlled, laboratory setting. Over time, PCR has evolved, spawning various iterations to suit diverse applications.

The first generation of PCR, known as qualitative PCR, employs standard PCR instruments to amplify target genes. Subsequent analysis entails agarose gel electrophoresis to scrutinize the resulting product.

Advancing the field, the second generation introduces fluorescent quantitative PCR (qPCR). This technique allows real-time monitoring of amplification products by incorporating fluorescent reagents. Despite its capability for relative quantification using standard curves, qPCR falls short in providing absolute quantification.

Another notable advancement is the Mutation Amplification Blocking System (ARMS), which leverages specific primers to amplify mutant target sequences with precision. Coupled with probe detection and real-time fluorescence quantitative PCR, ARMS enables the detection of rare mutations with heightened specificity and sensitivity. However, its scope is limited to known mutation types, rendering it unable to identify novel mutations.

Principles and Procedures of PCR

The PCR process entails several fundamental components:

• DNA Template: This refers to the sample DNA containing the target sequence.
• DNA Polymerase: Utilized to synthesize a new DNA strand complementary to the target sequence. Commonly used polymerases include Taq DNA polymerase.
• Nucleotides: These are the building blocks, specifically deoxyribonucleotide triphosphates (dNTPs) (A/T/C/G), essential for constructing the new DNA strand.
• Reverse Transcriptase: In reverse transcription PCR (RT-PCR), reverse transcriptase is crucial for converting sample RNA into complementary DNA (cDNA).

Sanger Sequencing

Sanger Sequencing stands out for its ability to directly identify both known and unknown mutations, making it a valuable asset in genetic analysis. However, its sensitivity is notably limited when it comes to detecting mutations within the EGFR gene.

This method involves sequentially reading the DNA bases over a stretch of approximately 800 base pairs, earning it the status of the gold standard in both traditional and next-generation sequencing. Notably, Sanger sequencing played a pivotal role in completing the groundbreaking Human Genome Project, spanning over 13 years.

In practice, Sanger sequencing entails designing primers targeting mutation sites within known disease-associated genes, followed by PCR amplification for direct sequencing. Unlike comprehensive exon amplification, only the specific mutation site or a nearby portion of the exon requires amplification. Despite its utility, Sanger sequencing's sensitivity is limited; it may fail to detect mutations occurring at frequencies below 5%.

Next Generation Sequencing (NGS)

NGS, or Next Generation Sequencing, offers remarkable exploratory power, generating copious amounts of data from a single DNA starting point, thus enabling higher sample throughput. This DNA sequencing technology, which builds upon PCR and GeneChip methodologies, represents a significant advancement over traditional sequencing techniques.

While first-generation sequencing employed synthetic termination sequencing, NGS introduces reversible termination ends, enabling sequencing concurrently with synthesis. NGS functions by capturing specific tags, typically fluorescent molecular markers, carried by newly added bases during DNA replication to determine the DNA sequence.

Also referred to as Massively Parallel Sequencing (MPS) or High-Throughput Sequencing (HTS), NGS stands out for its scalability, speed, and accessibility in analyzing genomic or partial nucleic acid sequences. It has the capability to sequence millions or even billions of DNA molecules simultaneously, revolutionizing large-scale sequencing endeavors.

NGS encompasses two main types: short-read sequencing and long-read sequencing. Short-read sequencing, characterized by its maturity in accuracy and cost-effectiveness, finds widespread use in basic research and clinical applications. Conversely, long-read-long sequencing, while still undergoing optimization in accuracy and cost, addresses the limitations of short-read sequencing in detecting structural variations in large fragments.

Advantages of NGS include its ability to amplify DNA through nanoscale PCR reactions, resulting in massive data generation through parallel reactions. The decreasing cost of data generation has democratized large-scale gene sequencing, facilitating population-level research and enhancing applications in clinical diagnostics, drug discovery, and pathogen tracing.

Table 1 NGS vs. PCR

Furthermore, advancements such as PacBio's SMRT technology and ONT's Nanopore sequencing have expanded NGS capabilities. PacBio's technology facilitates de novo assembly of complex species, fostering biodiversity studies, while ONT's Nanopore sequencing, with its PCR-independent approach and long read lengths exceeding 4 Mb, enhances structural variant detection. Portable NGS devices extend sequencing applications from laboratories to field settings and even into space, broadening the scope of genomic research and its practical applications.

Choosing Between NGS and qPCR: Factors and Considerations

Determining whether to utilize NGS or qPCR hinges on several factors, including the sample size, the extent of sequences in the target region, budget constraints, and the study's objectives. qPCR typically proves advantageous when dealing with a limited number of target regions (≤20) and when the study aims at screening or identifying known variants. Conversely, NGS emerges as the preferred choice in most other scenarios.

In contrast to traditional iterative testing approaches, targeted NGS streamlines processes by enabling concurrent sequencing of multiple genes across numerous samples. Moreover, NGS offers superior exploratory capabilities, facilitating the detection of novel variants. Thus, it's often the method of choice for studies requiring broader genomic insights or large-scale sequencing endeavors.