Understanding the key differences and when next-generation sequencing can be a more effective option than qPCR

Differences between NGS and qPCR

When comparing next-generation sequencing (NGS) vs quantitative PCR (qPCR) technologies, the key difference is discovery power. While both offer highly sensitive and reliable variant detection, qPCR can only detect known sequences. In contrast, NGS is a hypothesis-free approach that does not require prior knowledge of sequence information. NGS provides higher discovery power to detect novel genes and higher sensitivity to quantify rare variants and transcripts.

NGS vs qPCR technologies also differ in scalability and throughput. While qPCR is effective for low target numbers, the workflow can be cumbersome for multiple targets. NGS is preferable for studies with many targets or samples. A single NGS experiment can identify variants across thousands of target regions with single-base resolution.

NGS vs qPCR: A detailed comparison

NGS vs qPCR: A detailed comparison

When compared to qPCR, certain NGS methods can:

  • Detect both known and novel transcripts
  • Quantify individual sequence reads to produce absolute, not just relative, expression values
  • Detect subtle changes in gene expression, down to 10%
  • Identify novel transcripts, alternatively spliced isoforms, splice sites, and small and noncoding RNA species
  • Profile > 1000 target regions in a single assay
qPCR and targeted NGS comparison
qPCR allows for the analysis of particular variants at specific locations.
Targeted NGS
Targeted NGS simultaneously sequences several hundreds to thousands of genes.
  • Familiar workflow
  • Accessible equipment available in most labs
  • High sequencing depth enables high sensitivity (down to 1%)
  • Higher discovery power*
  • Higher mutation resolution†
  • Massively parallel sequencing enables high-throughput workflows and large datasets
  • Detects gene expression changes down to 10%
  • Only accesses a predefined set of transcripts
  • Detects only known transcripts, limiting discovery power
  • Limited throughput and mutation resolution
  • Not always as efficient for simple detection of a low number of targets

* Discovery power is the ability to identify novel variants.

† Mutation resolution is the size of the mutation identified. NGS can identify large chromosomal rearrangements down to single nucleotide variants.

It became obvious how hit-and-miss gene association studies were in identifying variants. They were more like fishing expeditions. We realized that NGS would enable us to look at much larger portions of the genome simultaneously.

more than 1000 target regions profiled in a single assay

Advantages of RNA-Seq vs qPCR

While qPCR is useful for quantifying the expression of a few genes, it can only detect known sequences. In contrast, RNA sequencing (RNA-Seq) using NGS can detect both known and novel transcripts. Because RNA-Seq does not require predesigned probes, the data sets are unbiased, allowing for hypothesis-free experimental design.

Key advantages of RNA-Seq over qPCR:

  • Increased discovery power for detecting novel transcripts
  • Enhanced sensitivity for detecting rare variants and lowly expressed genes
  • Higher throughput for simultaneous sequencing of multiple genes across multiple samples
  • Wider dynamic range for quantifying expression of genes without background noise or signal saturation
Next-gen sequencing is revolutionizing research around the globe

When to use NGS vs qPCR

The choice between NGS and qPCR depends on several factors, including the number of samples, the total amount of sequence in the target regions, budgetary considerations, and study goals. qPCR is typically a good choice when the number of target regions is low (≤ 20 targets) and when the study aims are limited to screening or identification of known variants.

Otherwise, NGS is more likely to suit your needs. With the ability to sequence multiple genes across multiple samples simultaneously, targeted NGS methods save time and resources compared to traditional iterative methods. NGS also provides higher discovery power, enabling detection of novel variants.

View video: Using NGS instead of qPCR

Transitioning from qPCR to NGS

The MiSeq System makes it easier and more affordable than ever to bring the power of next-generation sequencing to your lab. After sequencing is complete, tools such as Correlation Engine enable comparison of prior qPCR data with NGS data.

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Using qPCR but curious about NGS?

If you have questions about NGS for your specific research focus, we’d love to help. Our specialists can answer any questions and recommend the best solution for your setup.

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  4. Illumina. High-impact discovery through gene expression and regulation research. Accessed March 23, 2023.
  5. Illumina. Advantages of next-generation sequencing vs qPCR. Accessed May 22, 2023.