Advantages of next-generation sequencing vs. qPCR

Higher discovery power and wider dynamic range for quantifying variation

NGS vs. qPCR

When comparing next-generation sequencing (NGS) vs. 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.

Choosing Targeted NGS vs. qPCR

Explore the benefits and limitations of each method to understand which one best suits your needs.

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  qPCR Targeted NGS
  • Familiar workflow
  • Capital equipment already placed in most labs
  • Higher discovery power*
  • Higher sample throughput
  • Can only interrogate a limited set of variants
  • Virtually no discovery power
  • Low scalability
  • Less cost-effective for sequencing low numbers of targets (1–20 targets)
  • Time-consuming for sequencing low numbers of targets (1–20 targets)

* Discovery power is the ability to identify novel variants.

Cost-Effectiveness of NGS vs. qPCR

“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.”

Linda S. Pescatello, PhD
Distinguished Professor of Kinesiology, University of Connecticut

While qRT-PCR 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.

For read-counting methods, such as gene expression profiling, the digital nature of NGS allows a virtually unlimited dynamic range. RNA-Seq quantifies individual sequence reads aligned to a reference sequence, producing absolute rather than relative expression values. This broad dynamic range enables detection of subtle changes in expression, down to 10%. Beyond quantifying gene expression, RNA-Seq can identify novel transcripts, alternatively spliced isoforms, splice sites, and small and noncoding RNA.1,2

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NGS vs. qPCR
RNA-Seq vs. qPCR for Differential Gene Expression

An Illumina scientist evaluates how custom targeted RNA-Seq compares to qPCR for differential gene expression analysis.

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The choice between NGS vs. 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.

Guide to Targeted Resequencing

Learn how targeted NGS can help you gain insight, save time, and be more confident in your results compared to qPCR and other traditional methods.

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Beginner's Guide to Next-Generation Sequencing

Considering bringing next-generation sequencing to your lab, but unsure where to start? These resources cover key topics in NGS and are designed to help you plan your first experiment.

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Beginner's Guide to Next-Generation Sequencing
Genetic Variants Linked to Blood Pressure and Exercise

When qPCR genotyping approaches provided “hit-and-miss” results, researchers switched to NGS, which enabled them to examine larger portions of the genome.

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Targeted RNA Expression Analysis

Frank Middleton, PhD used RNA-Seq to analyze 370 genes of interest in a single assay, a study that would have been cost-prohibitive with custom qPCR or qPCR arrays.

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Targeted Resequencing
Targeted Resequencing

With targeted resequencing, a subset of genes or a genomic region is isolated and sequenced, which can conserve lab resources. Learn more about targeted resequencing.

Targeted RNA Sequencing
Advantages of Whole-Genome Sequencing

Targeted RNA-Seq enables researchers to sequence specific transcripts of interest, and provides both quantitative and qualitative information. Learn more about targeted RNA-Seq.

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See Illumina sequencing technology in action and learn how it works.

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Global Perspectives on the Impact of NGS

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  1. Ozsolak F, Milos PM. RNA-Sequencing: advances, challenges and opportunities. Nat Rev Genet. 2011;12:87-98.
  2. Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet. 2009;10:57-63.