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.
* Discovery power is the ability to identify novel 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.”
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.1,2Read Application Note
An Illumina scientist evaluates how custom targeted RNA-Seq compares to qPCR for differential gene expression analysis.View Video
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.
When qPCR genotyping approaches provided “hit-and-miss” results, researchers switched to NGS, which enabled them to examine larger portions of the genome.Read Interview
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.Read Interview
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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-Seq enables researchers to sequence specific transcripts of interest, and provides both quantitative and qualitative information. Learn more about targeted RNA-Seq.