NGS vs qPCR
NGS vs qPCR
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
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 qPCR allows for the analysis of particular variants at specific locations. |
Targeted NGS Targeted NGS simultaneously sequences several hundreds to thousands of genes. |
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| Benefits1-5 |
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| Challenges1-5 |
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* 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.
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
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.
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.
Prep
Illumina Stranded mRNA Prep
A simple, scalable, cost-effective, rapid single-day solution for analyzing the coding transcriptome.
RNA Prep with Enrichment + targeted panel
Achieve rapid, targeted interrogation of an expansive number of target genes with exceptional capture efficiency and coverage uniformity.
Sequence
MiSeq System
For smaller panels: targeted resequencing, metagenomics, small genome sequencing, targeted gene expression profiling, and more.
NextSeq 1000 & 2000 Systems
For larger panels: Targeted sequencing, RNA-Seq, single-cell methods, exome sequencing, and more.
Benchtop sequencers
Explore and compare benchtop sequencers to find the one that’s right for your research needs.
Analyze
DRAGEN RNA App
DRAGEN RNA app performs NGS secondary analysis of RNA transcripts.
Correlation Engine
Correlation Engine is an interactive omics knowledgebase that puts private omics data in biological context with highly-curated public data.
Getting started with NGS: The complete guide
This 20+ page eBook is a comprehensive, yet easy-to-follow guide all about NGS methods and applications, workflows, data analysis solutions, and a step-by-step guide to getting started with NGS.
Download eBookRelated content
NGS vs other methods
NGS offers the power to sequence vast amounts of genetic material at a fraction of the time and cost of traditional methods. Learn more about how NGS compares to traditional molecular biology methods.
NGS applications
Thanks to its scale and unbiased discovery power, researchers can use NGS in a variety of basic and translational research areas. Learn more about the methods and applications of NGS.
Using custom RNA panels for targeted gene expression profiling
Watch as a product team evaluates how AmpliSeq for Illumina Custom RNA assay compares to qPCR for analyzing differential gene expression.
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.
References
- Ozsolak F, Milos PM. RNA-Sequencing: advances, challenges and opportunities. Nat Rev Genet. 2011;12:87-98.
- Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet. 2009;10:57-63.
- Myllykangas S and Hanlee JP. Targeted deep resequencing of the human cancer genome using next-generation technologies. Biotechnol Genet Eng Rev. 2010; 27: 135–158.
- Illumina. High-impact discovery through gene expression and regulation research. https://www.illumina.com/content/dam/illumina-marketing/documents/gated/gene-expression-profiling-e-book-web.pdf. Accessed March 23, 2023.
- Illumina. Advantages of next-generation sequencing vs qPCR. https://www.illumina.com/science/technology/next-generation-sequencing/ngs-vs-qpcr.html. Accessed May 22, 2023.