If you’re wondering about the key differences between next generation sequencing, or NGS, compared to qPCR technologies, there’s one key difference to keep in mind: discovery power. Though both provide very sensitive, reliable detection of variants, qPCR is only capable of picking up known sequences, while NGS’ approach requires no prior knowledge of the sequence in question. This makes it an optimal tool for detecting novel variants while providing higher sensitivity in quantifying rare variations and transcriptions.
These technologies also differ in their throughput and scalability. As an example, qPCR is effective when low target numbers are used, but workflows become cumbersome when multiple targets are approached. NGS is preferred for multi-target or multi-sample studies, as one NGS experiment can find variants across thousands of targets using single-base resolution.
NGS vs qPCR
Generally speaking, qPCR is beneficial in that it has capital equipment already available in most labs and a familiar workflow, but is limited in that it’s only able to check limited variant sets, has almost no discovery power and is difficult to scale. By comparison, targeted next generation sequencing has a much higher discovery power and throughput of samples, but is less cost-effective and more time-consuming when sequencing low target numbers.
What About RNA-Seq vs. qPCR
Grt-PCR can be used for quantifying gene expression in small numbers, it’s only able to detect sequences already known. RNA sequencing (RNA-Seq), when handled with a next generation sequencing approach detects both known and novel genetics. Because redesigned probes aren’t required, data sets are unbiased for experiment design.
When using read-counting methods, NGS’ digital nature provides a near-unlimited dynamic range. RNA-Seq quantifies the individual sequences as aligned to a reference, delivering absolute values rather than relative. The broad range provides easier detection of small changes in gene expression to as low as 10%. RNA-Seq can also identify novel transcripts, splice sites, alternatively spliced isoforms and non-coding RNA.
When Should You Use NGS or qPCR?
Choosing between next generation sequencing and qPCR tests will depend on several factors that can impact your operation. This can include how many samples you have, the total amount of the genetic sequence within the targeted regions, budget concerns and the overall goals of your study.
Generally speaking, qPCR is a good choice when you’re using a low number of target regions, generally considered to be equal to or less than 20 targets. However, your study should also be limiting your samples to only screening or identifying known variants. If your study moves outside of these constraints, NGS should be considered.
If your study has over 20 target regions, may encounter unknown or novel variants, needs to sequence multiple genes across several different samples at the same time, targeted NGS methods will provide you with significant time and resource savings when compared to qPCR and similar iterative approaches.
Though there is room for both NGS and qPCR testing in medical and genetic science, both approaches have their own strengths and drawbacks that should be carefully considered prior to making a decision for your study or testing facility.