Researchers studying multi-factorial genetic disorders, such as cardiomyopathy, are using next-generation sequencing, or NGS, to rapidly and effectively analyze disease-associated genes. If you’re thinking about using NGS for this application, you’ve probably asked, “should I use a targeted panel or do exome sequencing?” Targeted sequencing has gained popularity in clinical research, but how do you know if this is right for your research needs?
Let’s dig a little deeper into targeted sequencing and find out. Targeted sequencing introduces an enrichment step where specific genes or gene regions are selectively amplified using gene panels in a simple PCR reaction. A gene panel consists of a pool of oligonucleotide primer pairs, each pair designed to amplify a specified region by PCR—producing a library of DNA amplicons, ready for sequencing. A library corresponds to a single sample, and multiple libraries can be pooled together to be sequenced in the same run. So what are some factors to consider when deciding on the right approach for your research?
First, since NGS generates an abundance of data, it’s important to consider the implications of managing large datasets. When you’re uncertain of which genes to look for, exome-sequencing helps you look at thousands of genes to get a comprehensive view of the genetic landscape. However, this can create a huge amount of data. When analyzing genes with known or suspected associations with a disease, large datasets can make bioinformatics challenging, and add cost with little to no benefit. For such applications, targeted sequencing can generate smaller and more manageable datasets based on specific genes, helping you save time, cost, and data-analysis effort.
Next, by focusing on a small genomic region, the targeted sequencing approach achieves much deeper coverage: up to 5,000 times compared to 100 times the coverage of the exome-sequencing approach.This enables identification of low-frequency variants associated with a particular disease—helping to ensure that every underlying variant in a subset of genes is detected. With the exome-sequencing approach, some important variants may go undetected; however, when the causative genes are not completely known, it may be worth the risk of missing certain regions in support of research.
Another important question to ask is: “how much do I really want to know?” For clinical researchers focusing on the variants that matter, targeted panels can be customized to only include genomic regions of their interest. Unlike exome sequencing, which generates a large number of variants—a sizable proportion of which can be of unknown significance—targeted sequencing helps reduce the risk of stumbling upon these variants.
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