When starting out with Next Generation Sequencing, you might encounter a little puzzle: How do I sequence specific regions of DNA or RNA rather than the whole genome?.
The answer to your puzzle comes in the form of NGS Enrichment strategies.
Enrichment strategies for Next Generation Sequencing are methods to increase the amount of target DNA or RNA in a sample. They are used when researchers are only interested in some regions of the genome. The types of enrichment strategies are the capture-based method and PCR-based method.
For instance, as a researcher, you are not always interested in sequencing an organism's complete genome (WGS). Although you extract total DNA as your input sample, you might want to only sequence regions of interest using known sequences that bind to those regions.
In other cases, you are more interested in understanding the gene expression, and for this, you only need the mRNA, which is only 2% of the total RNA. So how do you separate your target mRNA from the rest of the sample? This is where enrichment strategies come into play.
Enrichment strategies are useful because they not only increase the depth of sequencing for your target molecules but these strategies also help you to save time and money.
In this article, you will see the different enrichment strategies for target DNA/RNA molecules, the steps of each strategy, advantages, disadvantages, and applications.
Table of Contents
An enrichment strategy is a method designed in molecular biology to target regions within a DNA or RNA sample. Enrichment is often used when the analysis focuses only on one section of the genome or transcriptome.
Enrichment strategies are convenient because they are cost-effective and save time since you only sequence a fraction of the genome.
An important concept in this process is sequencing depth. Sequencing depth is the number of times the DNA polymerase has to read the same region in the genome. Depending on the technology, this can be 1 to 100 times. There is a positive relationship between the amount of times the polymerase reads the same region and the accuracy. The less time spent reading, the less accurate it is.
Because enrichment enhances the sequencing depth in target molecules, more data can be obtained from your target regions in the genome, which reduces the downstream data analysis.
There are two commonly used types of enrichment strategies. They are the capture-based method and PCR-based method.
The capture-based method is an enrichment strategy where oligos with biotinylated ends bind to your DNA/RNA library target sequences. The capture-based method is used after the standard library preparation to enrich libraries with the molecules of your interest. The library can be prepared in advance using ligation or tagmentation processes.
In the capture method, oligos are complementary to your target sequences. The biotin molecule attached to the oligos is used to pull the target molecules down with beads; therefore, you end with an enriched library with the molecules of interest. This process also significantly reduces the size of the libraries that go for sequencing.
The PCR-based method is an enrichment strategy that uses different primers to amplify the region of interest.
Here, primers bind to your target region in your genomic DNA or input sample. Then, PCR amplification is performed to end with an enriched library. Unlike the capture-based method, the PCR-based method is done before the library preparation.
The PCR primers that bind to your target region of genomic DNA contain partial adapters. Then, several rounds of PCR amplification is done, and a second PCR step is necessary to attach the final NGS adapters to your target molecules. Finally, library prep proceeds as usual and allows you to end with an enriched library ready for sequencing.
Comparison of enrichment strategies
Enrichment strategies make NGS cheaper, more efficient, and reliable because it only focuses on target molecules. Each enrichment strategy has its advantages and disadvantages.
Applications of Enrichment Strategies in NGS
The capture-based method is commonly used for the following applications
- Studying Human diseases (Parasites, fungi, bacteria, and viruses)
- Detection of sequence variation
- Single nucleotide polymorphism detection (SNP)
- Insertion/deletion (Iindel) detection
- Copy number variation (CNV) detection
- Screening multiple markers in multiple samples
- Disease detection (Oncology)
- Germline variants detection
- Genome editing validation
The PCR-based method is commonly used for the subsequent applications
For more resources, check our website https://goldbio.com/
García-García, G., Baux, D., Faugère, V., Moclyn, M., Koenig, M., Claustres, M., & Roux, A.-F. (2016). Assessment of the latest NGS enrichment capture methods in clinical context. Scientific Reports, 6(1), 20948. https://doi.org/10.1038/srep20948
Gaudin, M., & Desnues, C. (2018). Hybrid Capture-Based Next Generation Sequencing and Its Application to Human Infectious Diseases. Frontiers in Microbiology, 9, 2924. https://doi.org/10.3389/fmicb.2018.02924
Herman, D. S., Hovingh, G. K., Iartchouk, O., Rehm, H. L., Kucherlapati, R., Seidman, J. G., & Seidman, C. E. (2009). Filter-based hybridization capture of subgenomes enables resequencing and copy-number detection. Nature Methods, 6(7), 507-510. https://doi.org/10.1038/nmeth.1343
Kozarewa, I., Armisen, J., Gardner, A. F., Slatko, B. E., & Hendrickson, C. L. (2015). Overview of Target Enrichment Strategies. Current Protocols in Molecular Biology, 112(1). https://doi.org/10.1002/0471142727.mb0721s112
Singh, R. R. (2022). Target Enrichment Approaches for Next-Generation Sequencing Applications in Oncology. Diagnostics, 12(7), 1539. https://doi.org/10.3390/diagnostics12071539