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

What are enrichment strategies in NGS?

Types of enrichment strategies

Applications of enrichment strategies

Keywords

References


What are enrichment strategies in NGS?

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.


The 2 types of NGS enrichment strategies

There are two commonly used types of enrichment strategies. They are the capture-based method and PCR-based method.

Enrichment sequencing strategy flow chart



Capture-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.

Capture-based sequencing technology



PCR-based method

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.

PCR-based sequencing mehod




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.


Capture-based method

PCR-based method

Advantages

  • Cost-effective
  • Greater sequencing depth on target regions
  • High uniformity in coverage
  • Design personalization
  • Cost-effective
  • Greater sequencing depth on target regions
  • High specificity
  • Low DNA input needed

Disadvantages

  • Need to design an array
  • Large amount of DNA needed
  • High expertise required
  • PCR biases
  • Validation of the target sequences and primers needed
  • Reaction conditions need adjustment



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
  • Paleomicrobiology
  • Single nucleotide polymorphism detection (SNP)
  • Genotyping
  • Insertion/deletion (Iindel) detection
  • Copy number variation (CNV) detection
  • Genotyping
  • 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/



Keywords

Library prep, enrichment strategies, capture-based method, PCR-based method.



References

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