Introduction

Sequencing technologies have revolutionized biology and medicine, making it possible to study genomes at an unprecedented level of detail. However, these techniques are often expensive and time-consuming. In recent years, targeted next generation sequencing (NGS) methods have grown increasingly popular because of their ability to selectively sequence regions of interest, providing many of the same benefits as whole-genome sequencing without the cost and timeframe. In addition, targeted NGS can provide greater accuracy than whole-genome sequencing, due to the increased depth of coverage possible with less total sequencing. 1

Outlining Two Targeted NGS Methods

There are two main methods of targeted NGS: amplicon-based sequencing, which involves the use of PCR, and hybridization-capture, which uses complementary probes to pull down target sequences.

PCR-based amplicon sequencing is a powerful tool used to amplify short pieces of DNA and, with its many advantages, it is no wonder why it has been such a widely used diagnostic technique since before the advent of NGS. PCR is considered the “gold standard method” for clinical diagnosis of infectious diseases like COVID-19 because it can rapidly detect the presence of pathogens within a sample at low cost and with a very simple workflow.

However, most PCR-based methods only allow for amplification of a small number of targets in each reaction. For projects that need to analyze thousands of targets over many kilobases or even megabases of territory, PCR can quickly become impractical. Additionally, PCR primer design—especially for multiplexed reactions—can be difficult to optimize, and amplification bias can lead to uneven coverage or loss of coverage over targets of interest. Finally, if the target is sufficiently divergent from the PCR primers (for example, in newly evolved COVID-19 strains or due to base changes in cancer samples), then PCR has the potential to not amplify those sequences, leading to false negatives.2

 To assist in overcoming these challenges, capture methods like target hybridization followed by NGS, also known as hybrid capture sequencing, can be used. This approach allows for a large number of genomic targets to be captured in a single reaction, up to many megabases of content. There is a wide array of pre-designed panels available from a variety of commercial providers, and most also offer the opportunity to design custom panels. Human whole exome and pan-cancer panels are widely used, but hybrid capture can also be used to facilitate investigations into phylogenetics, evolution, epidemiology, and drug resistance. 2 

Which Targeted NGS Method is Right for You?

PCR amplicon-based sequencing is widely used for highly targeted sequencing of a relatively small number of regions that are enriched by amplification with sequence-specific PCR primers. Depending on the method used, researchers can amplify up to 100s of targets in the same reaction.3

This method allows researchers to discover, validate, and screen genetic variants using a highly targeted approach. It can be used to sequence viruses (such as SARS-CoV-2) as well as identify and classify bacteria in metagenomic samples via 16S amplification and sequencing.

Highlights of PCR amplicon-based sequencing include:

  • Smaller gene content, typically < 50 genes
  • Ideal for analyzing single nucleotide variants and insertions/deletions (indels)
  • More affordable
  • Simpler workflow

Hybrid capture enables thorough sequencing of a larger number of targets than PCR. Typical hybrid capture panels enrich mega bases of content using many thousands of probes in a single reaction. It can be used for human exome or targeted panel enrichment, such as cancer hotspots and inherited disease gene panels, and to target nonhuman panels, such as murine or plant samples.

Highlights of hybrid capture include:

  • Larger gene content, typically > 50 genes
  • Thorough profiling for all variant types
  • More comprehensive method
  • Longer hands-on time and longer turnaround time

Overcoming Library Prep Challenges

For high scale amplicon sequencing, once PCR products are generated, they need to be processed through a library preparation workflow prior to sequencing. Our plexWell™ 96 and plexWell™ 384 library prep kits employ transposase-mediated tagging of PCR products with Illumina® adapters in a unique, sequential manner that enables pooling early in the workflow. This cost-effective, streamlined, and highly scalable workflow yields pools of normalized libraries with > 2000 unique barcode combinations. Uniform read distributions and high data quality further reduces overall sequencing costs. 

Before hybrid capture can take place, library prep must first be carried out—and this is a crucial step in the process. Unfortunately, the methods currently available for library prep before hybrid capture are very time-consuming, labor-intensive, and cost-prohibitive. However, seqWell is developing a new method that leverages highly optimized Tn5 transposase tagmentation to produce high-quality multiplexed libraries that are compatible with multiple hybrid capture technologies and panel sizes. While this kit is still in product development, it is built off our purePlex™ unique dual index technology.

This method, currently dubbed purePlex HC, is a highly streamlined workflow that permits pooling of samples into plexes for hybrid capture immediately following transposase-mediated tagging, saving both time and cost while generating libraries with high molecular complexity.

Highlights of purePlex include:

  • Streamlined workflow that dramatically reduces labor, time, and consumables
  • 5-hour workflow for 96 samples, 45 min. hands-on time
  • Generates libraries with high molecular complexity that are compatible with multiple commercially available hybrid capture technologies, including human exome panels
  • Exome data generated using purePlex HC libraries is equivalent or higher quality than other commercially available library prep methods

Conclusion

Sequencing technologies continue to rapidly evolve, and targeted NGS is no exception. One of the newest developments in the field is long-read targeted capture, which can provide more accurate and complete data compared to short-read technologies. This uses either PCR or hybrid capture methods to enrich targets that are thousands of bases long. Long-read targeted capture allows for sequencing on commonly used sequencing instruments like those from PacBio or Oxford Nanopore.

The future of targeted sequencing is filled with endless possibilities. At seqWell, we continue to advance and innovate library prep so our customers can get the most out of their data, no matter the method.

References

  1. https://sequencing.roche.com/us/en/article-listing/targeted-sequencing.html#:~:text=What%20advantages%20does%20targeted%20sequencing%20offer%20over%20WGS%3F
  2. https://www.frontiersin.org/articles/10.3389/fmicb.2018.02924/full
  3. https://www.illumina.com/techniques/sequencing/dna-sequencing/targeted-resequencing/amplicon-sequencing.html