Introduction

While whole human genome sequencing has advanced discovery and human health, challenging regions of the genome are difficult to analyze using this approach, resulting in a population sequencing bias, and existing databases are noted to be neither complete nor accurate (1). For many research applications, the cost of whole genome sequencing can still be a burden, particularly when you take into account the computational processing and informatics needs for whole genome analysis. This added cost and complexity would be of little benefit when studying a specific region of interest for disease and translational research applications. To help address this issue, many researchers have adopted a targeted sequencing approach to improve coverage, to simplify analysis and interpretation, and to lower their total sequencing workflow costs.

Hybridization capture versus amplicon-based enrichment

By leveraging current genomic knowledge, a targeted NGS approach utilizes molecular biology methods to enrich for specific genetic sequences, allowing researchers to focus their studies on individual genes or genomic regions. Obtaining sequence coverage of challenging genomic regions is now possible, including regions from difficult sample types such as samples with degraded DNA or RNA, or circulating cell-free DNA from blood. By NOT sequencing what you do not need, there are cost benefits beyond wasted computational processing and informatics. Focusing on specific regions of interest allows researchers to sequence at a much higher depth of coverage for rare variant discovery. Many more samples can also be processed simultaneously in a single sequencing run for a faster time to result for both an individual sample and a cohort.

There are two general techniques used to enrich for specific genetic regions for sequencing (Figure 1). Hybridization capture can be either solution-based or performed on a solid-substrate such as a microarray. Both require the use of synthesized oligonucleotide probes (also known as baits) that are complementary to the genetic sequence of interest. In solution-based methods, the probes are biotinylated and added to the genetic material in solution to hybridize with the desired regions of interest. Magnetic streptavidin beads are used to capture and isolate the hybridized probes from the unwanted genetic material.


Figure 1. Workflows for target enrichment. (A) Hybridization capture can be performed in solution or on a solid substrate. (B) Amplicon-based target enrichment using Ion AmpliSeq technology.

With array-based capture, the probes are attached directly to the solid surface. The genetic material is applied to the microarray and target regions hybridize to the surface. Any unbound material is washed away, leaving the desired target regions isolated on the substrate. For both methods, the isolated and purified target regions are subsequently amplified and prepared for sequencing.

As the name implies, amplicon-based enrichment uses carefully designed PCR amplicons to flank targets and specifically amplify regions of interest. The amplified products are then purified from the sample material and used for sequencing, bypassing the need for enrichment by hybridization.

Advantages of amplicon-based approaches

There are many advantages of using amplicon-based enrichment techniques compared to hybridization capture methods (Table 1). Amplicon-based approaches offer a simpler, faster workflow with unmatched PCR specificity, allowing researchers to enrich for target gene regions from low sample input amounts. Genetic material from limited sample sources, such as fine needle aspirates or circulating tumor DNA can be sequenced for biomarker discovery and clinical trials.

 Advantage

Example

Why is it important?

Low sample input
Liquid biopsies
fine needle aspirates
Enables sequencing of limited samples, ensuring appropriate sequencing coverage
Better enrichment for homologous regions
Pseudogenes
PTEN gene is a known tumor suppressor gene, with an associated pseudogene (2).
Paralogs
Monogenic disease genes have frequently functionally redundant paralogs (3).
Hypervariable regions
T-cell receptor
Predictive biomarker discovery for immunotherapy (4)
Target and enrich low complexity regions
Di- and tri-nucleotide repeats
Microsatellite instability studies for disease such as prostate cancer (5)
Fusion detection
ETV6-NTRK3
Known fusion oncogene for secretory cancers (6) and other cancer types. NTRK3 inhibitors may be therapeutic for cancers exhibiting this biomarker

Table 1. Advantages of amplicon-based enrichment compared to hybridization capture methods

A targeted gene of interest may share homology with another segment of the genome. Hybridization capture would have difficulty distinguishing between the two regions, resulting in non-specific enrichment. This is not an issue with amplicon-based enrichment, where PCR primers can be uniquely designed to target the desired region. For example, the PTEN gene is a known tumor suppressor gene and is one of the most commonly lost suppressor genes in cancer. PTENP1 is a processed pseudogene very similar in sequence, but a mutation prevents translation of the normal PTEN protein. Being able to distinguish and target the right gene and avoid the pseudogene clearly plays an important role in cancer research. The same concept applies when trying to target low complexity regions that are prevalent in whole genomes, such as di- and tri-nucleotide repeats.

Since capture hybridization requires the development of complementary capture probes against a known reference genome, genetic mutations that one may be interested in potentially disrupt the hybridization itself and so result in a failure to enrich for a target region of interest. Again, since PCR primers are uniquely designed to flank and then amplify target regions, amplicon-based enrichment can better detect known and novel insertions, deletions, and fusion events. This is particularly true in genetic regions that have many variants in close proximity to one another. Hypervariable regions such as the T-cell immune repertoire can be sequenced using amplicon-based enrichment, providing translational researchers a tool to discover predictive biomarkers for immunotherapy.

Ion AmpliSeq technology—the industry leader in amplicon-based enrichment

Thermo Fisher Scientific has advanced amplicon-based enrichment with Ion AmpliSeq technology (Figure 2). Unique to this technology is the ability to multiplex up to 24,000 PCR primer pairs in a single reaction, allowing researchers to sequence hundreds of genes from multiple samples in a single sequencing run with fast turnaround time and low cost. Following PCR amplification of the targeted genomic regions, remaining primers are digested, and barcoded adapters are ligated to each sample, encoding each sample with a unique ID, so they can be sequenced at the same time.  The library is then purified using magnetic beads and it is ready for sequencing.


Figure 2. Capabilities of Ion AmpliSeq technology

This robust library preparation method enables targeted enrichment from as little as 1 ng of low-quality DNA or RNA, including nucleic acids extracted from FFPE slides or DNA circulating in the blood. This approach also generates more full-length PCR products and thus better coverage compared to other amplicon-based methods. Additionally, a single Ion AmpliSeq assay enables simultaneous analysis of multiple genomic features—including single nucleotide variants, indels, copy number variants, and RNA fusions. Both analytical and clinical validity has been demonstrated with Ion AmpliSeq technology, as proven in thousands of peer-reviewed publications across a broad range of applications, including oncology, inherited disease, and microbial research.

Ion AmpliSeq technology is powered by an assay design pipeline (Figure 3) that leverages our leadership in both PCR amplification and in comprehensive NGS. The Ion AmpliSeq pipeline allows us to rapidly design and build custom assays to best address the needs for each researcher, allowing them to focus on what is important for them and enabling sequencing and analysis in as little as one day. Recent advances with the design pipeline allow researchers to do more with less in difficult-to-enrich genetic regions (Figure 4). The faster design pipeline has improved in silico coverage, resulting in assay designs with higher coverage and greater uniformity. This means that genomic targets can be analyzed more efficiently, helping to lower costs, reduce throughput requirements, and decrease turnaround time from initial design to obtaining sequencing data.

Fig3-Schematic-IonAmpliSeqPipeline

Figure 3. Schematic of the Ion AmpliSeq pipeline. The pipeline starts with (A) the identification of the genome of interest and targets of interest. For human DNA designs, the targets of interest may be genes, genomic regions, or hotspots (SNVs or small indels of interest). (B) The pipeline then generates a large number of candidate amplicons, and (C) selects the best amplicons that fully cover the targets of interest, in the number of pools desired. For gene and region designs, two pool designs are often preferred. For hotspot designs, one pool design may be preferred. (D) The amplicons are tiled so that they can be optimally combined in the available pools, maximizing the coverage of the requested targets.

Figure 4. Percent in silico coverage comparison of new pipeline versus old pipeline. The comparison was performed for the (A) single-pool assay design and (B) two-pool assay design for 150 target genes. For each pool, designs were created for 150 bp and 275 bp amplicon lengths. The 150 target genes selected for this analysis are the most common genes designed with our Ion AmplliSeq designer pipeline, and each gene is represented by a single dot in each plot. The diagonal line in each plot notes equivalent coverage performance between the new designer pipeline and the old designer pipeline. Overall design performance is improved with the new pipeline, with most genes having higher coverage as indicated by the dots being on the left and above the diagonal. In particular, the improvement in coverage is significant for single-pool assay designs and for designs that require shorter amplicons. For researchers, this translates to greater sequencing efficiency and higher confidence in sequencing lower-quality samples.

Ready-to-use and pre-tested Ion AmpliSeq panels are available for a wide range of diseases, and research applications, from comprehensive cancer panels covering key genes and hotspots, to panels focused on inherited diseases and the immune repertoire. Additionally, a free design tool (Ion AmpliSeq Designer) can be used to create custom targeted panels that best address your individual research needs.

Learn more about Ion AmpliSeq technology and the breadth of applications.

Summary

Appropriate sequencing coverage across the genome is an important aspect that should not be overlooked and is needed to ensure the scientific community has complete and accurate genomic data to further discovery and improve human health with precision medicine. Researchers have turned towards targeted enrichment approaches to help sequence specific regions of interest at a much higher depth of coverage and at lower cost.

Compared to hybridization capture, amplicon-based enrichment methods have a better ability to target difficult genomic regions with lower input amounts of DNA and RNA to enable discovery. With Ion AmpliSeq technology, Ion Torrent sequencing is the industry leader in target enrichment providing higher multiplexing and coverage in a single assay compared to other methods. Continuing innovation with our comprehensive solutions, including our unique design pipeline, means we have a robust, customizable set of tools designed to work together to help answer your unique and important scientific questions faster and with less effort.


References

  • Watson et al., J Immuno 198:3371 (2017)
  • Chu and Taranawski, Med Sci Monit 10:RA235 (2004)
  • Chen et al. PLOS Comp Biol 9:e1003075 (2013)
  • Zhang et al. SITC Poster (2018)
  • Le et al., Science, 357:409 (2017)
  • Stenmann, Head and Neck Pathol 7:S12 (2013)

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