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Improved Targeted Sequencing: A Comprehensive Guide to Amplicon Sequencing




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  • Improved Targeted Sequencing: A Comprehensive Guide to Amplicon Sequencing

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    Amplicon sequencing is a targeted approach that allows researchers to investigate specific regions of the genome. This technique is routinely used in applications such as variant identification, clinical research, and infectious disease surveillance. The amplicon sequencing process begins by designing primers that flank the regions of interest. The DNA sequences are then amplified through PCR (typically multiplex PCR) to produce amplicons complementary to the targets. RNA targets are also appropriate for amplicon sequencing, but the RNA must be converted into cDNA prior to amplification. After PCR, sequencing libraries are prepared from the amplicons and then the libraries are pooled and loaded onto the appropriate sequencer. In this article, we will cover the benefits, popular applications, and important strategies of amplicon sequencing.

    Benefits of amplicon sequencing

    “Amplicon panels are the technology of choice for small target panels ranging anywhere from 5–50 kb of sequence,” said Pedro Echave, Global Leader and NGS Product Expert at PerkinElmer. “Due to the fact that they are PCR-based, their workflow is much faster (~4 h TAT) and simpler than hybridization capture.”

    Echave added that amplicon sequencing panels, “tend to be the most cost-efficient for sequencing as they require a lower amount of reads per sample for most applications, such as in the detection of germline mutations that only typically require coverage of 30x–100x.” The sequencing costs are further reduced due to the low reagent requirements (i.e., primers and PCR mix) and the need for only a thermocycler to perform amplicon-based experiments.

    Many users also choose this method when working with difficult samples. “Amplicon sequencing is widely popular for any application where the amount of starting nucleic acids is limited, or its quality is compromised,” said Jose Luis Costa, Global Director of Scientific Affairs at Thermo Fisher Scientific. Researchers have successfully employed amplicon sequencing to obtain useful data from low-input DNA, cell-free DNA (cfDNA), and other difficult samples like those from preserved tissues.
    Another attractive characteristic is that users can easily customize their own panels to fit their needs. As explained by Costa, “Not only do customized amplicon sequencing panels allow for a faster and more cost-effective turnaround time, but they also simplify workflow and allow for easier setup in the lab as less expertise is required to run the tests.”

    Common applications

    Cancer and inherited disease
    “The most popular use is for molecular profiling of cancer samples,” said Costa “These are, most of the time, small tissue samples that are formalin-fixed and paraffin-embedded (FFPE), a preservation method important to facilitate visualization of the tissue structure and components, but very aggressive to the nucleic acids, which result in a poor quality and limited amount of initial material.” Due to amplicon sequencing’s ability to handle such difficult samples, they’re a perfect option for studying these types of tissues.

    In addition to cancer research, amplicon sequencing is utilized for the detection of many other diseases. “There is often interest in sequencing some genes associated with inherited disorders (for example CFTR1 in the context of cystic fibrosis), or hotspots associated with particular diseases,” added Echave. “At PerkinElmer, we have our line of NEXTFLEX® amplicon panels, covering genes associated with cancer and cancer predisposition, newborn syndromes, and inherited disorders.” These types of panels enable researchers to focus their efforts on key genes and identify the important mutations linked to disease.

    16S rRNA gene/ITS
    Studies involving microbial detection and identification have greatly benefited from the application of amplicon sequencing. Microbiologists routinely use amplicon sequencing with primers targeted to variable regions (V1–V9) within the highly conserved 16S rRNA gene in prokaryotes. By analyzing the amplicons produced in these regions, scientists can identify the bacteria or archaea present. Similarly, internal transcribed spacers (ITS), DNA sequences situated between rRNA genes, are often used as targets in amplicon sequencing experiments for fungal identification.

    Using these amplicon-based applications for the identification of microorganisms is preferable because researchers can examine many samples at the same time, it doesn’t require extensive culturing, and it’s possible to determine individual genera from a mixed culture. This is valuable for studies investigating the microbiome, mycobiome, and also for environmental DNA (eDNA) research.

    CRISPR genome editing
    CRISPR-Cas9 technologies have revolutionized gene editing and have been remarkably helpful for investigating phenotypes across the genome. Due to the speed and accuracy of amplicon sequencing, many researchers choose this method to evaluate the results of their CRISPR-based experiments. Primers can be designed to target multiple mutation sites as well as detect any off-target effects. While Sanger sequencing has been ideal for detecting CRISPR edits in the past, evaluating amplicons through next-generation sequencing has significantly increased the throughput of CRISPR screening work.

    Often referred to as genotyping by sequencing (GBS), scientists frequently use amplicon sequencing for their genotyping work. By examining the sequence variations, it is possible to identify important genetic markers associated with a specific trait. GBS is particularly valuable in agricultural work such as plant and animal breeding programs. The data generated from these experiments can be used to select beneficial traits in crop improvement projects or it can be used to identify and determine disease susceptibility for plants and animals.

    Infectious disease surveillance
    High specificity and scalability have made amplicon sequencing especially suitable for rapidly detecting and characterizing pathogens from a wide variety of sample types. Amplicon sequencing is frequently used for monitoring pathogens through wastewater1 and has been influential in tracking the spread of specific variants during the coronavirus pandemic2,3. Utilization of this technology has enabled researchers and clinicians to track different viral outbreaks across the globe and rapidly assess key mutations4,5.

    Difficult genomic regions
    There are many sections within the genome that have been traditionally difficult to sequence such as homologous, hypervariable, low complexity, and G-C-rich regions. Other targeted methods, like hybridization capture, can often have difficulties distinguishing these regions due to non-specific enrichment. “As a PCR-based technology, different tactics can be employed that would not be viable using other sequencing methods to tackle the resolution of difficult genomic regions,” said Echave. Amplicon sequencing differs by employing highly specific flanking primers to amplify these difficult regions allowing researchers to obtain the important sequence information they need.

    Liquid biopsy
    “Another popular application for amplicon sequencing is for the study of cell-free DNA/RNA in bodily fluids, better known as liquid biopsy,” said Costa. These types of samples are critical for understanding disease and can help track molecular changes within the body. “Due to the minimal requirements for the amount and quality of initial material, amplicon sequencing can detect genetic variants of cfDNA/RNA from multiple sources like blood, CSF, urine, and bile.” This is because amplicon sequencing uses PCR to amplify the regions of interest, and despite the low input material, can selectively produce many necessary copies of DNA.

    Strategies for successful amplicon sequencing experiments

    Users have many considerations when planning an amplicon sequencing experiment and each is crucial for ensuring on-target reads with sufficient coverage to properly analyze the data. Below are some valuable concepts for optimizing an amplicon sequencing experiment.

    High-specificity primers
    “Primers should maintain high levels of specificity to minimize read loss that may be caused by primer dimers or unaligned low-quality and/or chimeric reads,” stated Echave. High-specificity primers are what differentiates this method from hybridization capture and allow users to sequence difficult targets. “An ideal amplicon panel ensures no amplicon dropouts, creating a gap guard for your target region that is verified by sequencing. High uniformity is also required to get high coverage even with a low number of reads, thereby allowing high-level multiplexing or the use of a smaller sequencing cartridge.”

    Input material
    While the primer design is crucial for experimental success, the source material will also dictate the approach to the sequencing panel. As mentioned by Echave, “It is important to consider the input amount and the integrity of the DNA that the researcher will have available for the experiment. For instance, you must consider if this material is coming from blood or tissue, or from an FFPE sample. The degree of fragmentation of the material might have a direct impact on the performance of the panel and results tend to be better with shorter and overlapping amplicons.” Furthermore, Costa added, “To ensure optimal multiplex sequencing, samples with similar quality and quantity should be sequenced together in order to avoid bias toward simply sequencing the best samples in the batch.”

    Custom panels
    “A custom amplicon sequencing panel is recommended if the panels that are already available do not cover the specific targets a researcher is interested in,” suggested Costa. In many instances, an amplicon panel for a solution already exists, but the ease of design makes custom amplicon sequencing a valuable option when researchers have different requirements for their targets. Costa added, “There are also cases where researchers may want to select a set of targets to include additional internal controls or perhaps there is research being conducted surrounding a genetic variant from a specific population that is not captured in the already available assays.”

    Moreover, custom amplicon panels allow users to tailor the design to fit not only their targets but also their specific samples. “By utilizing a custom amplicon sequencing panel, researchers can adjust the amplicon size to the quality of the original sample,” added Costa. “For example, the more degraded samples used in a study, the shorter the amplicons can be designed.” Costa also believes that custom amplicon sequencing panels may be a good cost-saving measure. “There are situations in which researchers are just interested in a subset of targets present in the assay. In this case, it would be financially beneficial to design a custom panel for the specific targets of interest in order to reduce sequencing costs.”

    Tiling primers
    Another useful strategy when planning an amplicon sequencing experiment is utilizing primer pairs that produce overlapping amplicons, known as tiling. Using tiling can be beneficial when amplifying regions of varying lengths and GC content because it ensures complete coverage of target regions; however, the multiplex primer schemes may need to be evaluated to fully optimize the coverage of the targets1. Custom amplicon panels that include tiled primers should be designed using professional amplicon sequencing design software to reduce dimers and other unwanted interactions.

    Degenerate primers
    While it is generally beneficial to use high-specificity primers, alternatively, researchers may choose to design primers with varying levels of degeneracy. Degenerate primers are oligonucleotides that contain a mix of possible bases in various positions. Utilizing primers with degenerate bases can increase the sequencing coverage of variable regions or targets that are highly likely to have mutations. This strategy may not always be recommended for sensitive applications where any off-target amplification would disrupt the analysis. It may also be difficult to predict the extent of off-target amplification when the degeneracy is high in the primers. For experiments with degenerate primers, it may be necessary to split groups of primers into different reactions and combine the amplicons to reduce cross-reactivity.

    Final thoughts
    Amplicon sequencing projects have many requirements for performing successful sequencing experiments. Researchers using this technique should review each of the mentioned strategies and also follow traditional guidelines like utilizing sequencing controls and optimizing the PCR to improve the experiment. Regardless of the intended application, amplicon sequencing continues to benefit scientific research and should be considered for your next targeted sequencing experiment.

    1. Lin X, Glier M, Kuchinski K, et al. Assessing multiplex tiling PCR sequencing approaches for detecting genomic variants of SARS-CoV-2 in municipal wastewater. mSystems. 2021;6(5):e01068-21. doi: 10.1128/mSystems.01068-21
    2. Gohl DM, Garbe J, Harris D, et al. A rapid, cost-effective tailed amplicon method for sequencing SARS-CoV-2. BMC Genomics. 2020;21(1):1-10. doi: 10.1186/s12864-020-07283-6
    3. Nasereddin A, Daas A, Daboul J, et al. Identification of SARS-CoV-2 variants of concern using amplicon next-generation sequencing. Microbiology Spectrum. 2022;10(4):e00736-22. doi: 10.1128/spectrum.00736-22
    4. Quick J, Loman NJ, Duraffour S, et al. Real-time, portable genome sequencing for Ebola surveillance. Nature. 2016;530(7589):228-232. doi: 10.1038/nature16996.
    5. Watson SJ, Welkers MRA, Depledge DP, et al. Viral population analysis and minority-variant detection using short read next-generation sequencing. Philosophical Transactions of the Royal Society B: Biological Sciences. 2013;368(1614):20120205-20120205. doi:10.1098/rstb.2012.0205

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