Amplicon sequencing: 5 tips for a successful experiment

Amplicon sequencing is a targeted sequencing method that uses oligonucleotide primers to amplify specific regions of the genome. This approach provides researchers with the opportunity to obtain genetic information about only those parts of the genomic code that they care about the most. Amplicon sequencing employs polymerase chain reaction (PCR), one of the most widely used techniques in molecular biology. PCR consists of creating multiple copies-a process known as amplification-of a specific nucleic acid sample in a short time.

Amplicon sequencing is a perfect tool for (i) investigating microbial diversity in complex natural and artificial environments, (ii) targeted pathogen sequencing to track transmission routes and study pathogen evolution, and (iii) variant identification. Refer to IDT's amplicon sequencing page for more information.

Amplicon sequencing is widely used for identification of various pathogens and tracking their evolution and transmission patterns. This technology has significantly contributed to tracking and controlling the spread of the SARS‑CoV-2 virus during the COVID-19 pandemic. IDT offers SARS CoV 2 Amplicon Panels that can identify SARS-CoV-2 variants from a wide array of sample types and covers 99.7% of the SARS‑CoV-2 genome. Amplicon sequencing is also actively applied to identify a range of other pathogenic agents, from monkeypox virus to bacteria causing infection in individuals with knee prosthetic joint infection (PJI).

16S rRNA sequencing is one of the most popular amplicon sequencing methods. It is routinely used to identify and compare taxonomy composition of bacteria and archaea in a given sample. This powerful and cost-effective culture-independent approach gives scientists the opportunity to identify strains that would be overlooked when using more traditional wet laboratory techniques. The prokaryotic 16S rRNA gene is approximately 1500 bp long and contains 9 variable regions (V1-V2) interspaced by conserved regions. Sequencing these variable regions of the 16S rRNA gene is widely applied for deep taxonomic classification (at the genus or sometimes even at the species level) of complex microbial communities. In addition, 16S rRNA sequencing benefits from intrinsic advantages of amplicon sequencing including the ability to combine multiple samples (multiplex) in a single sequencing run.

1. Nucleic acid sample
   Use a fresh, high-quality nucleic acid template. Given very high resolution of the PCR based methods (which is usually considered an advantage) including amplicon sequencing, using a degraded or contaminated template may lead to inconclusive or controversial results.
    
2. Master mix and high-standard primer design
   Use a commercially available master mix instead of mixing all the components [DNA polymerase, nucleotides (dNTPs), specific ions (MgCl2)] individually. This helps to minimize human error as well as sample-to-sample variation and improves consistency. Choose the right master mix for your experiments from a selection of products offered by IDT. Use dedicated amplicon sequencing solutions designed to efficiently target genes of interest. For example, IDT’s ARTIC SARS-CoV-2 Amplicon Panel may help to elucidate the presence of mutations consistent with known SARS‑CoV-2 variants or identify new variants. Use a dedicated primer design software that gives the opportunity to adjust design parameters depending on amplicon size, desired melting temperature (Tm), and other factors. For example, the PrimerQuest™ Tool provides flexible design options based on your nucleic acid sequences to help you design your custom experiments.
    
3. Contamination
   High resolution of amplicon sequencing means that even a very small amount of input template can lead to amplification of contaminating sequences not present in the analyzed sample. This is why all surfaces in the PCR area should be regularly decontaminated either by 5% bleach solution or UV sterilization. If at all possible, prepare your PCR reactions in a template-free area (ideally, a separate room) and then add your nucleic acid samples in another room.
    
4. Controls
   One of the most effective approaches to minimize the possibility of contamination is the efficient use of controls throughout the experiment.
   • A negative or a no template control (NTC) is particularly important to confirm the lack of cross-contamination. The NTC includes all the reagents with the exception of the nucleic acid template; usually the template is replaced with an equal amount of nuclease-free water.
   • A positive control that contains a known nucleic acid sequence and primers designed to detect it. This control is used to identify false-negative results. If your positive control produces a signal, it means that all the reagents in your PCR are working properly. A positive control can be particularly useful to investigate the effects of barcode mismatching.
      
5. Barcode mismatches
   Since modern NGS platforms generate massive amounts of data, multiple libraries are often pooled—or multiplexed—and sequenced in a single run to reduce the sequencing costs. To be able to identify each read post sequencing, all DNA fragments are tagged—or barcoded—using sample-specific DNA indexes during the library preparation step. This sample multiplexing sometimes results in index misassignment due to various mechanisms including index hopping or barcode contamination. As a result, sequencing reads may be assigned to the wrong index (and consequently, to the wrong sample) and will be discarded from downstream bioinformatic analyses. To minimize the risk of barcode misassignment, it is recommended to carefully choose the multiplexing strategy, to use either unique dual indexed (UDI) adapters or indexing primers.

Amplicon sequencing is a high-throughput targeted sequencing approach that gives researchers the opportunity to sequence up to thousands of genetic regions of interest in a single run. This highly targeted method is cost efficient and provides shorter turnaround times compared to untargeted approaches. To learn more about how amplicon sequencing can help you advance your research, visit IDT’s information and product pages, where you will find additional resources that will navigate you through a variety of amplicon sequencing options.

*RUO—For research use only. Not for use in diagnostic procedures. Unless otherwise agreed to in writing, IDT does not intend for these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations. RUO23-1938_001