Amplicon sequencing is a method of sequencing that targets specific genomic regions or conserved regions within the organisms to characterize them and study genetic variations.
Unlike whole-genome sequencing which covers the entire genome, amplicon sequencing selectively amplifies and sequences regions of interest, using polymerase chain reaction (PCR). PCR amplifies the target DNA regions and produces multiple copies of the target DNA called amplicons.
It is a targeted sequencing method and is widely used for identifying genetic variants, studying microbial diversity, and detecting pathogens in clinical samples. The sequencing process becomes more efficient and targeted by focusing only on the specific regions of interest. This allows detailed study of the specific regions without the need to sequence the entire genome.
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Principle of Amplicon Sequencing
Amplicon sequencing works on the principle of sequencing specific DNA regions of interest that have been amplified using PCR. Specific oligonucleotide primers are designed to amplify specific genomic regions of interest. These regions are then amplified using PCR to produce short, specific DNA segments called amplicons, which are then sequenced using high-throughput sequencing technologies. This highly targeted method ensures that only the regions of interest are sequenced providing high sensitivity for characterizing organisms and detecting genetic variants. The amplicon sequencing process involves extracting genetic regions of interest, enriching selected regions with PCR, adding adapters, creating libraries, and then pooling multiple samples for sequencing in a single run.
Process/Steps of Amplicon Sequencing
1. Sample Preparation and DNA Extraction
- The first step is sample preparation which begins with sample collection and DNA extraction from the samples of interest.
- DNA can be extracted using different methods including chemical lysis and mechanical methods.
- At first, the cells are lysed to release the DNA and then the cellular debris is removed from the mixture to separate the DNA from other contaminants.
- This is followed by purification, precipitation, and resuspension steps to finally obtain purified DNA of interest.
2. PCR Amplification
- The PCR process includes several cycles of denaturation, annealing, and elongation to amplify the desired DNA regions.
- Primers specific to the genomic regions of interest are designed which amplifies the target genomic regions and produces amplicons.
- These primers also add barcodes necessary for identifying amplicons from different samples during sequencing.
- This amplification step is necessary for enriching the regions of interest to increase their presence in the final data.
- After amplification, the PCR products are purified to remove any unreacted primers, primer dimers, nucleotides, and other contaminants to ensure that only high-quality amplicons are sequenced.
3. Library Construction
- After amplification, the next step involves adding sequencing adapters to the amplicons and constructing libraries to prepare the DNA for sequencing.
- Adapters are necessary for the sequencing process as they allow the amplicons to attach to the sequencing flow cell.
- After adding the adapters, the libraries need to be checked to ensure proper adapter ligation. Gel electrophoresis can be used to check the size and purity of the amplified products and confirm the presence of the target amplicons.
4. Sequencing
- Once the libraries are prepared, they are ready for sequencing. This step involves loading the libraries onto a sequencing platform.
- Sequencing platforms such as Illumina HiSeq, MiSeq, and PacBio can be used to sequence the amplicons. Illumina platforms are widely used for amplicon sequencing.
- HiSeq provides high-throughput sequencing and is suitable for large-scale projects but takes a longer processing time. On the other hand, MiSeq is a more cost-effective and faster option for small-scale sequencing projects.
5. Data Analysis
- The final step is bioinformatics analysis of the sequencing data.
- The sequencing reaction produces reads stored in FASTQ files which are demultiplexed to separate samples by barcodes and remove the barcodes using tools like sabre.
- Then, low-quality bases, adapters, and contaminants are filtered and trimmed using tools such as Trimmomatic.
- Then, identical sequences are merged and duplicates are removed using dereplication. During PCR amplification, fragments from different DNA sequences can join together incorrectly. Chimera removal identifies and eliminates such chimeric sequences.
- The sequences are then either clustered into Operational Taxonomic Units (OTUs) or resolved into high-resolution Amplicon Sequence Variants (ASVs) depending on the chosen workflow.
- Standard outputs including count tables, FASTA files, and taxonomy assignments are generated. Taxonomic assignment involves classifying sequences based on their phylogenetic information using reference databases.
- Then, diversity analysis studies both alpha diversity and beta diversity using different metrics like species richness, evenness, dissimilarity, and phylogenetic diversity. Alpha diversity measures variation within a single sample while beta diversity compares the differences between samples. This is done using tools such as phyloseq and DESeq2.
Types of Amplicon Sequencing
Based on the target sequences to be sequenced, amplicon sequencing can be divided into 3 types:
a. 16S rRNA sequencing
16S rRNA sequencing involves sequencing the 16S ribosomal RNA (rRNA) gene that is present in prokaryotes. It is the most widely used method for identifying and studying bacteria and archaea. This sequence consists of variable regions and conserved regions. The conserved regions provide binding sites for the primers in the PCR amplification step, ensuring that the amplification process focuses on the gene of interest. The variable regions contain unique sequences that differ between different species providing the information needed for accurate identification and classification.
b. 18S rRNA sequencing
18S rRNA sequencing targets the 18S rRNA gene specific to eukaryotic organisms and includes both conserved and variable regions. Eukaryotic organisms can be identified and classified by targeting this gene. The conserved regions ensure that the primers bind effectively, while the variable regions provide the necessary differentiation to distinguish different eukaryotic species.
c. ITS sequencing
ITS sequencing targets the ITS (Internal Transcribed Spacer) region located between the 18S, 5.8S, and 28S rRNA genes in the ribosomal DNA of fungi. It is used for fungal identification and taxonomy. The ITS regions are known for their high variability compared to other regions of the ribosomal RNA genes which makes them highly effective for distinguishing closely related fungal species.
Advantages of Amplicon Sequencing
- Amplicon sequencing allows the efficient identification and screening of genetic variants.
- Amplicon sequencing can support multiplexing multiple amplicons per reaction which ensures high coverage of the targeted regions.
- This method reduces sequencing costs and turnaround time compared to methods like whole-genome sequencing as it requires fewer reads per sample and fewer reagents.
- Amplicon sequencing requires low DNA input.
- The PCR-based workflow of amplicon sequencing is faster and simpler compared to hybridization capture methods.
Limitations of Amplicon Sequencing
- Amplicon sequencing uses PCR amplification that can introduce bias including uneven amplification which affects the accuracy of sequencing.
- Poor primer design can result in reduced specificity and off-target amplification.
- This method of sequencing only focuses on specific regions and might miss important genetic variations outside of the targeted regions.
- Degraded or contaminated starting material can lead to poor amplification and false results.
Applications of Amplicon Sequencing
- Amplicon sequencing is used to detect mutations associated with different traits and inherited diseases. It can be used to identify rare genetic mutations in complex samples and detect low-frequency mutations missed by other techniques.
- Amplicon sequencing is used to identify and characterize pathogens in clinical samples. It has been used in monitoring pathogens in wastewater and tracking viral variants.
- Amplicon sequencing can also be used in agriculture to identify genetic variations in crops and detect plant pathogens to manage crop diseases.
- Amplicon sequencing is used in phylogenetics to classify and identify new species by comparing amplicon sequences to known databases. It is also useful to study the genetic diversity and evolutionary relationships between different species.
- Amplicon sequencing is used to identify and classify microorganisms in various environments.
- Amplicon sequencing can be used for DNA profiling in forensic investigations.
References
- https://tuftsdatalab.github.io/Research_Technology_Bioinformatics/tutorials/AmpliconSeq/16STutorial.pdf
- Bybee, S. M., Bracken-Grissom, H., Haynes, B. D., Hermansen, R. A., Byers, R. L., Clement, M. J., Udall, J. A., Wilcox, E. R., & Crandall, K. A. (2011). Targeted amplicon sequencing (TAS): a scalable next-gen approach to multilocus, multitaxa phylogenetics. Genome biology and evolution, 3, 1312–1323. https://doi.org/10.1093/gbe/evr106
- Liu, Y. X., Qin, Y., Chen, T., Lu, M., Qian, X., Guo, X., & Bai, Y. (2021). A practical guide to amplicon and metagenomic analysis of microbiome data. Protein & cell, 12(5), 315–330. https://doi.org/10.1007/s13238-020-00724-8
- Amplicon and metagenomics overview. (n.d.). Retrieved from https://astrobiomike.github.io/misc/amplicon_and_metagen
- NASA GeneLab. (n.d.). Retrieved from https://genelab.nasa.gov/amplicon-sequencing
- Amplicon sequencing solutions. (n.d.). Retrieved from https://www.illumina.com/techniques/sequencing/dna-sequencing/targeted-resequencing/amplicon-sequencing.html
- Improved Targeted Sequencing: A comprehensive guide to amplicon sequencing. (n.d.). Retrieved from https://www.seqanswers.com/articles/324095-improved-targeted-sequencing-a-comprehensive-guide-to-amplicon-sequencing
- Principles and Workflow of 16S/18S/ITS Amplicon Sequencing – CD Genomics. (n.d.). Retrieved from https://www.cd-genomics.com/resourse-principels-and-workflow-of-16s-18s-its-amplicon-sequencing.html
- DeWitt, J., PhD. (n.d.). Targeted NGS Amplicon Sequencing | IDT. Retrieved from https://eu.idtdna.com/pages/technology/next-generation-sequencing/dna-sequencing/targeted-sequencing/amplicon-sequencing?cDEE
- The workflow and Applications of Amplicon Sequencing – CD Genomics. (n.d.). Retrieved from https://www.cd-genomics.com/the-workflow-and-applications-of-amplicon-sequencing.html
- Schmerker, J. (2024, February 12). Here’s what you need to know about amplicons and amplicon sequencing. Retrieved from https://eu.idtdna.com/pages/community/blog/post/here-s-what-you-need-to-know-about-amplicons-and-amplicon-sequencing