What are the key steps in the amplicon sequencing process?

Genomic revolution has made DNA sequencing more accessible than ever before. Amplicon sequencing offers a streamlined approach to analyze specific genetic regions across multiple samples. This targeted method requires carefully executing several critical steps to generate reliable results.

Sample prep essentials

• Every successful sequencing project begins with proper sample preparation. A good starting material has a direct impact on the downstream results. Scientists extract DNA or RNA from biological samples to minimize contamination and degradation.
• For environmental samples, this might involve filtering water or processing soil. Clinical samples require protocols that preserve nucleic acid integrity while removing PCR inhibitors. Regardless of source, the goal remains to obtain pure, high-quality genetic material suitable for amplification.
• Once extracted, scientists assess the quality and quantity of nucleic acids. Spectrophotometry measures purity, while fluorometric methods determine concentration. This quality control step prevents wasted resources on poor samples and ensures consistent results across the experiment.

PCR amplification strategy

The heart of amplicon sequencing lies in PCR amplification. This step uses primers designed to target specific genomic regions of interest. Primer design requires careful consideration of target specificity, GC content, melting temperature, and potential secondary structures. The PCR reaction conditions must be optimized for each primer set. This optimisation involves adjusting temperatures, cycle numbers, and reagent concentrations to maximise specificity and yield. Many protocols employ a touchdown PCR approach to reduce non-specific amplification.

• Primer selection considerations – Researchers must choose primers that capture the genetic diversity they wish to study while avoiding bias. Universal primers target conserved regions flanking variable sequences, making them ideal for biodiversity studies.
• PCR optimization techniques – When working with challenging samples, additives like DMSO or bovine serum albumin help overcome inhibitors. Nested PCR approaches can increase specificity for challenging targets.

Library preparation details

• Library preparation transforms PCR products into a form suitable for sequencing. This process attaches adapters to amplicons, enabling them to bind to the sequencing flow cell and providing unique identifiers for each sample.
• Modern protocols often incorporate dual indexing strategies where unique barcodes are added to both ends of each amplicon. This approach allows hundreds of samples to be pooled together and sequenced simultaneously while maintaining the ability to trace each sequence back to its source.
• The completed libraries undergo quality control using capillary electrophoresis or real-time PCR. These checks assess library size distribution, adapter dimer contamination, and quantification for optimal loading onto sequencing platforms.

Sequencing and data analysis

The prepared libraries move to sequencing platforms where machines read the nucleotide sequence of each amplicon. Different platforms offer various trade-offs between read length, accuracy, and throughput. Research questions vary according to their specific requirements. After sequencing comes the computational challenge of data analysis, Bioinformatics pipelines process raw sequence data through quality filtering, denoising, and chimaera removal steps. These processed sequences then undergo clustering into operational taxonomic units or amplicon sequence variants.

The final analysis steps depend on the research question. They might include taxonomic assignment, diversity analysis, or variant calling. Visualization tools help transform complex datasets into interpretable results that address the original research questions. Mastering these key steps enables researchers to harness the full power of amplicon sequencing. While the technology continues to evolve, these fundamental processes remain the backbone of successful sequencing projects across diverse fields of genomic research.