The Guide to Understanding Amplicon Sequencing

DNA sequencing is a technique for reading the sequence of nucleotides in a DNA molecule. The most innovative and effective method of amplicon sequencing is the Next Generation Sequencing (NGS) – in other words: deep DNA sequencing. This technology is used to analyze entire genomes, transcriptomes or to study protein-DNA interactions. NGS is the future of not only genetics but also medicine, nutrition, and general clinical applications. DNA sequencing is a method that is extremely efficient in providing data at a relatively low cost. The dynamic development of personal genomics makes deep sequencing technology an indispensable tool in diagnosing many diseases.

Next-generation sequencing (NGS) has taken all fields of biomedical sciences by storm. It is currently challenging to publish any vital research on genetics and molecular biology without the use of NGS research and analysis. Next-Generation Sequencing (NGS) is a group of methods allowing for the large-scale determination of the sequence of the genome, epigenome, transcriptome (RNA-Seq), and the study of protein-DNA interactions (ChIP-sequencing). The development of such extensive and innovative techniques made it possible to significantly reduce the costs of obtaining whole genome sequences.

Next-Generation Sequencing and Its Applications

The main principle of Next-Generation Sequencing technology is similar to Sanger sequencing and is based on signals emitted by individual bases during template DNA resynthesis. Proper preparation of the DNA library, especially the enrichment step, allows for the extension of this process by simultaneously conducting millions of reactions and obtaining genomic data gigabytes from one sequencing.

The NGS method is particularly useful in diagnosing diseases with similar clinical symptoms, caused by mutations of many different genes, such as in neuromuscular diseases. These diseases’ clinical symptoms are often not specific, and it is challenging to select one particular gene for testing on their basis. The NGS method is able to analyze many genes in one test, allowing in most cases to establish a specific diagnosis.

High bandwidth, gigabytes of data, immense precision, and quality of readings prove to be efficient in almost unlimited applications!

Next-Generation Sequencing application examples include:

  • de novo sequencing of entire prokaryotic and eukaryotic genomes
  • exome sequencing
  • re-sequencing of selected fragments of genomes and amplicons
  • sequencing of transcriptomes
  • sequencing and analysis of metagenomes, based on the 16S RNA fragment (bacterial composition) and the ITS sequence (fungal population) from the environmental DNA pool

Multiplex PCR-based Enrichment

Amplicon is a DNA fragment created as a result of natural or artificial amplification. The amplicon can arise, for example, by PCR or LCR and natural gene duplication.

The growing wealth of information about the sequence of the genome means that genetic analyses are carried out on an increasing scale; these findings are an invaluable tool in clinical application. Polymerase chain reaction (PCR) is a method of selectively amplifying DNA fragments. One of the main applications of PCR is the detection and quantification of mRNA through highly sensitive and specific sequence amplification. The term amplicon is often interchangeably used with PCR. 

Some of the Advantages of Multiplex PCR include:

  • Diversity: various applications from the primary pathway of gene expression profiling to the diagnostic identification of pathogens.
  • Saving of genetic material: only one reaction is needed to amplify multiple genes.
  • Efficiency: one reaction is sufficient to amplify many genes simultaneously.

The main fields of application of Polymerase chain reaction are:

  • Determination of the titer of viruses and bacteria
  • Detection and identification of pathogens and rare variants
  • Differential gene expression
  • Monitoring the progress of therapy
  • SNP genotyping
  • RNAi validation
  • Analysis of genetic diseases
  • Detection of pathogens in food and the environment
  • Forensic research
  • Identification of somatic mutations
  • Taxonomic classification of microorganisms

The Advantages of Amplicon Sequencing Application 

The traditional amplicon sequencing method requires lower DNA input, which falls within the range of 1-10 ng. The typical workflow of amplicon-based target enrichment is shorter and more manageable (three to six hours) than that of hybrid capture-based target enrichment. Due to the precision of primer design, the amplicon sequencing has naturally higher on-target rates (>95%) compared to other known sequencing methods. No special equipment is required while utilizing the amplicon sequencing; for instance, DNA fragmentation is unnecessary. The technique is also known for its better performance on difficult clinical samples.

CleanPlex® Target Enrichment Solution 

Paragon Genomics launched a novel ultra-high multiplex PCR-based NGS target enrichment technology, which overpasses many downsides to the amplicon sequencing method while synchronously utilizing its significant advantages. The CleanPlex technology reduces the typical background noise by removing nonessential DNA fragments, such as primer dimers and other longer nonspecific PCR elements, with the help of innovative and patented enzymatic background cleaning step. This technique results in pure and pristine libraries. The traditional multiplex PCR has a specific panel size limitation, which can be overcome by CleanPlex technology due to its background cleaning properties. The CleanPlex Amplicon Sequencing technology can directly amplify DNA from a single cell (about 6pg of DNA), and it enables the detection of novel fusion genes. 

CleanPlex NGS panels offer various efficient advantages to researchers and assay developers, providing ultra-high multiplexing capabilities, low input requirements, single-tube workflow, and reduced sequencing cost resulting in high-quality libraries. With inputs as low as 10ng and fast and simple workflow, the panels offer multiplex 20,000+ amplicons per reaction with prime target design rate, high coverage uniformity, and increased sensitivity.