Targeted amplification of DNA by PCR is an integral part of daily work in laboratories around the world. It is used in research, diagnostics, forensics and even food analysis. The resulting DNA amplicons often need to be checked by amplicon sequencing, especially when they are further used for molecular cloning for instance. The Oxford Nanopore technology led to quite an advancement in amplicon sequencing due to outstanding read lengths and, therefore, sequencing of amplicons in single reads.

Key takeaways:

  • Amplicon sequencing is used in research on genetic variations causing and affecting diseases and disorders.
  • Amplicon sequencing is used to classify microorganisms and assess the functional capabilities of microbiomes.
  • Amplicon sequencing is used to analyse and monitor environmental DNA (eDNA).
  • Oxford Nanopore technology generates very long reads of more than 4,000,000 bp and can sequence an entire amplicon in one single read.

Amplicon sequencing application in research

Amplicon sequencing is a relatively simple way to detect and analyse hot-spot mutations, copy number variations (CNV), inserts and deletions (InDels), and (SNPs). It is used in basic research on animals, plants, and microorganisms.

Amplicon sequencing is also used in clinical research on genetic variations that cause or affect diseases and disorders. Many diseases and disorders have been linked to genetic variations in exons and introns, including diseases with high morbidity such as cancer and cardiovascular diseases. In terms of cancer, the same type of cancer can show different genetic variations in different patients. Therefore, the use of amplicon sequencing could be beneficial for the development of treatment strategies.

Similarly, the risk for coronary heart disease could potentially be increased by genetic variations affecting atherosclerosis, inflammatory responses, LDL levels, etc., thus, by individual genetic differences. Amplicon sequencing is a great tool to identify somatic mutations in complex samples and even identify rare genetic variations.

Genome editing studies also greatly benefit from amplicon sequencing to confirm the success of the genome edit.

Amplicon sequencing for microbiome and eDNA investigations

Amplicon sequencing is widely utilised to analyse microbial communities, aid taxonomic classification of microorganisms, and determine the functional capabilities of microbiomes (e.g. nutrient cycle). Here, targeted amplification of the 16S rRNA gene for bacteria and archaea, and the ITS region for fungi are the preferred methods. The subsequent amplicon sequencing allows researchers to gain insight in the microbiome of the gastrointestinal tract for instance.

Environmental DNA (eDNA) analyses is another interesting field where amplicon sequencing is utilised. eDNA is genetic material that is shed by organisms into the surrounding environment. eDNA can be found in tissue fragments and excretions such as urine. Thus, amplicon sequencing can contribute to the analysis and monitoring of the biodiversity, including the presence and abundance of organisms, of terrestrial and aquatic ecosystems. For instance, it has been shown that wastewater analysis could predict disease outbreaks several weeks before disease symptoms occur in people.

Amplicon sequencing with Oxford Nanopore technology

Oxford Nanopore technology is based on single-molecule sequencing and can generate unparalleled read lengths of more than 4,000,000 bp and sequencing data of up to 50 Gb per sequencing run. This outstanding capacity allows to capture entire genes such as the bacterial 16S rRNA gene, which is about 1,500 bp long. As mentioned before, the 16S rRNA gene is used to identify bacteria. In clinical diagnostics, the 16SrRNA gene is used to identify bacterial pathogens that are not detected by standard diagnostic tests. Here, Oxford Nanopore technology has been shown to keep up with Illumina technology that generates much shorter reads (150bp) when it comes to pathogen detection and identification.

The high read length allows for sequencing of genomic regions that are often rather challenging for technologies yielding shorter reads. Challenging genomic regions include complex regions with tandemly arrayed genes (TAGs), which are clusters with duplicated genes where copies are in close proximity. TAGs account for a large proportion of genes, and a prominent example are RNA encoding genes that can be transcribed much faster that way compared to a single copy.

Illustration of the eukaryotic ribosomal DNA in tandem arrangement. Each unit contains a rRNA coding part (18S, 5.8S, 28S rRNA, internal transcribed spacers (ITS) and external transcribed spacers (ETS)) and intergenic spacer.

Due to the gene duplication and sequence similarity, characterisation of these complex regions often proves difficult and misassemblies are common. This increasingly becomes a problem as it seems that complex genomic regions are involved in diseases and disorders among others. Oxford Nanopore technology-based amplicon sequencing with its very long reads is much more suitable for correct analysis of complex regions.

Furthermore, the Oxford Nanopore technology is highly suitable for low-input DNA samples and samples with varying starting material due to the single-molecule sequencing technology. Amplicon sequencing can now be used in a wide range of research studies.

What you can expect for the amplicon sequencing solution

The Oxford Nanopore technology-based amplicon sequencing solution utilises the latest v14 library prep chemistry to generate long-read sequencing libraries, and, therefore, greatly reduces fragmentation of the input DNA.

The Eurofins Genomics amplicon sequencing solution:

  • Is designed for starting material with the size of 600bp up to 20,000 bp
  • Allows for multiplexing (maximum of 96 samples per flow cell)
  • Does not require primers
  • Has an accuracy of 99%
  • Delivers amplicon sequencing data – approximately 7 Gb per flow cells – as FastQ files

Eurofins Genomics also offers additional BioIT services:

  • Sequence quality assessment
  • Amplicon consensus generation
  • Amplicon variant sites analysis

The multiplexing capabilities of the Oxford Nanopore technology is especially interesting for high-throughput amplicon sequencing.

Find out more about Oxford Nanopore technology and its use for sequencing of long DNA and RNA molecules.

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Image source of Copy number variants:, Created by: NHS National Genetics and Genomics Education Centre, Licence: CC BY-NC 2.0

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