AMPLICONS

AMPLICON SEQUENCING

Overview – What is amplicon sequencing?

Amplicons are DNA products of a polymerase chain reaction (PCR). The term amplicon is often used interchangeably with PCR product. High-throughput next-generation sequencing (NGS) can be used to obtain the sequence of a PCR fragment that targets a specific genomic region. In NGS amplicon sequencing, amplicons are generated by PCR, pooled and finally sequenced. Since NGS-based targeted sequencing results in very high coverage of a specific region of interest, amplicon sequencing can detect variants at very low levels and frequencies. The method allows for multiplexing of samples, where hundreds of PCR fragment sequences can be determined simultaneously. These multiplexing capabilities have made amplicon sequencing efficient at covering large genomic regions. Amplicon sequencing also makes data interpretation during downstream processing more manageable in comparison to data analysis following whole genome sequencing.

Workflow amplicon sequencing

Amplicon sequencing is particularly useful for genome targeting and detection of hot-spot mutations, copy number variations, gene fusions, InDels and single-nucleotide polymorphisms (SNPs). Due to its many uses, the sequencing of amplicons can be successfully applied to a variety of disciplines like nutrition, bacterial metagenomics, gene editing and clinical research. 

Applications – What are the advantages of amplicon sequencing?

Amplicon sequencing is an ideal technique for the following applications:

  • Detection of rare variants
  • Phase variation analysis
  • Haplotype characterisation of complex immune regions
  • Identification of somatic mutations in complex samples
  • Taxonomic classification of microorganisms
  • Validation and follow-up of whole-genome sequencing studies 
  • Genome editing

Workflow – amplicon sequencing methods & technologies

Next-generation amplicon sequencing often employs PCR-based methods with primers that target numerous genes of interest simultaneously. The primer sets that flank the target DNA regions typically contain adaptor and barcode sequences to directly prepare the resulting amplification products for NGS. 

Sanger sequencing vs. NGS-based targeted sequencing

Amplicon sequencing involves either Sanger sequencing or NGS approaches. Both techniques have inherent advantages and disadvantages. Typically, the method chosen depends on the size of the targeted sequencing study.

In Sanger sequencing, the obtained sequencing reads are an average of all DNA present in the PCR mixture. Sanger sequencing is particularly useful for confirming results from larger amplicon sequencing projects. 

NGS approaches to targeted sequencing allow for simultaneous multiplexed sequencing of thousands of amplicons. For research objectives that require targeting hundreds of genes at time, next-generation-based amplicon sequencing provides fast and cost-efficient throughput per run. 

Scientific expertise: amplicon sequencing

GATC Biotech has several next-generation-sequencing-based services for amplicons that have been readily adopted by numerous researchers.

GATC Biotech offers exceptional flexibility by providing different services to meet a wide range of sequencing needs. One product, INVIEW AMPLICON ULTRA-DEEP, uses Illumina’s platform to provide extensive coverage of DNA fragments with reads of up to 570 bp in length. The service is ideal for in-depth analysis of complex and heterogeneous samples and for pinpointing rare mutations and SNPs. Another product, INVIEW AMPLICON ULTRA-LONG, takes advantage of the PacBio system to interrogate target regions up to 3 kb in size. These ultra-long reads are perfect for studies on bacterial taxonomic classification, co-variation or haplotyping of immune system components, like human leukocyte antigen (HLA).

GATC Biotech offers specialised amplicon sequencing services for exon targeting for variant discovery in the human exome or for 16S rRNA gene sequencing for microbiome profiling purposes. For research projects with a small number of samples and with a small number of target sites, our Sanger sequencing can also be conveniently used.  

Publications

Find here, a list of selected research articles supported by GATC Biotech’s sequencing products, as well as the latest publications on targeted sequencing.

Related products to amplicon sequencing

Interested in trying an NGS approach for targeted sequencing? Then discover our end-to-end solutions for amplicon sequencing. All products offer DNA isolation, PCR establishment, , sequencing on the leading Illumina or PacBio platform, BioIT analysis and a GATC Data Analysis report of all results in different formats and with accompanying explanations.

See how our INVIEW AMPLICON product can help advance your targeted sequencing project or contact us for more information.

Are you analysing microbial communities via 16S rRNA gene amplicon sequencing? Then try one of our popular predesigned INVIEW MICROBIOME products for taxonomic characterisation down to the species level.

Are you working with human DNA samples and need to interrogate only the protein-coding genomic sequences? Then explore our standardised INVIEW HUMAN EXOME service for generating and analysing all exon sequences. 

For cases where only a few genes of interest from a smaller number of samples need to be characterised, see how our Sanger sequencing services can be used to your advantage.  

Further reading on amplicon sequencing

For further information on NGS targeted sequencing, try the following reviews:

Murray, D.C., Coghlan, M.L., Bunce, M. From benchtop to desktop: important considerations when designing amplicon sequencing workflows. PLoS One 10(4):e0124671 (2015).

Han, S.W. et al. Targeted sequencing of cancer-related genes in colorectal cancer using next-generation sequencing. PLoS One. 8(5):e64271 (2013).