Amplicon sequencing is a type of targeted sequencing that can be used for various purposes. Some common types of amplicon sequencing are 16S and ITS sequencing, which are used in phylogeny and taxonomy studies for the identification of bacteria and fungi, respectively. When there is a need to explore the genome more generally, amplicon sequencing can be used to discover rare somatic mutations, detect and characterize variants, and identify germline single nucleotide polymorphisms (SNPs), insertions/deletions (INDELs), and known fusions [1, 2]. Targeted gene sequencing panel projects are another example of amplicon sequencing, where these panels include genes that are often associated with a certain disease or phenotype-of-interest [3].

In this article, we will go over what amplicon sequencing is, describe the advantages and disadvantages of short- and long-read sequencing, and then explain how Genohub can help support your project.

Amplicon Sequencing

Amplicon sequencing is targeted sequencing that involves specific primer design in order to achieve high on-target rates. It’s called amplicon sequencing, because a crucial step of the process is polymerase chain reaction (PCR), which is a method that amplifies specific DNA sequences based on the primers used. Primers are small DNA oligos that are specifically designed to target only the genes/regions-of-interest. When the amplification part of PCR occurs, only these specific genes are multiplied. The final products of PCR are called amplicons, hence amplicon sequencing [1].

It’s important to think about what type of sequencing (short vs. long read) needs to be done for your specific project, because in order to sequence amplicon samples, the appropriate adapters need to be added to help them adhere to sequencing flow cells [2]. These adapters will differ depending on the flow cell, and in some cases, it may even be more cost-effective to send DNA samples and have one of our NGS partners perform all the library prep themselves.

Short read sequencing (Illumina)

Short-read amplicon sequencing is done with Illumina platforms, often the MiSeq, and has been the standard for 16S, ITS and other microbial profiling projects for many years. Being the standard for so long has advantages, as there are many targeted gene panels created and validated already for use with Illumina sequencing, which can make the workflow much easier on researchers who are new to targeted sequencing. There is also an abundance of literature with Illumina sequencing, so it’s easy for researchers to compare their findings to those of other groups. The biggest advantage is that researchers can sequence hundreds of genes in a single run, which lowers sequencing costs and turnaround time, especially if the researcher is interested in many different genes [1].

A disadvantage with short-read sequencing is that the sequencing resolution may not be as high as long-read sequencing. A comparison of short-read to long-read 16S amplicon sequencing showed that only long-read sequencing could provide strain-level community resolution and insight into novel taxa. Then for the metagenomics portion, a greater number of and more complete bacterial metagenome-assembled genomes (MAGs) were recovered from the data generated from long reads [4].

Long read sequencing (PacBio and Nanopore)

Long-read amplicon sequencing is done with either the PacBio or Oxford Nanopore platforms. They both offer complete, contiguous, uniform, and non-biased coverage across long amplicons up to 10 kb. Advantages of this type of long-read amplicon sequencing is that it’s more efficient, accurate and sensitive than short-read sequencing.

PacBio sequencing can obtain up to 99.999% single-molecule base calling accuracy and has been used to sequence full-length 16S and ITS sequences with very high accuracy as well [3].

Nanopore sequencing can provide accurate variant calling as well as robust coverage of larger targeted regions, which can help enhance the analysis of repetitive regions and improve taxonomic assignment [5]. Nanopore sequencing also tends to allow a bit more flexibility than PacBio sequencing when it comes to scaling amplicon projects at a cost-effective price [6].

The disadvantages to using long-read sequencing for amplicon projects is that it tends to be much more expensive and time-consuming than short-read sequencing, and sometimes long reads may not even be needed if the targeted amplicons themselves are already very short.

How can Genohub help you?

Genohub’s amplicon sequencing partners are experts in every step of the amplicon sequencing process, including extraction, PCR amplification, adapter ligation, library prep and data analysis. Our partners have experience extracting from many different types of environmental and biological samples, but they can work just as well with your DNA or amplicons if you prefer to extract and/or perform PCR in your own lab. From our experience, it’s more cost-effective to send DNA samples rather than amplicons, unless you can attach Illumina adapters yourself.

We know that each research project is unique, so we have partners who are also open to working with your custom primers, custom gene panels and custom bioinformatics needs! Get started today by letting us know about your amplicon sequencing project here: https://genohub.com/ngs/ .