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Sequencing

General steps for sequencing in eDNA metabarcoding are:

Selection of barcode region to amplify: The choice depends on the target micro- or macro-biome (look at the table shown earlier)

Selection of primer pair for amplification: There are typically multiple primer options available for each barcode region, with the selection depending on the specific taxonomic target or desired amplicon length.

Library preparation: Library preparation for metabarcoding begins with PCR from the extracted DNA using primers selected for your barcode region of choice. In order that samples can be multiplexed and sequenced together a short (usually 8–10bp), unique index sequence (also known as a Multiplex IDentifier or MID-tag) needs to be added. Lastly, adapter sequences for the technology to be used are added. For Illumina sequencing these are known as the P5 and P7 adapters.

There are two key PCR methods used to amplify the DNA barcode region:

  • Dual Index method 1 involves a single PCR with barcode-specific primers followed by a ligation of indexed P5 & P7 adapters (Figure 3).
  • Dual index method 2 involves a two-step PCR (Figure 3). In this second method, the first PCR is performed with the barcode-specific primers that also include a known linker sequence (shown in orange and green in the Figure 3). And the second PCR includes the indexed P5 & P7 adapters using primers that also contain the linker sequence.
Diagram illustrating different steps in library preparation for DNA sequencing. The image shows multiple sequences with colored blocks representing various components: primers, adapters, and barcodes. The sequences progress through stages where different sections are added, such as barcode-specific primers, linker sequences, and indexed P5 and P7 adapters, highlighting both single and two-step PCR processes for dual indexing.
Figure 3 The image shows two most common methods used for preparing sequence constructs for metabarcoding, showing single-step PCR(Fusion primers) and a two-step PCR approach. (Image credit: Joe Taylor, UKCEH; This image is licensed under a Creative Commons Attribution 4.0 International (CC BY-NC-SA 4.0) license)

Alternatively, a third method (not shown) involves a single PCR using indexed primers for the target region, followed by the addition of adapters via a ligation reaction. However, this ligation step can be costly and technically challenging. Similarly, the long primers used in method 1 can be more expensive, especially when multiple tubes are required for unique indexes during multiplexing. As a result, dual index method 2 is the most flexible (since one set of indexed adapters can be used for multiple PCR targets) and is the most commonly applied approach.

Selecting a sequencing technology:

Sequencing in eDNA metabarcoding is typically performed using Illumina sequencing technology (Bohmann et al 2022), however, explore the following options before selecting the sequencing technology suitable for your research. Click on the tabs to expand:

After generating your sequencing data, you’ll need to interpret the results through bioinformatics analysis to identify the diverse species in your samples. You can now proceed to the next page to explore a variety of open resources available for this type of data analysis.