Intraspecies-Resolution Metabarcoding: automated primer design and a plant pathology case study

Leighton Pritchard, Sonia Humphris, Emma Campbell, Lauren Watts, Ed Haynes, Hollie Pufal, Greig Cahill, Triona Davey, John Elphinstone, Ian Toth

Research output: Contribution to conferencePoster

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We present results demonstrating species-specificity and sub-species resolution by novel, automatically-designed metabarcoding primers for environmental DNA analysis.

Conventional metabarcoding remains a cornerstone of rapid, high-throughput environmental DNA (eDNA) community analysis and biodiversity assessment. Standard barcodes such as 16S (prokaryotes) and ITS1 (fungi/oomycetes) have been instrumental in identifying the complex composition of communities using total eDNA. However, standard barcodes have limitations in terms of resolution and quantitation and, though genus-level identification can be reliable, species-level identification is often not possible.

To overcome the limitations of resolution, we implemented extensions to the diagnostic primer design tool pdp ( that enable automated design of metabarcoding markers and corresponding primers that are (i) specific to a prescribed taxon at species level and (ii) capable of discriminating between members of the same species. This allows for rapid, high-throughput measurement of diversity below species level for a target organism.

We aimed to survey geographical distribution and pathogen transfer of the widepread plant pathogenic bacterium Pectobacterium atrosepticum (Pba). This organism has considerable sub-species taxonomic structure identifiable using MLST and with whole-genome methods, but which is not accessible using standard barcodes. We designed metabarcoding primers (202bp) specific to Pba using `pdp`, and established that these have resolution comparable to eight-gene MLST, revealing sub species-level diversity within single fields, and on the same individual plant host.
Original languageEnglish
Number of pages1
Publication statusPublished - 6 Apr 2021
EventMicrobiology Society Annual Conference 2021 - Online
Duration: 26 Apr 202130 Apr 2021


ConferenceMicrobiology Society Annual Conference 2021
Internet address


  • metabarcoding
  • DNA
  • eDNA
  • Pectobacterium atrosepticum (Pba)
  • pathogens


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