PacBio amplicon sequencing for metabarcoding of mixed DNA samples from lichen herbarium specimens

By

Gueidan, Cécile 1974-
Elix, John A. (John Alan), 1941-
McCarthy, Patrick Martin 1955-
Roux, Claude
Mallen-Cooper, Max
Kantvilas, Gintaras

Type

Article

Date of Publication

2019

Original Publication

NeoBiota : advancing research on alien species and biological invasions

Volume

53

Pages

73-91

Subjects

high-throughput sequencing , lichenised fungi , long amplicon analysis (LAA) , SMRT sequencing

Contributed by

Pensoft Publishers

Language

English

Published by

Sophia, Bulgaria Pensoft Publishers

Identifiers

DOI: https://doi.org/10.3897/mycokeys.53.34761

 

Abstract

The detection and identification of species of fungi in the environment using molecular methods heavily depends on reliable reference sequence databases. However, these databases are largely incomplete in terms of taxon coverage, and a significant effort is required from herbaria and living fungal collections for the mass-barcoding of well-identified and well-curated fungal specimens or strains. Here, a PacBio amplicon sequencing approach is applied to recent lichen herbarium specimens for the sequencing of the fungal ITS barcode, allowing a higher throughput sample processing than Sanger sequencing, which often required the use of cloning. Out of 96 multiplexed samples, a full-length ITS sequence of the target lichenised fungal species was recovered for 85 specimens. In addition, sequences obtained for co-amplified fungi gave an interesting insight into the diversity of endolichenic fungi. Challenges encountered at both the laboratory and bioinformatic stages are discussed, and cost and quality are compared with Sanger sequencing. With increasing data output and reducing sequencing cost, PacBio amplicon sequencing is seen as a promising approach for the generation of reference sequences for lichenised fungi as well as the characterisation of lichen-associated fungal communities.