Internal transcribed spacer primer evaluation for vascular plant metabarcoding

By

Kolter, Andreas
Gemeinholzer, Birgit 1968-

Type

Article

Date of Publication

2021-9-2

Original Publication

Metabarcoding and metagenomics

Volume

5

Pages

1--20

Subjects

Barcoding , DMSO , internal transcribed spacer , mock community , PCR , Spermatophyta , Tracheophyta

Contributed by

Pensoft Publishers

Language

English

Published by

Bulgaria Pensoft Publishers 2017-

Identifiers

DOI: https://doi.org/10.3897/mbmg.5.68155

 

Abstract

The unprecedented ongoing biodiversity decline necessitates scalable means of monitoring in order to fully understand the underlying causes. DNA metabarcoding has the potential to provide a powerful tool for accurate and rapid biodiversity monitoring. Unfortunately, in many cases, a lack of universal standards undermines the widespread application of metabarcoding. One of the most important considerations in metabarcoding of plants, aside from selecting a potent barcode marker, is primer choice. Our study evaluates published ITS primers in silico and in vitro, through mock communities and presents newly designed primers. We were able to show that a large proportion of previously available ITS primers have unfavourable attributes. Our combined results support the recommendation of the introduced primers ITS-3p62plF1 and ITS-4unR1 as the best current universal plant specific ITS2 primer combination. We also found that PCR optimisation, such as the addition of 5% DMSO, is essential to obtain meaningful results in ITS2 metabarcoding. Finally, we conclude that continuous quality assurance is indispensable for reliable metabarcoding results.