Pooled samples bias fungal community descriptions

• Published in: Molecular ecology resources Vol. 10; no. 1; pp. 135 - 141
• Main Authors: AVIS, P.G, BRANCO, S, TANG, Y, MUELLER, G.M
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford, UK : Blackwell Publishing Ltd 2010; Blackwell Publishing Ltd; Wiley Subscription Services, Inc
• Subjects: ARISA; clone sequence; Deoxyribonucleic acid; DNA; Ecological studies; environmental sample; Extraction procedures; Fungi; Polymorphism; pooled samples; Restriction fragment length polymorphism; Species richness; T-RFLP
• ISSN: 1755-0998; 1755-098X; 1755-0998
• DOI: 10.1111/j.1755-0998.2009.02743.x

We tested the accuracy of molecular analyses for recovering the species richness and structure of pooled fungal communities of known composition. We constructed replicate pools of 2-20 species and analysed these pools by two separate pooling-DNA extraction procedures and three different molecular analyses (Automated Ribosomal Intergenic Spacer Analysis (ARISA), terminal restriction fragment length polymorphism (T-RFLP) and clone library-sequencing). None of the methods correctly described the known communities. Only clone library-sequencing with high sequencing per pool (~100 clones) recovered reasonable estimates of richness. Frequency data were skewed with all procedures and analyses. These results indicate that the error introduced by pooling samples is significant and problematic for ecological studies of fungal communities.