title: Accurate Estimation of Fungal Diversity and Abundance through Improved Lineage-Specific Primers Optimized for Illumina Amplicon Sequencing.
abstract: While high-throughput sequencing methods are revolutionizing fungal ecology, recovering accurate estimates of species richness and abundance has proven elusive. We sought to design internal transcribed spacer (ITS) primers and an Illumina protocol that would maximize coverage of the kingdom Fungi while minimizing nontarget eukaryotes. We inspected alignments of the 5.8S and large subunit (LSU) ribosomal genes and evaluated potential primers using PrimerProspector. We tested the resulting primers using tiered-abundance mock communities and five previously characterized soil samples. We recovered operational taxonomic units (OTUs) belonging to all 8 members in both mock communities, despite DNA abundances spanning 3 orders of magnitude. The expected and observed read counts were strongly correlated (r = 0.94 to 0.97). However, several taxa were consistently over- or underrepresented, likely due to variation in rRNA gene copy numbers. The Illumina data resulted in clustering of soil samples identical to that obtained with Sanger sequence clone library data using different primers. Furthermore, the two methods produced distance matrices with a Mantel correlation of 0.92. Nonfungal sequences comprised less than 0.5% of the soil data set, with most attributable to vascular plants. Our results suggest that high-throughput methods can produce fairly accurate estimates of fungal abundances in complex communities. Further improvements might be achieved through corrections for rRNA copy number and utilization of standardized mock communities.
  
  
  
  Fungi play numerous important roles in the environment. Improvements in sequencing methods are providing revolutionary insights into fungal biodiversity, yet accurate estimates of the number of fungal species (i.e., richness) and their relative abundances in an environmental sample (e.g., soil, roots, water, etc.) remain difficult to obtain. We present improved methods for high-throughput Illumina sequencing of the species-diagnostic fungal ribosomal marker gene that improve the accuracy of richness and abundance estimates. The improvements include new PCR primers and library preparation, validation using a known mock community, and bioinformatic parameter tuning.
authors.affiliation: Department of Biology, University of New Mexico, Albuquerque, New Mexico, USA fflt@unm.edu.
authors.forename: D Lee
authors.identifier: 0000-0002-5985-9210
authors.initials: DL
authors.lastname: Taylor
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.
authors.forename: William A
authors.initials: WA
authors.lastname: Walters
authors.affiliation: Broad Institute of MIT & Harvard, Cambridge, Massachusetts, USA.
authors.forename: Niall J
authors.initials: NJ
authors.lastname: Lennon
authors.affiliation: Broad Institute of MIT & Harvard, Cambridge, Massachusetts, USA.
authors.forename: James
authors.initials: J
authors.lastname: Bochicchio
authors.affiliation: Department of Biological Sciences, Northern Arizona University, Flagstaff, Arizona, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Krohn
authors.affiliation: Department of Biological Sciences, Northern Arizona University, Flagstaff, Arizona, USA.
authors.forename: J Gregory
authors.identifier: 0000-0002-8865-1670
authors.initials: JG
authors.lastname: Caporaso
authors.affiliation: Natural Resources Institute Finland (Luke), Vantaa, Finland.
authors.forename: Taina
authors.initials: T
authors.lastname: Pennanen
mesh_terms: D044822:Biodiversity; D017931:DNA Primers / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D005658:Fungi / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D014644:Genetic Variation; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013045:Species Specificity
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal; D021903:DNA, Ribosomal Spacer