Accurate estimation of fungal diversity and abundance through improved lineage-specific primers optimized for Illumina amplicon sequencing

D. Lee Taylor, William A. Walters, Niall J. Lennon, James Bochicchio, Andrew Krohn, James G Caporaso, Taina Pennanen

Research output: Contribution to journalArticle

68 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)7217-7226
Number of pages10
JournalApplied and Environmental Microbiology
Volume82
Issue number24
DOIs
StatePublished - 2016

Fingerprint

Soil
soil sampling
ribosomal RNA
gene dosage
Large Ribosome Subunits
vascular plants
internal transcribed spacers
Gene Dosage
eukaryotic cells
soil
gene
cluster analysis
eukaryote
Ecology
Eukaryota
methodology
vascular plant
rRNA Genes
clones
Blood Vessels

ASJC Scopus subject areas

  • Biotechnology
  • Food Science
  • Applied Microbiology and Biotechnology
  • Ecology

Cite this

Accurate estimation of fungal diversity and abundance through improved lineage-specific primers optimized for Illumina amplicon sequencing. / Taylor, D. Lee; Walters, William A.; Lennon, Niall J.; Bochicchio, James; Krohn, Andrew; Caporaso, James G; Pennanen, Taina.

In: Applied and Environmental Microbiology, Vol. 82, No. 24, 2016, p. 7217-7226.

Research output: Contribution to journalArticle

Taylor, D. Lee ; Walters, William A. ; Lennon, Niall J. ; Bochicchio, James ; Krohn, Andrew ; Caporaso, James G ; Pennanen, Taina. / Accurate estimation of fungal diversity and abundance through improved lineage-specific primers optimized for Illumina amplicon sequencing. In: Applied and Environmental Microbiology. 2016 ; Vol. 82, No. 24. pp. 7217-7226.
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