Dataset Information

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.

Importance

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.

SUBMITTER: Taylor DL

PROVIDER: S-EPMC5118932 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Publications

Accurate Estimation of Fungal Diversity and Abundance through Improved Lineage-Specific Primers Optimized for Illumina Amplicon Sequencing.

Taylor D Lee DL Walters William A WA Lennon Niall J NJ Bochicchio James J Krohn Andrew A Caporaso J Gregory JG Pennanen Taina T

Applied and environmental microbiology 20161121 24

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 ...[more]

PMID: 27736792

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Project description:UnlabelledThe use of high-throughput sequencing technologies with the 16S rRNA gene for characterization of bacterial and archaeal communities has become routine. However, the adoption of sequencing methods for eukaryotes has been slow, despite their significance to natural and engineered systems. There are large variations among the target genes used for amplicon sequencing, and for the 18S rRNA gene, there is no consensus on which hypervariable region provides the most suitable representation of diversity. Additionally, it is unclear how much PCR/sequencing bias affects the depiction of community structure using current primers. The present study amplified the V4 and V8-V9 regions from seven microalgal mock communities as well as eukaryotic communities from freshwater, coastal, and wastewater samples to examine the effect of PCR/sequencing bias on community structure and membership. We found that degeneracies on the 3' end of the current V4-specific primers impact read length and mean relative abundance. Furthermore, the PCR/sequencing error is markedly higher for GC-rich members than for communities with balanced GC content. Importantly, the V4 region failed to reliably capture 2 of the 12 mock community members, and the V8-V9 hypervariable region more accurately represents mean relative abundance and alpha and beta diversity. Overall, the V4 and V8-V9 regions show similar community representations over freshwater, coastal, and wastewater environments, but specific samples show markedly different communities. These results indicate that multiple primer sets may be advantageous for gaining a more complete understanding of community structure and highlight the importance of including mock communities composed of species of interest.ImportanceThe quantification of error associated with community representation by amplicon sequencing is a critical challenge that is often ignored. When target genes are amplified using currently available primers, differential amplification efficiencies result in inaccurate estimates of community structure. The extent to which amplification bias affects community representation and the accuracy with which different gene targets represent community structure are not known. As a result, there is no consensus on which region provides the most suitable representation of diversity for eukaryotes. This study determined the accuracy with which commonly used 18S rRNA gene primer sets represent community structure and identified particular biases related to PCR amplification and Illumina MiSeq sequencing in order to more accurately study eukaryotic microbial communities.

Dataset Information

Accurate Estimation of Fungal Diversity and Abundance through Improved Lineage-Specific Primers Optimized for Illumina Amplicon Sequencing.

Importance

Publications

Accurate Estimation of Fungal Diversity and Abundance through Improved Lineage-Specific Primers Optimized for Illumina Amplicon Sequencing.

Similar Datasets

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