274
Comparative Analysis of Sanger and Illumina Miseq Sequencing for Determining Indoor Fungal Diversity
Saturday, March 4, 2017: 2:45 PM
Rooms B303-B304 (Georgia World Congress Center, Building B)
Angela R. Lemons, M.S., ,
Rationale: Diversity studies utilizing sequencing technologies have elucidated a wider array of fungal species, particularly those belonging to the phylum Basidiomycota, typically overlooked using traditional methods of assessment. With the emergence of next-generation sequencing technologies, we aimed to compare the fungal diversity captured using Sanger and Illumina MiSeq sequencing approaches to determine the reproducibility of the datasets.

Methods: Genomic DNA originating from a previous study (n=4) were submitted for Sanger and Illumina MiSeq sequencing of the internal transcribed spacer (ITS) rRNA regions. Sanger sequencing utilized the primer pair ITS1F-ITS4R while Illumina combined the paired reads of primer sets ITS1F-ITS2R and ITS3F-ITS4R. The fungal diversity acquired from each sequencing method were compared.  

Results: Sequencing the ITS1-2 regions showed differences in fungal species distribution captured using Sanger and Illumina MiSeq. Sanger sequencing elucidated fungi placed in the phyla Ascomycota (54%) and Basidiomycota (46%). In contrast, Illumina sequencing of the ITS1-2 regions (ITS1F-ITS4R) revealed 129,859 fungal sequences from which 98% were placed in the Ascomycota. Examination of the sequences showed a lack of assembly of the paired reads that resulted in the removal of Basidiomycete sequences from the analysis. Limiting Illumina sequencing to the ITS1 region (ITS1F-ITS2R) showed a distribution similar to that observed with Sanger sequencing, with 243,589 sequences placed in the Ascomycota (67%) and Basidiomycota (33%).

Conclusions: This study demonstrates the importance of evaluating limitations of new sequencing technologies before implementation in an environmental or epidemiological survey. The results of this study additionally suggest that the ITS1 region provides the most reproducibility between sequencing platforms.