Fungal DNA extraction for Nanopore sequencing v1

This protocol is intended for extraction of high molecular weight DNA from fungal samples.

Characterization and Phylogeny of Fungi from Industrial Wastewater Using Highly Conserved Multiple Gene Loci

The aim of this study was isolation and molecular characterization of fungi from untreated industrial effluent by multigene phylogenetic analyses. The Fungi isolated were characterized based on PCR amplification and genomic sequencing of the internal transcribed spacer region (ITS), partial β-tubulin (Ben A), calmodulin (CaM), and DNA-directed RNA polymerase second large subunit (RPB2) genes, along with morphological characterization and species diversity. Fungal DNA extraction kits and primers sets for the selected genes were purchased and used following the manufacturer’s instructions. The obtained sequences were subjected to BLAST analysis and the corresponding fungal isolates were assigned species names after comparison with representative sequences available in GenBank. All the sequences from this study were deposited in GenBank and the accession number assigned. Phylogenetic trees of the fungal isolates were drawn for each gene by the Maximum Likelihood method using MEGA 7.0 software. Fifteen (15) Fungi species belonging to four genera of Aspergillus, Penicillium, Fusarium and Trichoderma with Aspergillus as the predominant genus were identified.

Assessment of fecal DNA extraction protocols for metagenomic studies

Background Shotgun metagenomic sequencing has improved our understanding of the human gut microbiota. Various DNA extraction methods have been compared to find protocols that robustly and most accurately reflect the original microbial community structures. However, these recommendations can be further refined by considering the time and cost demands in dealing with samples from very large human cohorts. Additionally, fungal DNA extraction performance has so far been little investigated. Results We compared 6 DNA extraction protocols, MagPure Fast Stool DNA KF Kit B, Macherey Nagel™ NucleoSpin™®Soil kit, Zymo Research Quick-DNA™ Fecal/Soil Microbe kit, MOBIO DNeasy PowerSoil kit, the manual non-commercial protocol MetaHIT, and the recently published protocol Q using 1 microbial mock community (MMC) (containing 8 bacterial and 2 fungal strains) and fecal samples. All samples were manually extracted and subjected to shotgun metagenomics sequencing. Extracting DNA revealed high reproducibility within all 6 protocols, but microbial extraction efficiencies varied. The MMC results demonstrated that bead size was a determining factor for fungal and bacterial DNA yields. In human fecal samples, the MagPure bacterial extraction performed as well as the standardized protocol Q but was faster and more cost-effective. Extraction using the PowerSoil protocol resulted in a significantly higher ratio of gram-negative to gram-positive bacteria than other protocols, which might contribute to reported gut microbial differences between healthy adults. Conclusions We emphasize the importance of bead size selection for bacterial and fungal DNA extraction. More importantly, the performance of the novel protocol MP matched that of the recommended standardized protocol Q but consumed less time, was more cost-effective, and is recommended for further large-scale human gut metagenomic studies.