ITS2 representative sequences of fungal OTUs from a metabarcoding study on decaying Norway spruce in southern Finland
CitationKorhonen A, Miettinen O, Kotze J, Hamberg L. ITS2 representative sequences of fungal OTUs from a metabarcoding study on decaying Norway spruce in southern Finland. PlutoF. Occurrence dataset https://doi.org/10.15468/6xe3m5 accessed via GBIF.org on 2023-03-21.
DescriptionThis dataset contains occurrence data of fungal operational taxonomic units (OTUs) across samples from decaying Norway spruce (Picea abies). Sampling locations are indicated with geographic coordinates. OTUs are associated with representative sequences resulting from a clustering step that was done across sequences from all samples collectively. Therefore, occurrence records imply detections of sequence variants closely resembling but not necessarily identical with the associated representative sequences. OTU was recorded as present when its read count accounted for ≥ 5‰ of the total reads in a sample. Original, raw sequence reads are available in NCBI under BioProject PRJNA732060. Representative sequences are linked to material samples. Altogether, there were 90 sampled spruce trunks, each sampled at 4 different points, resulting in 360 individual samples in total. Samples are labeled according to the following format: [numerical code of the tree trunk (1-90)]_[numerical code of subsample (1-4)]. Wood samples were collected from downed, decaying spruce trunks in October 2018. Four wood samples were taken from each tree trunk at 2, 4, 6, and 8 m distance from the base. Epiphytes, bark and loose rotten wood were removed with a flame-sterilized knife to expose a clean surface of solid wood on the side of the trunk. Wood shavings were extracted with a flame-sterilized 6 mm diameter drill and collected into paper bags and stored at -20°C until DNA extraction. DNA was extracted with a NucleoSpin Soil (Macherey-Nagel, Düren, DE) extraction kit from 112 ± 52 mg (mean ± SD) of sample material. Extraction was done according to the manufacturer’s instructions with lysis buffer SL2 and final elution in 30 µl volume. The ITS2 region was amplified using PCR with primers gITS7 and ITS4 with 6 bp dual index in 28 cycles. The resulting PCR fragments were sequenced with the MiSeq v3 (Illumina, San Diego, US-CA) 2 × 300 bp paired-end system yielding ca. 22 M raw sequence reads. Quality filtering and the removal of artefacts, primer‐dimers and primers from raw sequence reads (NCBI BioSample accessions SAMN19307225-SAMN19307584 under BioProject PRJNA732060) were conducted with the PipeCraft 1.0 pipeline. Raw ITS sequence reads were processed according to the manual with the following specifications. Assembly of paired-end reads and initial quality ﬁltering was conducted with vsearch v1.11.1 with the following parameters: minimum overlap 20, max differences 99, minimum length 150 bp, e_max 1, max ambiguous 0, and trunc qual 20. Chimera ﬁltering was performed for the reoriented reads using reference-based ﬁltering with Unite ITS2 ref. v7.1 as the database, also removing primers and primer artifacts from sequences at this step. In addition, the fungal ITS2 region was extracted from reads with ITSx. The remaining 7.876 M sequences were then clustered with Swarm v2 algorithm (d = 1, fastidious = TRUE).
- Korhonen, A., Miettinen, O., Kotze, D.J., Hamberg, L. (2022). Landscape context and substrate characteristics shape fungal communities of dead spruce in urban and semi-natural forests. Environmental Microbiology, Jan 20. - http://doi.org/10.1111/1462-2920.15903
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