Computational protocol: Diversity and distribution of fungal communities in the marine sediments of Kongsfjorden, Svalbard (High Arctic)

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Protocol publication

[…] Raw sequence data generated from pyrosequencing were processed using QIIME 1.8.0 software. In brief, the sequence libraries were split and denoised to avoid diversity overestimation caused by sequencing errors, including sequences with average quality score <20 over a 50 bp sliding window, sequences shorter than 200 bp, sequences with homopolymers longer than six nucleotides, and sequences containing ambiguous base calls or incorrect primer sequences. Operational Taxonomic Units (OTUs) were clustered with 97% similarity cutoff using UPARSE and chimeric sequences were identified and removed using UCHIME. The most abundant sequence for each OTU was chosen as a representative sequence. These OTUs were used to calculate alpha-diversity indices (i.e., Chao1, Good’s coverage estimator, and Shannon) and beta-diversity metrics using QIIME 1.8.0 software. [...] Sequences representing the OTUs were subjected to BLASTn search in GenBank (http://www.ncbi.nlm.nih.gov/genbank/) in order to determine their taxonomic affiliation. The following criteria were used to interpret the sequences. For sequence identities ≥97%, the genus and species were accepted, for sequence identities between 95% and 97%, only the genus was accepted, and for sequence identities <95%, OTUs were labeled at the order, family or phylum name or as ‘unassigned’. A phylogenetic tree was constructed to illustrate the relationships between the fungi in Arctic sediments from Kongsfjorden and those in Arctic waters from lands around Kongsfjorden (NCBI SRA Accession No. SRP049681) using MEGA v. 6.0 and the Neighbor-Joining algorithm, with bootstrap values calculated from 1,000 replicate runs. The ITS sequences were aligned using a multiple sequence alignment program MAFFT version 7 (http://mafft.cbrc.jp/alignment/server/). The relevance of environmental variables in explaining the distribution patterns of fungal communities in the eight sediments was analyzed by distance based redundancy analysis (db-RDA) using R 3.1.1 statistical software. A Venn diagram of shared and unique OTUs among the 3 basins was generated using Venny 2.0 (http://bioinfogp.cnb.csic.es/tools/venny/index. html). Network analysis was performed to visualize the distribution of 113 OTUs among the 8 sampling sites using Gephi 0.8.2 software. […]

Pipeline specifications

Software tools QIIME, UPARSE, UCHIME, BLASTN, MEGA-V, MAFFT, VENNY, Gephi
Databases SRA
Applications Phylogenetics, 16S rRNA-seq analysis, GWAS