Computational protocol: Seasonal variation in nifH abundance and expression of cyanobacterial communities associated with boreal feather mosses

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Protocol publication

[…] On the basis of the alignment of nifH sequences carried out by , specific primers targeting each cyanobacterial cluster were designed (that is, ‘Nostoc cluster I', ‘Nostoc cluster II', ‘Stigonema cluster' and ‘nifH2 cluster', respectively; ). Primer sequences were subjected to BLASTN () to confirm that they were targeting each cyanobacterial cluster in the GenBank database. To reconfirm the specificity of the individual primer sets, the qPCR products were purified using the QIAquick PCR Purification Kit (Qiagen GmbH, Minden, Germany) and sequenced using the respective forward primer from each product. The retrieved sequences were included in a phylogenetic analysis using the same nucleotide sequences as . Nucleotide sequences were aligned in Ugene (), using the MUSCLE algorithm and a phylogenetic dendrogram was inferred. Branch support was obtained with 100 bootstraps replicates.Cyanobacterial 16S rRNA primer pair () was evaluated using the TestPrime web tool (http://www.arb-silva.de/search/testprime). Primer specificity and coverage for the most commonly observed cyanobacteria genera on P. schreberi and H. splendens (Nostoc, Calothrix, Fischerella, Cylindrospermum and Stigonema) and cross-reactivity with non-cyanobacteria sequences was determined with ARB software package () using the SILVA RefNR SSU-123 sequences (). […]

Pipeline specifications

Software tools BLASTN, Unipro UGENE, ARB
Applications Phylogenetics, Sanger sequencing