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Pipeline publication

[…] SOC medium was added to the mixture after heat shock and incubated for 20 min in shaker (160 RPM) at 37°C, and the entire reaction volume was plated in LB plates containing 150 μg mL-1 of ampicillin., Plasmid extraction was performed using the QIAprep Spin Miniprep kit (Qiagen) and cycle sequencing was carried out using an ABI Prism® BigDye® Terminator v3.1 cycle sequencing kit. The oligonucleotide primers used to sequence PCR products are given in Additional file : Table S4. Sanger sequencing was performed in an ABI PRISM 310 Genetic Analyzer. Contigs were aligned in the program Phred/Phrap/Consed (Philip Green, University of Washington, Seattle, USA), accepting bases with quality >20. BLASTn was used to search the nr database at NCBI for related strains to be included in phylogenetic analysis. GenBank accession numbers are indicated in Additional file : Table S5. Maximum-likelihood trees were constructed in MEGA 5.0 []. The best substitution model for each sequence was chosen based on analysis in MEGA 5.0 (K2 + G for the small tree of 16S rRNA; GTR + G + I for mcyD concatenated with mcyE; K2 + G + I for long tree of 16S rRNA; JTT + G + I for condensation, adenylation, and epimerization domains sequences of McyA; and JTT + G for peptidyl carrier protein domain sequences of McyA). Neighbor-joining and maximum parsimony trees were constructed using the respective methods: JTT + G and CNI on random trees. The domains present in the McyA amino acid sequences were detected in the program PKS/NRPS Analysis []. Adenylation domain substrate specificity prediction was performed using the program NRPSpredictor2 [,]., The nucleotide sequences of 13 cyanobacterial strains (Additional file : Table S5) corresponding to second adenylation domain of mcyA (mcyA2) were aligned in ClustalW (MEGA 5.0). The recombination analyses were performed in the program RDP3 [,], which implements different methods to detect recombination. The methods used in this study were: original RDP [], BOOTSCAN [], GENECONV [], MAXCHI [], CHIMAERA [], SISCAN [] and 3Seq []. Default parameters were used and a P-value cutoff was chosen as 0.05. Nucleotide sequence comparison was performed in BLASTn (NCBI database) […]

Pipeline specifications

Software tools BLASTN, MEGA, PKS/NRPS