Diversity and phylogeny of Baltic Sea picocyanobacteria inferred from their ITS and phycobiliprotein operons.

Picocyanobacteria of the genus Synechococcus span a range of different colours, from red strains rich in phycoerythrin (PE) to green strains rich in phycocyanin (PC). Here, we show that coexistence of red and green picocyanobacteria in the Baltic Sea is widespread. The diversity and phylogeny of red and green picocyanobacteria was analysed using three different genes: 16S rRNA-ITS, the cpeBA operon of the red PE pigment and the cpcBA operon of the green PC pigment. Sequencing of 209 clones showed that Baltic Sea picocyanobacteria exhibit high levels of microdiversity. The partial nucleotide sequences of the cpcBA and cpeBA operons from the clone libraries of the Baltic Sea revealed two distinct phylogenetic clades: one clade containing mainly sequences from cultured PC-rich picocyanobacteria, while the other contains only sequences from cultivated PE-rich strains. A third clade of phycourobilin (PUB) containing strains of PE-rich Synechococcus spp. did not contain sequences from the Baltic Sea clone libraries. These findings differ from previously published phylogenies based on 16S rRNA gene analysis. Our data suggest that, in terms of their pigmentation, Synechococcus spp. represent three different lineages occupying different ecological niches in the underwater light spectrum. Strains from different lineages can coexist in light environments that overlap with their light absorption spectra.

Performance of amplicon-based next generation DNA sequencing for diagnostic gene mutation profiling in oncopathology.

PURPOSE: Next generation DNA sequencing (NGS) holds promise for diagnostic applications, yet implementation in routine molecular pathology practice requires performance evaluation on DNA derived from routine formalin-fixed paraffin-embedded (FFPE) tissue specimens. The current study presents a comprehensive analysis of TruSeq Amplicon Cancer Panel-based NGS using a MiSeq Personal sequencer (TSACP-MiSeq-NGS) for somatic mutation profiling. METHODS: TSACP-MiSeq-NGS (testing 212 hotspot mutation amplicons of 48 genes) and a data analysis pipeline were evaluated in a retrospective learning/test set approach (n = 58/n = 45 FFPE-tumor DNA samples) against 'gold standard' high-resolution-melting (HRM)-sequencing for the genes KRAS, EGFR, BRAF and PIK3CA. Next, the performance of the validated test algorithm was assessed in an independent, prospective cohort of FFPE-tumor DNA samples (n = 75). RESULTS: In the learning set, a number of minimum parameter settings was defined to decide whether a FFPE-DNA sample is qualified for TSACP-MiSeq-NGS and for calling mutations. The resulting test algorithm revealed 82% (37/45) compliance to the quality criteria and 95% (35/37) concordant assay findings for KRAS, EGFR, BRAF and PIK3CA with HRM-sequencing (kappa = 0.92; 95% CI = 0.81-1.03) in the test set. Subsequent application of the validated test algorithm to the prospective cohort yielded a success rate of 84% (63/75), and a high concordance with HRM-sequencing (95% (60/63); kappa = 0.92; 95% CI = 0.84-1.01). TSACP-MiSeq-NGS detected 77 mutations in 29 additional genes. CONCLUSION: TSACP-MiSeq-NGS is suitable for diagnostic gene mutation profiling in oncopathology.

KRAS and BRAF mutation analysis in routine molecular diagnostics: comparison of three testing methods on formalin-fixed, paraffin-embedded tumor-derived DNA.

Accurate mutation detection assays are strongly needed for use in routine molecular pathology analyses to aid in the selection of patients with cancer for targeted therapy. The high-resolution melting (HRM) assay is an ideal prescreening tool, and SNaPshot analysis offers a straightforward genotyping system. Our present study was determined to compare these mutation testing methods on formalin-fixed, paraffin-embedded (FFPE) tumor-derived DNA. We compared the performance of HRM, followed by cycle sequencing (HRM-sequencing); multiplex PCR assay, followed by SNaPshot analysis (multiplex mutation assay); and a successor assay using HRM, followed by SNaPshot (HRM-SNaPshot) for mutation analysis of both KRAS (codon 12/13/61) and BRAF (codon 600/601). In a series of 195 FFPE-derived DNA specimens, a high genotypic concordance between HRM-sequencing and multiplex mutation assay was found (κ, 0.98; 95% CI, 0.94 to 1), underlining the potential of a combined HRM-SNaPshot approach. In reconstruction experiments, the analytical sensitivity of HRM-SNaPshot was twofold to fourfold higher than HRM-sequencing and multiplex mutation assay, respectively. In addition, HRM-SNaPshot had a good performance rate (99%) on FFPE tumor-derived DNA, and mutation detection was highly concordant with the predecessor assays (κ for both, 0.98). The occurrence of BRAF and KRAS mutations is mutually exclusive. HRM-SNaPshot is an attractive method for mutation analysis in pathology, given its good performance rate on FFPE-derived DNA, high analytical sensitivity, and prescreening approach.

Colorful microdiversity of Synechococcus strains (picocyanobacteria) isolated from the Baltic Sea.

Synechococcus is a cosmopolitan genus of picocyanobacteria living in the photic zone of freshwater and marine ecosystems. Here, we describe the isolation of 46 closely related picocyanobacterial strains from the Baltic Sea. The isolates showed considerable variation in their cell size and pigmentation phenotypes, yielding a colorful variety of red, pink and blue-green strains. These pigmentation phenotypes could not be differentiated on the basis of their 16S rRNA-internal transcribed spacer (ITS) sequences. Thirty-nine strains, designated BSea, possessed 16S rRNA-ITS sequences almost identical with Synechococcus strain WH5701. Despite their similar 16S rRNA-ITS sequences, the BSea strains separated into several different clusters when comparing the phycocyanin (cpcBA) operon. This separation was largely consistent with the phycobiliprotein composition of the different BSea strains. The majority of phycocyanin (PC)-rich Bsea strains clustered with WH5701. Remarkably, the phycoerythrin (PE)-rich strains of BSea formed an as yet unidentified cluster within the cpcBA phylogeny, distantly related to other PE-rich groups. Detailed analysis of the cpcBA operon using neighbour-net analysis indicated that the PE-rich BSea strains are probably endemic for the Baltic Sea. Comparison of the phylogenies obtained by the 16S rRNA-ITS, the cpcBA, and the concatenated 16S rRNA-ITS and cpcBA operon sequences revealed possible events of horizontal gene transfer among different Synechococcus lineages. Our results show that microdiversity is important in Synechococcus populations and that it can reflect extensive diversification of different pigmentation phenotypes into different ecological niches.

Positive selection on transposase genes of insertion sequences in the Crocosphaera watsonii genome.

Insertion sequences (ISs) are mobile elements that are commonly found in bacterial genomes. Here, the structural and functional diversity of these mobile elements in the genome of the cyanobacterium Crocosphaera watsonii WH8501 is analyzed. The number, distribution, and diversity of nucleotide and amino acid stretches with similarity to the transposase gene of this IS family suggested that this genome harbors many functional as well as truncated IS fragments. The selection pressure acting on full-length transposase open reading frames of these ISs suggested (i) the occurrence of positive selection and (ii) the presence of one or more positively selected codons. These results were obtained using three data sets of transposase genes from the same IS family that were collected based on the level of amino acid similarity, the presence of an inverted repeat, and the number of sequences in the data sets. Neither recombination nor ribosomal frameshifting, which may interfere with the selection analyses, appeared to be important forces in the transposase gene family. Some positively selected codons were located in a conserved domain, suggesting that these residues are functionally important. The finding that this type of selection acts on IS-carried genes is intriguing, because although ISs have been associated with the adaptation of the bacterial host to new environments, this has typically been attributed to transposition or transformation, thus involving different genomic locations. Intragenic adaptation of IS-carried genes identified here may constitute a novel mechanism associated with bacterial diversification and adaptation.

Selection on protein-coding genes of natural cyanobacterial populations.

We examined the distribution of synonymous and non-synonymous changes in 12 protein-coding genes of natural populations of cyanobacteria to infer changes in gene functionality. By comparing mutation distributions within and across species using the McDonald-Kreitman test, we found data sets to contain evidence for purifying selection (hetR of Trichodesmium, nifH of Cylindrospermopsis raceborskii and rpoC1 of Anabaena lemmermannii) and positive selection (kaiC of Microcoleus chthonoplastes and rbcX of Anabaena and Aphanizomenon sp.). Other genes from the same set of clonal isolates (petB and rbcL in M. chthonoplastes and Anabaena/Aphanizomenon, respectively) did not harbour evidence for either form of selection. The results of branch models of codon evolution agreed fully with the results of the McDonald-Kreitman test in terms of significance and absolute value of the dN/dS estimates. The high frequency of gene-specific mutation patterns and their association with branches that separate closely related cyanobacterial genera suggest that evolutionary tests are suited to uncover gene-specific selective differentiation in cyanobacterial genomes. At the same time, given the lack of information about the history of cyanobacteria, analysis of larger numbers of protein-coding genes of clonal cyanobacterial isolates will produce more detailed pictures of the effects of natural selection.