Evaluation of Two Highly-Multiplexed Custom Panels for Massively Parallel Semiconductor Sequencing on Paraffin DNA

Background—Aim

Massively parallel sequencing (MPS) holds promise for expanding cancer translational research and diagnostics. As yet, it has been applied on paraffin DNA (FFPE) with commercially available highly multiplexed gene panels (100s of DNA targets), while custom panels of low multiplexing are used for re-sequencing. Here, we evaluated the performance of two highly multiplexed custom panels on FFPE DNA.

Methods

Two custom multiplex amplification panels (B, 373 amplicons; T, 286 amplicons) were coupled with semiconductor sequencing on DNA samples from FFPE breast tumors and matched peripheral blood samples (n samples: 316; n libraries: 332). The two panels shared 37% DNA targets (common or shifted amplicons). Panel performance was evaluated in paired sample groups and quartets of libraries, where possible.

Results

Amplicon read ratios yielded similar patterns per gene with the same panel in FFPE and blood samples; however, performance of common amplicons differed between panels (p<0.001). FFPE genotypes were compared for 1267 coding and non-coding variant replicates, 999 out of which (78.8%) were concordant in different paired sample combinations. Variant frequency was highly reproducible (Spearman’s rho 0.959). Repeatedly discordant variants were of high coverage / low frequency (p<0.001). Genotype concordance was (a) high, for intra-run duplicates with the same panel (mean±SD: 97.2±4.7, 95%CI: 94.8–99.7, p<0.001); (b) modest, when the same DNA was analyzed with different panels (mean±SD: 81.1±20.3, 95%CI: 66.1–95.1, p = 0.004); and (c) low, when different DNA samples from the same tumor were compared with the same panel (mean±SD: 59.9±24.0; 95%CI: 43.3–76.5; p = 0.282). Low coverage / low frequency variants were validated with Sanger sequencing even in samples with unfavourable DNA quality.

Conclusions

Custom MPS may yield novel information on genomic alterations, provided that data evaluation is adjusted to tumor tissue FFPE DNA. To this scope, eligibility of all amplicons along with variant coverage and frequency need to be assessed.     

Exploring the robustness of macrophyte-based classification methods to assess the ecological status of coastal and transitional ecosystems under the Water Framework Directive

Identifying and quantifying the factors that contribute to the potential misclassification of the ecological status of water bodies is a major challenge of the Water Framework Directive (WFD). The present study compiles extensive biomonitoring data from a range of macrophyte-based classification methods developed by several European countries. The data reflect spatial and temporal variation as well as inter-observer variation. Uncertainty analysis identified that factors related to the spatial scale of sampling generally contributed most to the uncertainty in classifying water bodies to their ecological status, reflecting the high horizontal and depth-related heterogeneity displayed by macrophyte communities. In contrast, the uncertainty associated with temporal variation was low. In addition, inter-observer variation, where assessed, did not contribute much to overall uncertainty, indicating that these methods are easily transferable and insensitive to observer error. The study, therefore, suggests that macrophyte-based sampling schemes should prioritize large spatial replication over temporal replication to maximize the effectiveness and reliability of water body classification within the WFD. We encourage conducting similar uncertainty analyses for new/additional ecological indicators to optimize sampling schemes and improve the reliability of classification of ecological status.     

Diversity of European seagrass indicators: patterns within and across regions

Seagrasses are key components of coastal marine ecosystems and many monitoring programmes worldwide assess seagrass health and apply seagrasses as indicators of environmental status. This study aims at identifying the diversity and characteristics of seagrass indicators in use within and across European ecoregions in order to provide an overview of seagrass monitoring effort in Europe. We identified 49 seagrass indicators used in 42 monitoring programmes and including a total of 51 metrics. The seagrass metrics represented 6 broad categories covering different seagrass organizational levels and spatial scales. The large diversity is particularly striking considering that the pan-European Water Framework Directive sets common demands for the presence and abundance of seagrasses and related disturbance-sensitive species. The diversity of indicators reduces the possibility to provide pan-European overviews of the status of seagrass ecosystems. The diversity can be partially justified by differences in species, differences in habitat conditions and associated communities but also seems to be determined by tradition. Within each European region, we strongly encourage the evaluation of seagrass indicator–pressure responses and quantification of the uncertainty of classification associated to the indicator in order to identify the most effective seagrass indicators for assessing ecological quality of coastal and transitional water bodies.     

Quantitative Imaging of Microtubule Alteration as an Early Marker of?Axonal Degeneration after Ischemia in Neurons

Neuronal death can be preceded by progressive dysfunction of axons. Several pathological conditions such as ischemia can disrupt the neuronal cytoskeleton. Microtubules are basic structural components of the neuronal cytoskeleton that regulate axonal transport and neuronal function. Up-to-date, high-resolution observation of microtubules in living neuronal cells is usually accomplished using fluorescent-based microscopy techniques. However, this needs exogenous fluorescence markers to produce the required contrast. This is an invasive procedure that may interfere with the microtubule dynamics. In this work, we show, for the first time to our knowledge, that by using the endogenous (label-free) contrast provided by second harmonic generation (SHG) microscopy, it is possible to identify early molecular changes occurring in the microtubules of living neurons under ischemic conditions. This is done by measuring the intensity modulation of the SHG signal as a function of the angular rotation of the incident linearly polarized excitation light (technique referred to as PSHG). Our experiments were performed in microtubules from healthy control cultured cortical neurons and were compared to those upon application of several periods of oxygen and glucose deprivation (up to 120 min) causing ischemia. After 120-min oxygen and glucose deprivation, a change in the SHG response to the polarization was measured. Then, by using a three-dimensional PSHG biophysical model, we correlated this finding with the structural changes occurring in the microtubules under oxygen and glucose deprivation. To our knowledge, this is the first study performed in living neuronal cells that is based on direct imaging of axons and that provides the means of identifying the early symptoms of ischemia. Live observation of this process might bring new insights into understanding the dynamics and the mechanisms underlying neuronal degeneration or mechanisms of protection or regeneration.     

BIODIVERSITY OF CORALLINE ALGAE IN THE NORTHEASTERN ATLANTIC INCLUDING CORALLINA CAESPITOSA SP. NOV. (CORALLINOIDEAE,RHODOPHYTA)1

The Corallinoideae (Corallinaceae) is represented in the northeastern Atlantic by Corallina officinalis L.; Corallina elongata J. Ellis et Sol.; Haliptilon squamatum (L.) H. W. Johans., L. M. Irvine et A. M. Webster; and Jania rubens (L.) J. V. Lamour. The delimitation of these geniculate coralline red algae is based primarily on morphological characters. Molecular analysis based on cox1 and 18S rRNA gene phylogenies supported the division of the Corallinoideae into the tribes Janieae and Corallineae. Within the Janieae, a sequence difference of 46–48 bp (8.6%–8.9%) between specimens of H. squamatum and J. rubens in the cox1 phylogeny leads us to conclude that they are congeneric. J. rubens var. rubens and J. rubens var. corniculata (L.) Yendo clustered together in both phylogenies, suggesting that for those genes, there was no genetic basis for the morphological variation. Within the Corallineae, it appears that in some regions, the name C. elongata has been misapplied. C. officinalis samples formed two clusters that differed by 45–54 bp (8.4%–10.0%), indicating species‐level divergence, and morphological differences were sufficient to define two species. One of these clusters was consistent with the morphology of the type specimen of C. officinalis (LINN 1293.9). The other species cluster is therefore described here as Corallina caespitosa sp. nov. This study has demonstrated that there is a clear need for a revision of the genus Corallina to determine the extent of “pseudocryptic” diversity in this group of red algae.     

<Emphasis Type="Italic">Petunia</Emphasis> × <Emphasis Type="Italic">hybrida</Emphasis> during transition to flowering as affected by light intensity and quality treatments

Petunia × hybrida was grown under high (H), medium (M) and low (L) light intensity [photoperiod; 16 h d⁻¹, photosynthetic photon flux density (PPFD); 360, 120 and 40 μmol m⁻² s⁻¹, respectively] as well as under end-of-day (EOD) red (R) and far-red (FR) light quality treatments [photoperiod; 14.5 h d⁻¹, PPFD; 30 μmol m⁻² s⁻¹ EOD; 15 min, Control (C) light; without EOD light treatment]. Shoot growth, leaf anatomical and photosynthetic responses as well as the responses of peroxidase (POD) isoforms and their specific activities following transition to flowering (1–6 weeks) were evaluated. Flower bud formation of Petunia × hybrida was achieved at the end of the 4th week for H light treatment and on the end of the 6th week for FR light treatment. No flower bud formation was noticed in the C and R light treatments. H and M light treatments induced lower chlorophyll (Chla, Chlb, Chla+b) concentrations in comparison to L light. On the other hand R and FR light chlorophyll content were similar to C light. Photosynthetic parameters [CO₂ assimilation rate (A), transpiration rate (E) and stomatal conductance (g _s) values] were higher in the H light treated plants in comparison to M and L light treated plants. A, E and g _s values of R and FR light were similar to C light plants. Leaf anatomy revealed that total leaf thickness, thickness of the contained tissues (epidermis, palisade and spongy parenchyma) and relative volume percentages of the leaf histological components were differently affected within the light intensity and the light quality treatments. POD specific activities increased from the 1st to the 6th week during transition to flowering. Native-PAGE analysis revealed the appearance of four anionic POD (A₁–A₄) isoforms in all light treatments. On the basis of the leaf anatomical, photosynthetic and plant morphological responses, the production of high quality Petunia × hybrida plants with optimal flowering times could be achieved through the control of both light intensity and light quality. The appearance of A₁ and A₂ anionic POD isoforms could be also used for successful scheduling under light treatments.     

Evidence for a LPS-binding protein in medfly hemocyte surface: mediation in LPS internalization but not in LPS signaling

A doublet of medfly hemocyte proteins with a molecular mass of about 55 and 50 kDa were precipitated with LPS. Antibodies raised against human CD14 recognize the same doublet of proteins. These results support that mammalian CD14 and the doublet of protein bands in medfly hemocytes share common epitopes. This doublet of protein bands is released from hemocytes upon LPS triggering. A portion of the released protein is clustered on the surface of a distinct hemocyte type and the other remains soluble. The membrane-bound LPS-binding protein is involved in LPS internalization and Escherichia coli phagocytosis but not in LPS signaling.     

MAP kinases mediate phagocytosis and melanization via prophenoloxidase activation in medfly hemocytes

E. coli phagocytosis by medfly hemocytes, in contrast to mammalian macrophages, associates with E. coli-challenged hemocyte secretion by mitogen activating protein (MAP) kinases. In the present work, we examined whether this system links with the proteolytic activation of prophenoloxidase (proPO). ProPO and prophenoloxidase-activating proteinases (PAPs) were initially identified within freshly isolated medfly hemocytes. Moreover, flow cytometry and immunocytochemical analysis revealed the constitutive expression of proPO and its stable association with hemocyte surface. The expression level of hemocyte surface proPO is not affected by E. coli infection. In addition, flow cytometry analysis in freshly isolated hemocytes showed that E. coli phagocytosis is markedly blocked by antibodies against proPO or PAPs, as well as by several serine protease inhibitors, strongly supporting the involvement of proPO cascade in the phagocytosis process. Similarly, it was shown that melanization process depends on proPO activation. MAP kinases appeared to control both phagocytosis and melanization, since they regulate PAPs secretion, a prerequisite for the conversion of proPO to active PO. From this and previous studies, hemocytes appear to be central to immune response in medfly.