Performance comparison of four exome capture systems for deep sequencing

Background

Recent developments in deep (next-generation) sequencing technologies are significantly impacting medical research. The global analysis of protein coding regions in genomes of interest by whole exome sequencing is a widely used application. Many technologies for exome capture are commercially available; here we compare the performance of four of them: NimbleGen’s SeqCap EZ v3.0, Agilent’s SureSelect v4.0, Illumina’s TruSeq Exome, and Illumina’s Nextera Exome, all applied to the same human tumor DNA sample.

Results

Each capture technology was evaluated for its coverage of different exome databases, target coverage efficiency, GC bias, sensitivity in single nucleotide variant detection, sensitivity in small indel detection, and technical reproducibility. In general, all technologies performed well; however, our data demonstrated small, but consistent differences between the four capture technologies. Illumina technologies cover more bases in coding and untranslated regions. Furthermore, whereas most of the technologies provide reduced coverage in regions with low or high GC content, the Nextera technology tends to bias towards target regions with high GC content.

Conclusions

We show key differences in performance between the four technologies. Our data should help researchers who are planning exome sequencing to select appropriate exome capture technology for their particular application.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-449) contains supplementary material, which is available to authorized users.     

Spatial patterns of fish communities along two estuarine gradients in southern Florida

In tropical and subtropical estuaries, gradients of primary productivity and salinity are generally invoked to explain patterns in community structure and standing crops of fishes. We documented spatial and temporal patterns in fish community structure and standing crops along salinity and nutrient gradients in two subtropical drainages of Everglades National Park, USA. The Shark River drains into the Gulf of Mexico and experiences diurnal tides carrying relatively nutrient enriched waters, while Taylor River is more hydrologically isolated by the oligohaline Florida Bay and experiences no discernable lunar tides. We hypothesized that the more nutrient enriched system would support higher standing crops of fishes in its mangrove zone. We collected 50 species of fish from January 2000 to April 2004 at six sampling sites spanning fresh to brackish salinities in both the Shark and Taylor River drainages. Contrary to expectations, we observed lower standing crops and density of fishes in the more nutrient rich tidal mangrove forest of the Shark River than in the less nutrient rich mangrove habitats bordering the Taylor River. Tidal mangrove habitats in the Shark River were dominated by salt-tolerant fish and displayed lower species richness than mangrove communities in the Taylor River, which included more freshwater taxa and yielded relatively higher richness. These differences were maintained even after controlling for salinity at the time of sampling. Small-scale topographic relief differs between these two systems, possibly created by tidal action in the Shark River. We propose that this difference in topography limits movement of fishes from upstream marshes into the fringing mangrove forest in the Shark River system, but not the Taylor River system. Understanding the influence of habitat structure, including connectivity, on aquatic communities is important to anticipate effects of construction and operational alternatives associated with restoration of the Everglades ecosystem.     

Epithelial-mesenchymal transition occurs after epidermal development in mouse skin

In the present study, we studied epithelial-mesenchymal transition (EMT) with fetal and postnatal serial skin sections. E-cadherin, occludin and zonula occludens 1 (ZO-1)-expressing cells appear in the dermal area from E18.5 to postnatal day 9 (P9), with highest expression from P2 to P5. The co-expression of mesenchymal marker alpha-smooth muscle (alpha-SMA), fibronectin and vimentin with E-cadherin in these dermal cells was further examined. Almost no dermal cells express alpha-SMA before P0. From P2 to P6, cells expressing both E-cadherin and alpha-SMA appear in the dermis. In contrast, fibronectin-releasing cells were detected in the dermis as early as on E15.5, although on P5, some dermal cells was found weakly expressing both fibronectin and E-cadherin, most cells strongly expressing fibronectin did not express E-cadherin. Vimentin was mainly expressed in both endothelial and blood-derived cells and did not show co-expression with E-cadherin. Confocal microscopy studies further found that during EMT, E-cadherin appears intracellularly, while the expression of alpha-SMA starts from the membrane area and moves to the cytosol of the cells. Our data are the first in vivo evidence that EMT occurs during mouse skin development. Dermal cells are derived from EMT and other origins, including blood, during skin development.     

Lack of anion effect on volume expansion natriuresis in the developing canine kidney

The renal response to volume expansion with sodium chloride or sodium bicarbonate was studied in 15 newborn and 13 adult dogs. Proximal and distal nephron function were estimated using the technique of distal nephron blockade. Fractional sodium reabsorption was 99.0 +/- 0.3% in newborn and 96.6 +/- 0.06% in adult during the NaCl expansion (P less than 0.01) and 98.1 +/- 0.7% in the newborn and 93.2 +/- 0.7% in the adult during NaHCO3 expansion (P less than 0.001). With either anion the higher fractional sodium reabsorption in the newborn was due to reabsorption of a greater fraction of the load presented to the distal nephron segment. The percent of the distal sodium load that was reabsorbed was 98.0 +/- 0.6% in the newborn and 92.2 +/- 1.0% in the adult during NaCl expansion, and 96.1 +/- 1.3% in the newborn and 81.5 +/- 2.4% in the adult during NaHCO3 expansion. Differences in distal nephron chloride, potassium and bicarbonate reabsorption among the groups support the hypothesis that the enhanced distal sodium reabsorption in the newborn occurred largely in the ascending loop of Henle with NaCl expansion, while it occurred in the late distal and cortical collecting tubules with NaHCO3 expansion. There was no difference between the natriuretic responses to NaCl or NaHCO3 in the newborn (P greater than 0.20); however, the natriuretic response to NaCl was less than that to NaHCO3 in the adult (P less than 0.001). This suggests that the bulk of the sodium that escaped reabsorption in Henle's loop during NaHCO3 expansion was reabsorbed in the late distal tubule in the newborn, but not in the adult.     

Commercial potential for Haematococcus microalgae as a natural source of astaxanthin

As a result of high production costs, commercial products from microalgae must command high prices. Astaxanthin produced by Haematococcus is a product that has become a commercial reality through novel and advanced technology. Cultivation methods have been developed to produce Haematococcus containing 1.5-3.0% astaxanthin by dry weight, with potential applications as a pigment source in aquaculture, poultry feeds and in the worldwide nutraceutical market.     

CD 95-independent mechanisms of IL-2 deprivation-induced apoptosis in activated human lymphocytes

Growth factor deprivation-induced apoptosis plays an important role in several cellular systems. However, knowledge of the molecular mechanisms involved are restricted to a few murine models or tumor cell lines. Therefore, we aimed studying signaling pathways leading to apoptosis in activated human peripheral T cells after IL-2 withdrawal. Lymphoblasts from patients with CD 95 (Fas/APO-1)-deficiency revealed that functional CD95 was not required to induce apoptosis after IL-2 withdrawal. Moreover, apoptosis induction in response to various cytotoxic stimuli was found to be mediated in the absence of functional CD95 but was affirmatorily influenced by IL-2 signaling. Immunoblots showed no downregulation of Bcl-2 or Bcl-xL and no upregulation of Bax, whereas decreased mitochondrial membrane potential was readily measurable 24 h after cytokine deprivation. Tetrapeptide inhibitors showed limited efficacy in preventing apoptosis whereas the caspase inhibitor zVAD-FMK potently blocked induction of apoptosis. Cleavage of different fluorogenic substrates revealed multiple caspase enzyme activities in lymphoblasts, which were not negatively affected by the fas mutation. Starting at 8 h after IL-2 withdrawal, upregulation of active caspase-3 but not of caspase-8 could be detected. Taken together, our data argue for molecular mechanisms of cytokine deprivation-induced apoptosis in activated human lymphocytes independent of CD95.     

Nitrification in terrestrial hot springs of Iceland and Kamchatka

Archaea have been detected recently as a major and often dominant component of the microbial communities performing ammonia oxidation in terrestrial and marine environments. In a molecular survey of archaeal ammonia monooxygenase (AMO) genes in terrestrial hot springs of Iceland and Kamchatka, the amoA gene encoding the alpha-subunit of AMO was detected in a total of 14 hot springs out of the 22 investigated. Most of these amoA-positive hot springs had temperatures between 82 and 97 degrees C and pH range between 2.5 and 7. In phylogenetic analyses, these amoA genes formed three independent lineages within the known sequence clusters of marine or soil origin. Furthermore, in situ gross nitrification rates in Icelandic hot springs were estimated by the pool dilution technique directly on site. At temperatures above 80 degrees C, between 56 and 159 mumol NO(3)(-) L(-1) mud per day was produced. Furthermore, addition of ammonium to the hot spring samples before incubation yielded a more than twofold higher potential nitrification rate, indicating that the process was limited by ammonia supply. Our data provide evidence for an active role of archaea in nitrification of hot springs in a wide range of pH values and at a high temperature.     

A systemic Pasteurella multocida toxin aggravates cardiac hypertrophy and fibrosis in mice

Pasteurella multocida toxin (PMT) persistently activates heterotrimeric G proteins of the Gα_q/11, Gα_12/13 and Gα_i family without interaction with G protein‐coupled receptors (GPCRs). We show that PMT acts on heart tissue in vivo and on cardiomyocytes and cardiac fibroblasts in vitro by deamidation of heterotrimeric G proteins. Increased normalized ventricle weights and fibrosis were detected after intraperitoneal administration of PMT in combination with the GPCR agonist phenylephrine. In neonatal rat cardiomyocytes, PMT stimulated the mitogen‐activated protein kinase pathway, which is crucial for the development of cellular hypertrophy. The toxin induced phosphorylation of the canonical phosphorylation sites of the extracellular‐regulated kinase 1/2 and, additionally, caused phosphorylation of the recently recognized autophosphorylation site, which appears to be important for the development of cellular hypertrophy. Moreover, PMT stimulated the small GTPases Rac1 and RhoA. Both switch proteins are involved in cardiomyocyte hypertrophy. In addition, PMT stimulated RhoA and Rac1 in neonatal rat cardiac fibroblasts. RhoA and Rac1 have been implicated in the regulation of connective tissue growth factor (CTGF) secretion and expression. Accordingly, we show that PMT treatment increased secretion and expression of CTGF in cardiac fibroblasts. Altogether, the data indicate that PMT is an inducer of pathological remodelling of cardiac cells and identifies the toxin as a promising tool for studying heterotrimeric G protein‐dependent signalling in cardiac cells.