Comparison of solution-based exome capture methods for next generation sequencing

Background

Techniques enabling targeted re-sequencing of the protein coding sequences of the human genome on next generation sequencing instruments are of great interest. We conducted a systematic comparison of the solution-based exome capture kits provided by Agilent and Roche NimbleGen. A control DNA sample was captured with all four capture methods and prepared for Illumina GAII sequencing. Sequence data from additional samples prepared with the same protocols were also used in the comparison.

Results

We developed a bioinformatics pipeline for quality control, short read alignment, variant identification and annotation of the sequence data. In our analysis, a larger percentage of the high quality reads from the NimbleGen captures than from the Agilent captures aligned to the capture target regions. High GC content of the target sequence was associated with poor capture success in all exome enrichment methods. Comparison of mean allele balances for heterozygous variants indicated a tendency to have more reference bases than variant bases in the heterozygous variant positions within the target regions in all methods. There was virtually no difference in the genotype concordance compared to genotypes derived from SNP arrays. A minimum of 11× coverage was required to make a heterozygote genotype call with 99% accuracy when compared to common SNPs on genome-wide association arrays.

Conclusions

Libraries captured with NimbleGen kits aligned more accurately to the target regions. The updated NimbleGen kit most efficiently covered the exome with a minimum coverage of 20×, yet none of the kits captured all the Consensus Coding Sequence annotated exons.     

Contribution of genetic and dietary insulin resistance to Alzheimer phenotype in APP/PS1 transgenic mice

According to epidemiological studies, type‐2 diabetes increases the risk of Alzheimer’s disease. Here, we induced hyperglycaemia in mice overexpressing mutant amyloid precursor protein and presenilin‐1 (APdE9) either by cross‐breeding them with pancreatic insulin‐like growth factor 2 (IGF‐2) overexpressing mice or by feeding them with high‐fat diet. Glucose and insulin tolerance tests revealed significant hyperglycaemia in mice overexpressing IGF‐2, which was exacerbated by high‐fat diet. However, sustained hyperinsulinaemia and insulin resistance were observed only in mice co‐expressing IGF‐2 and APdE9 without correlation to insulin levels in brain. In behavioural tests in aged mice, APdE9 was associated with poor spatial learning and the combination of IGF‐2 and high‐fat diet further impaired learning. Neither high‐fat diet nor IGF‐2 increased β‐amyloid burden in the brain. In male mice, IGF‐2 increased β‐amyloid 42/40 ratio, which correlated with poor spatial learning. In contrast, inhibitory phosphorylation of glycogen synthase kinase 3β, which correlated with good spatial learning, was increased in APdE9 and IGF‐2 female mice on standard diet, but not on high‐fat diet. Interestingly, high‐fat diet altered τ isoform expression and increased phosphorylation of τ at Ser202 site in female mice regardless of genotype. These findings provide evidence for new regulatory mechanisms that link type‐2 diabetes and Alzheimer pathology.     

Complement Factor H Binds to Human Serum Apolipoprotein E and Mediates Complement Regulation on High Density Lipoprotein Particles

The alternative pathway of complement is an important part of the innate immunity response against foreign particles invading the human body. To avoid damage to host cells, it needs to be efficiently down-regulated by plasma factor H (FH) as exemplified by various diseases caused by mutations in its domains 19–20 (FH19–20) and 5–7 (FH5–7). These regions are also the main interaction sites for microbial pathogens that bind host FH to evade complement attack. We previously showed that inhibition of FH binding by a recombinant FH5–7 construct impairs survival of FH binding pathogens in human blood. In this study we found that upon exposure to full blood, the addition of FH5–7 reduces survival of, surprisingly, also those microbes that are not able to bind FH. This effect was mediated by inhibition of complement regulation and subsequently enhanced neutrophil phagocytosis by FH5–7. We found that although FH5–7 does not reduce complement regulation in the actual fluid phase of plasma, it reduces regulation on HDL particles in plasma. Using affinity chromatography and mass spectrometry we revealed that FH interacts with serum apolipoprotein E (apoE) via FH5–7 domains. Furthermore, binding of FH5–7 to HDL was dependent on the concentration of apoE on the HDL particles. These findings explain why the addition of FH5–7 to plasma leads to excessive complement activation and phagocytosis of microbes in full anticoagulated blood. In conclusion, our data show how FH interacts with apoE molecules via domains 5–7 and regulates alternative pathway activation on plasma HDL particles.     

Comprehensive taxon sampling reveals unaccounted diversity and morphological plasticity in a group of dimitic polypores (Polyporales,Basidiomycota)

The phylogeny of the poroid and hydnoid genera Antrodiella, Junghuhnia, and Steccherinum (Polyporales, Basidiomycota) was studied utilizing sequences of the gene regions ITS, nLSU, mtSSU, atp6, rpb2, and tef1. Altogether 148 taxa, represented by 549 sequences, were included in analyses. Results show that most species of these genera form a well supported clade in the Polyporales, called Steccherinaceae, along with 12 other hydnoid and poroid genera. Within the Steccherinaceae, generic concepts need to be revised: no fewer than 15 new genera are needed to accommodate existing and new species. At least 16 transitions have taken place between poroid and hydnoid hymenophore types within the Steccherinaceae, and similar plasticity can be seen in microscopic characters. Nevertheless, natural genera revealed in the analysis can mostly be characterized morphologically and, with few exceptions, poroid and hydnoid species belong to separate genera. The genus Steccherinum is shown to contain both hydnoid and poroid species. Species of the former Antrodiella belong to at least 10 genera within the Steccherinaceae. © The Willi Hennig Society 2011.     

What is the type species of <Emphasis Type="Italic">Phanerochaete</Emphasis> (Polyporales,Basidiomycota)?

Phanerochaete velutina and the closely related species P. alnea, P. alnea subsp. lubrica, and P. rhodella are identified as distinct species based on morphological criteria and molecular analyses. Besides differences in macroscopic and microscopic basidiocarp features, the taxa differ in distribution and ITS sequence. Phanerochaete alnea is widely distributed in Eurasia and North America, whereas P. alnea subsp. lubrica is limited to the Pacific Coast of North America. Phanerochaete velutina is known from Eurasia and Alaska, and P. rhodella is found in North America only. Nomenclatural problems with the name Phanerochaete P. Karst. are discussed, and type specimens for Thelephora alnea Fr., the generic type of Phanerochaete, and Peniophora karstenii Massee are selected. Phanerochaete alnea, P. alnea subsp. lubrica, and P. velutina are described and illustrated. In addition, the synonymy of Phanerochaete robusta and P. aurantiobadia is confirmed.     

The metabolic syndrome and incident diabetes: assessment of four suggested definitions of the metabolic syndrome in a Chinese population with high post-prandial glucose.

AIMS: To assess the sensitivity and specificity of the four definitions of the metabolic syndrome for incident diabetes in both men and women. METHODS: The screening survey for type 2 diabetes was conducted in 1994. A follow-up study on 627 high-risk non-diabetic individuals at baseline was carried out in 1999 in Beijing area. 70 men and 76 women developed diabetes during the five-year follow-up. Sensitivity and specificity of four definitions of the metabolic syndrome based on the NCEP, WHO, EGIR and AACE recommendations were compared by McNemar's test. RESULTS: The metabolic syndrome based on all four definitions identified men at a 3.7-4.5-fold and women at a 1.6-2.8-fold risk of developing diabetes during 5-year follow-up. The AACE definition had the highest sensitivity for predicting diabetes (men: 0.61; women: 0.58) and lowest specificity (men: 0.71; women: 0.70). The WHO definition identified 53 % of male and 42 % female incident diabetes. The NCEP definition of adiposity as waist girth > 102 cm was the least sensitive, detecting only 27 % of incident diabetes in men; however, it was the most specific (0.91). The EGIR definition identified the lowest number of female cases (28 %) and fewer male cases (28 %) of incident diabetes, but was specific (women: 0.87; men: 0.91). CONCLUSIONS: Further studies on definition of the metabolic syndrome should focus on the potential ethnic differences in insulin resistance and anthropometric indicators for obesity.     

Formyl peptide receptor-mediated ERK1/2 activation occurs through G(i) and is not dependent on beta-arrestin1/2

Formyl peptide receptor (FPR) and C5a receptor (C5aR) are chemoattractant G protein-coupled receptors (GPCRs) involved in the innate immune response against bacterial infections and tissue injury. Like other GPCRs, they recruit beta-arrestin1/2 to the plasma membrane and activate the extracellular signal-regulated kinases 1 and 2 (ERK1/2). Previous studies with several GPCRs have suggested that beta-arrestins play an important role as signal transducers by scaffolding signaling molecules such as ERK1/2. This function of the beta-arrestins was not discovered until several years after their role in desensitization and endocytosis had been reported. In this study, we investigated the role of the beta-arrestins in the activation of ERK1/2 and receptor endocytosis. We took advantage of previously described mutants of FPR that have defects in G(i) coupling or beta-arrestin recruitment. The results obtained with the mutant FPRs, as well as experiments using an inhibitor of G(i) and cells overexpressing beta-arrestin2, showed that activation of ERK1/2 takes place through G(i) and is not affected by beta-arrestins. However, overexpression of beta-arrestin2 does enhance FPR sequestration from the cell surface, suggesting a role in desensitization, as shown for many other GPCRs. Experiments with CHO C5aR cells showed similar sensitivity to the G(i) inhibitor as CHO FPR cells, suggesting that the predominant activation of ERK1/2 through G protein may be a common characteristic among chemoattractant receptors.     

Fate of intravenously administered squalene and plant sterols in human subjects

We have studied metabolism of plant sterols and squalene administered intravenously in the form of lipid emulsion mimicking chylomicrons (CM). The CM-like lipid emulsion was prepared by dissolving squalene in commercially available Intralipid. The emulsion was given as an intravenous bolus injection of 30 ml containing 6.3 mg of cholesterol, 1.9 mg of campesterol, 5.7 mg of sitosterol, 1.6 mg of stigmasterol, 18.1 mg of squalene, and 6 g of triglycerides in six healthy volunteers. Blood samples were drawn from the opposite arm before and serially 2.5 -180 min after the injections. The decay of CM squalene, plant sterols, and triglycerides was monoexponential. The half-life of CM squalene was 74 +/- 8 min, that of campesterol was 37 +/- 5 min (P < 0.01 from squalene), and those of sitosterol, stigmasterol, and triglycerides were 17 +/- 2, 15 +/- 1, and 17 +/- 2 min, respectively (P < 0.01 from squalene and campesterol). The CM squalene concentration still exceeded the baseline level 180 min after injection (P = 0.02), whereas plant sterols and triglycerides returned to the baseline level between 45 and 120 min after injection. The half-lives of squalene and campesterol were positively correlated with their fasting CM concentrations. In addition, VLDL squalene, campesterol, and triglyceride concentrations, VLDL, LDL, and HDL sitosterol concentrations, as well as VLDL and LDL stigmasterol concentrations were increased significantly. Cholesterol concentrations increased in VLDL (P < 0.05), but were unchanged in CM after injection. These data suggest that squalene clearance occurs more slowly than that of plant sterols and triglycerides from CM, and that squalene is more tightly associated with triglyceride-rich lipoproteins than are plant sterols in injected CM-like emulsions.     

(35)S]GTPgammaS autoradiography reveals a wide distribution of G(i/o)-linked ADP receptors in the nervous system: close similarities with the platelet P2Y(ADP) receptor

No G(i)-linked P2Y receptors have been cloned to date but the presence of such receptors is thought to be restricted to platelets and certain clonal cell lines. Using the functional approach of [(35)S]guanosine 5'-[gamma-thio]-triphosphate autoradiography, we uncovered the widespread presence of such receptors in the CNS. Under conditions in which the prominent signal due to tonic adenosine receptor activity is masked, ADP and ATP stimulated G-protein activity in multiple grey and white matter regions. Localization in the grey matter suggests inhibitory auto-/heteroreceptor function. In the white matter, activated G proteins appeared as 'hot spots' (presumed oligodendrocyte progenitors) with scattered distribution along the main fibre tracts. Responses to ATP were diminished under conditions that inhibited degradation, suggesting that prior conversion to ADP explained agonist action. Uracil nucleotides were ineffective but 2-methylthio-ADP activated G proteins approximately 500-fold more potently than ADP, although both were similarly degraded. Throughout the brain, ADP-dependent G-protein activity was reversed by 2-hexylthio-AdoOC(O)Asp(2), a non-phosphate ATP analogue, whereas selective P2Y(1) receptor antagonists proved ineffective. A similar receptor was also disclosed from the adrenal medulla. These data witness a hitherto unrecognized abundance of G(i/o)-linked ADP receptors in the nervous system. Biochemical and pharmacological behaviour suggests striking similarities to the elusive platelet P2Y(ADP) receptor.