Towards pan-genome read alignment to improve variation calling

Background

Typical human genome differs from the reference genome at 4-5 million sites. This diversity is increasingly catalogued in repositories such as ExAC/gnomAD, consisting of >15,000 whole-genomes and >126,000 exome sequences from different individuals. Despite this enormous diversity, resequencing data workflows are still based on a single human reference genome. Identification and genotyping of genetic variants is typically carried out on short-read data aligned to a single reference, disregarding the underlying variation.

Results

We propose a new unified framework for variant calling with short-read data utilizing a representation of human genetic variation – a pan-genomic reference. We provide a modular pipeline that can be seamlessly incorporated into existing sequencing data analysis workflows. Our tool is open source and available online: https://gitlab.com/dvalenzu/PanVC.

Conclusions

Our experiments show that by replacing a standard human reference with a pan-genomic one we achieve an improvement in single-nucleotide variant calling accuracy and in short indel calling accuracy over the widely adopted Genome Analysis Toolkit (GATK) in difficult genomic regions.

     

Quantitative detection of cell surface protein expression by time-resolved fluorimetry.

A method is introduced for quantitative detection of cell surface protein expression. The method is based on immunocytochemistry, the use of long decay time europium(III) chelate and platinum(II) porphyrin labels, and detection of photoluminescence emission from adhered cells by time-resolved fluorimetry. After immunocytochemistry, the assay wells are evaporated to dryness and measured in the dry state. This protocol allows repeated and postponed analysis and microscopy imaging. In order to investigate the performance of the method, we chose expression of intercellular adhesion molecule-1 (ICAM-1) of endothelial cell line EAhy926 as a research target. The expression of ICAM-1 on the cells was enhanced by introduction of a cytokine, tumour necrosis factor-alpha (TNFalpha). The method gave signal:background ratios (S:B) of 20 and 9 for europium and platinum labels, respectively, whereas prompt fluorescent FITC label gave a S:B of 3. Screening window coefficients (=Z'-factor) were >0.5 for all the three labels, thus indicating a score for an excellent screening assay. In conclusion, the method appears to be an appropriate choice for protein expression analysis, both in high-throughput screening applications, and for detailed sample investigation by fluorescent microscopy imaging.     

The high road and the low road: trafficking choices in plants

Polar transport of the signaling molecule auxin is critical for plant development and depends on both the polar distribution of auxin efflux carriers, which pump auxin out of the cell and the alignment of these polarized cells. Two papers in this issue of Cell (Michniewicz et al., 2007; Jaillais et al., 2007) address how polar transport of these carriers occurs and describe the endosomal pathways involved.     

Volatile constituents of Erodium cicutarium (L.) L’ Hérit. (Geraniaceae)

Essential oils from Erodium cicutarium were obtained by hydrodistillation (samples consisting of entire plants (ec1), leaves and stems (ec2)) and analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS), resulting in a total of 177 components being identified. The essential oils were of a very similar chemical composition and consisted mainly of aliphatic compounds and their derivatives. Fatty acids and fatty acid derived compounds were the most common, 51.3% (ec1) and 60.1% (ec2), followed by carotenoid derived compounds, 12.6% (ec1) and 20.2% (ec2), and then terpenoids, 14.9% (ec1) and 14.2% (ec2). The main constituents in the oils were hexadecanoic acid, 22.8% (ec2) and 35.9% (ec1) and hexahydrofarnesyl acetone, 10.8% (ec2) and 11.6% (ec1). The results obtained differ markedly from those previously reported for the same species.     

New separation-free assay technique for SNPs using two-photon excitation fluorometry

A new separation-free method for detection of single nucleotide polymorphisms (SNPs) is described. The method is based on the single base extension principle, fluorescently labeled dideoxy nucleotides and two-photon fluorescence excitation technology, known as ArcDia™ TPX technology. In this assay technique, template-directed single base extension is carried out for primers which have been immobilized on polymer microparticles. Depending on the sequence of the template DNA, the primers are extended either with a labeled or with a non-labeled nucleotide. The genotype of the sample is determined on the basis of two-photon excited fluorescence of individual microparticles. The effect of various assay condition parameters on the performance of the assay method is studied. The performance of the new assay method is demonstrated by genotyping the SNPs of human individuals using double-stranded PCR amplicons as samples. The results show that the new SNP assay method provides sensitivity and reliability comparable to the state-of-the-art SNaPshot™ assay method. Applicability of the new method in routine laboratory use is discussed with respect to alternative assay techniques.     

The quantitative analysis of the vascularization following two basic auditory canal skin incisions

Three groups of nine patients each were analyzed. The first two groups consisted of those that underwent tympanoplastic due to chronic inflammation of middle ear. Two different standard auditory canal skin incisions were applied, i.e. tympanomeatal flap (TMF) or vascular strip (VS). The third control group consisted of non-operated patients. All the operated patients were subjected to a quantitative analysis of the auditory canal revascularization by means of the Weibel stereological test method, i.e. the B 100 double network system. The density of capillaries, arterioles, venulolymphatic spaces and a total volume density of all vascular elements of the auditory canal skin were measured. The obtained results of vascularization were compared with those of the target control group. It was found out that there were no significant differences in vascularization of auditory canal skin between TMF and VS patients from one side and the control group on the other side.     

Changes in antioxidant status of heart muscle tissue in experimental diabetes in rabbits

The present study was designed to evaluate the oxidative stress-related parameters in alloxan-induced diabetes in rabbits. After 3, 6, 12 and 24 weeks of hyperglycaemia the enzymatic and non-enzymatic factors were measured in heart tissue of diabetic and control groups. Superoxide dismutase and glutathione peroxidase activities and the contents of total sulfhydryl compounds significantly increased at all time intervals. Catalase activity increased initially (after 3 and 6 weeks), decreased after 12 weeks and increased again at the 24th week of the experiment. Glutathione reductase activity increased initially (at 3rd week), decreased below control level after 6 and 12 weeks, then increased again. Ascorbic acid concentration decreased after 3 and 6 weeks, and increased at the 12th and 24th weeks. The level of lipid peroxidation products was reduced after 3, 6 and 12 weeks of the experiment. After 24 weeks it was significantly elevated. These data suggest that hyperglycaemia induces oxidative stress in the heart but the defense mechanisms in the heart tissue are fairly efficacious against oxidative injury.     

uPA-silica-Particles (SP-uPA): a novel analytical system to investigate uPA-uPAR interaction and to test synthetic uPAR antagonists as potential cancer therapeutics

The urokinase-type plasminogen activation system, including the serine protease uPA (urokinase-type plasminogen activator) and its cell surface receptor (uPAR, CD87), are important key molecules in tumor invasion and metastasis. Besides its proteolytic function, binding of uPA to uPAR on tumor cells exerts various cell responses such as migration, adhesion, proliferation, and differentiation. Hence, the uPA/uPAR system is a potential target for tumor therapy. We have designed a new generation of uPA-derived synthetic cyclic peptides suited to interfere with the binding of uPA to uPAR and present a new technology involving micro silica particles coated with uPA (SP-uPA) and reacting with recombinant soluble uPAR (suPAR), to rapidly assess the antagonistic potential of uPA-peptides by flow cytofluorometry (FACS). For this, we used silica particles of 10 microm in diameter to which HMW-uPA is coupled using the EDC/NHS method. Soluble, recombinant suPAR was added and the interaction of SP-uPA with suPAR verified by reaction with monoclonal antibody HD13.1 directed to uPAR, followed by a cyan dye (cy5)-labeled antibody directed against mouse IgG. Thereby it was possible to test naturally occurring ligands of uPAR (HMW-uPA, ATF) as well as highly effective, synthetic cyclic uPA-derived peptides (cyclo21,29[D-Cys21Cys29]-UPA21-30, cyclo21,29[D-Cys21Nle28Cys29]-uPA21-30, cyclo21,29[D-Cys(21)2-Nal24Cys29]-uPA21-30, and cyclo21,29[D-Cys21Orn23Thi24Thi25Cys29]-uPA21-30. The results obtained with the noncellular SP-uPA/uPAR system are highly comparable to those obtained with a cellular system involving FITC-uPA and the promyeloid cell line U937 as the source of uPAR.     

World Health Organization Estimates of the Global and Regional Disease Burden of 11 Foodborne Parasitic Diseases, 2010: A Data Synthesis

Background

Foodborne diseases are globally important, resulting in considerable morbidity and mortality. Parasitic diseases often result in high burdens of disease in low and middle income countries and are frequently transmitted to humans via contaminated food. This study presents the first estimates of the global and regional human disease burden of 10 helminth diseases and toxoplasmosis that may be attributed to contaminated food.

Methods and Findings

Data were abstracted from 16 systematic reviews or similar studies published between 2010 and 2015; from 5 disease data bases accessed in 2015; and from 79 reports, 73 of which have been published since 2000, 4 published between 1995 and 2000 and 2 published in 1986 and 1981. These included reports from national surveillance systems, journal articles, and national estimates of foodborne diseases. These data were used to estimate the number of infections, sequelae, deaths, and Disability Adjusted Life Years (DALYs), by age and region for 2010. These parasitic diseases, resulted in 48.4 million cases (95% Uncertainty intervals [UI] of 43.4–79.0 million) and 59,724 (95% UI 48,017–83,616) deaths annually resulting in 8.78 million (95% UI 7.62–12.51 million) DALYs. We estimated that 48% (95% UI 38%-56%) of cases of these parasitic diseases were foodborne, resulting in 76% (95% UI 65%-81%) of the DALYs attributable to these diseases. Overall, foodborne parasitic disease, excluding enteric protozoa, caused an estimated 23.2 million (95% UI 18.2–38.1 million) cases and 45,927 (95% UI 34,763–59,933) deaths annually resulting in an estimated 6.64 million (95% UI 5.61–8.41 million) DALYs. Foodborne Ascaris infection (12.3 million cases, 95% UI 8.29–22.0 million) and foodborne toxoplasmosis (10.3 million cases, 95% UI 7.40–14.9 million) were the most common foodborne parasitic diseases. Human cysticercosis with 2.78 million DALYs (95% UI 2.14–3.61 million), foodborne trematodosis with 2.02 million DALYs (95% UI 1.65–2.48 million) and foodborne toxoplasmosis with 825,000 DALYs (95% UI 561,000–1.26 million) resulted in the highest burdens in terms of DALYs, mainly due to years lived with disability. Foodborne enteric protozoa, reported elsewhere, resulted in an additional 67.2 million illnesses or 492,000 DALYs. Major limitations of our study include often substantial data gaps that had to be filled by imputation and suffer from the uncertainties that surround such models. Due to resource limitations it was also not possible to consider all potentially foodborne parasites (for example Trypanosoma cruzi).

Conclusions

Parasites are frequently transmitted to humans through contaminated food. These estimates represent an important step forward in understanding the impact of foodborne diseases globally and regionally. The disease burden due to most foodborne parasites is highly focal and results in significant morbidity and mortality among vulnerable populations.