Validation and assessment of variant calling pipelines for next-generation sequencing

Background

The processing and analysis of the large scale data generated by next-generation sequencing (NGS) experiments is challenging and is a burgeoning area of new methods development. Several new bioinformatics tools have been developed for calling sequence variants from NGS data. Here, we validate the variant calling of these tools and compare their relative accuracy to determine which data processing pipeline is optimal.

Results

We developed a unified pipeline for processing NGS data that encompasses four modules: mapping, filtering, realignment and recalibration, and variant calling. We processed 130 subjects from an ongoing whole exome sequencing study through this pipeline. To evaluate the accuracy of each module, we conducted a series of comparisons between the single nucleotide variant (SNV) calls from the NGS data and either gold-standard Sanger sequencing on a total of 700 variants or array genotyping data on a total of 9,935 single-nucleotide polymorphisms. A head to head comparison showed that Genome Analysis Toolkit (GATK) provided more accurate calls than SAMtools (positive predictive value of 92.55% vs. 80.35%, respectively). Realignment of mapped reads and recalibration of base quality scores before SNV calling proved to be crucial to accurate variant calling. GATK HaplotypeCaller algorithm for variant calling outperformed the UnifiedGenotype algorithm. We also showed a relationship between mapping quality, read depth and allele balance, and SNV call accuracy. However, if best practices are used in data processing, then additional filtering based on these metrics provides little gains and accuracies of >99% are achievable.

Conclusions

Our findings will help to determine the best approach for processing NGS data to confidently call variants for downstream analyses. To enable others to implement and replicate our results, all of our codes are freely available at http://metamoodics.org/wes.

     

Innate immune responses of a scleractinian coral to vibriosis

Scleractinian corals are the most basal eumetazoan taxon and provide the biological and physical framework for coral reefs, which are among the most diverse of all ecosystems. Over the past three decades and coincident with climate change, these phototrophic symbiotic organisms have been subject to increasingly frequent and severe diseases, which are now geographically widespread and a major threat to these ecosystems. Although coral immunity has been the subject of increasing study, the available information remains fragmentary, especially with respect to coral antimicrobial responses. In this study, we characterized damicornin from Pocillopora damicornis, the first scleractinian antimicrobial peptide (AMP) to be reported. We found that its precursor has a segmented organization comprising a signal peptide, an acidic proregion, and the C-terminal AMP. The 40-residue AMP is cationic, C-terminally amidated, and characterized by the presence of six cysteine molecules joined by three intramolecular disulfide bridges. Its cysteine array is common to another AMP and toxins from cnidarians; this suggests a common ancestor, as has been proposed for AMPs and toxins from arthropods. Damicornin was active in vitro against Gram-positive bacteria and the fungus Fusarium oxysporum. Damicornin expression was studied using a combination of immunohistochemistry, reverse phase HPLC, and quantitative RT-PCR. Our data show that damicornin is constitutively transcribed in ectodermal granular cells, where it is stored, and further released in response to nonpathogenic immune challenge. Damicornin gene expression was repressed by the coral pathogen Vibrio coralliilyticus. This is the first evidence of AMP gene repression in a host-Vibrio interaction.     

A click procedure with heterogeneous copper to tether technetium-99m chelating agents and rhenium complexes. Evaluation of the chelating properties and biodistribution of the new radiolabelled glucose conjugates

An efficient protocol was developed to tether chelating agents and rhenium complexes onto a glucoside scaffold with a heterogeneous copper catalyst via click chemistry. The supported catalyst avoids the formation of unwanted copper complexes during the cyclisation step. The possibility to graft a pre-chelated M(CO)₃ core by click chemistry onto a biomolecule was highlighted for the first time. ^99mTc(CO)₃-glucoconjugates displayed excellent in vitro stability, a fast in vivo blood clearance and a low specific organ uptake or long-term retention in spleen and stomach.     

The source of human mesenchymal stromal cells influences their TLR profile as well as their functional properties

Mesenchymal stromal cells (MSC) can be expanded from different sources. We compared the influence of inflammation and TLR ligation on the phenotype and function of MSC derived from bone marrow (BM), adipose tissue (AT), and Wharton’s jelly (WJ). WJ-MSC were featured by a lack of TLR4 expression. While inflammation upregulated TLR3 in all three MSC types, TLR4 upregulation was observed only on BM-MSC. TLR ligation increased the production of inflammatory cytokines in BM- and AT-MSC but not in WJ-MSC and augmented anti-inflammatory cytokines in AT-MSC. Although inflammation increased in all MSC types the secretion of inflammatory cytokines, additional TLR triggering did not have further effect on WJ-MSC. The immunosuppressive potential of WJ-MSC on MLR was affected neither by inflammation nor by TLR triggering. This resistance was related to an overproduction of HGF. These data indicate that MSC source could be of importance while designing immunomodulating cell therapy in transplantation.     

Impact of 900 MHz electromagnetic field exposure on main male reproductive hormone levels: a Rattus norvegicus model

This work analyzes the effects of radiofrequency-electromagnetic field (RF-EMF) exposure on the reproductive system of male rats, assessed by measuring circulating levels of FSH, LH, inhibin B, activin B, prolactin, and testosterone. Twenty adult male Sprague–Dawley rats (180?±?10 g) were exposed to 900 MHz RF-EMF in four equal separated groups. The duration of exposure was 1, 2, and 4 h/day over a period of 30 days and sham-exposed animals were kept under the same environmental conditions as the exposed group except with no RF-EMF exposure. Before the exposure, at 15 and 30 days of exposure, determination of the abovementioned hormone levels was performed using ELISA. At the end of the experiment, FSH and LH values of the long time exposure (LTE) group were significantly higher than the sham-exposed group (p?p?p?相似文献   

Electrochemical detection of the MT-ND6 gene and its enzymatic digestion: Application in human genomic sample

A simple electrochemical biosensor was developed for the detection of the mitochondrial NADH dehydrogenase 6 gene (MT-ND6) and its enzymatic digestion by BamHI enzyme. This biosensor was fabricated by modification of a glassy carbon electrode with gold nanoparticles (AuNPs/GCE) and a probe oligonucleotide (ssDNA/AuNPs/GCE). The probe, which is a thiolated segment of the MT-ND6 gene, was deposited by self-assembling immobilization on AuNPs/GCE. Two indicators including methylene blue (MB) and neutral red (NR) were used as the electroactive indicators and the electrochemical response of the modified electrode was measured by differential pulse voltammetry. The proposed biosensor can detect the complementary sequences of the MT-ND6 gene. Also the modified electrode was used for the detection of an enzymatic digestion process by BamHI enzyme. The electrochemical biosensor can detect the MT-ND6 gene and its enzymatic digestion in polymerase chain reaction (PCR)-amplified DNA extracted from human blood. Also the biosensor was used directly for detection of the MT-ND6 gene in all of the human genome.     

ProDomAs,protein domain assignment algorithm using center‐based clustering and independent dominating set

Decomposition of structural domains is an essential task in classifying protein structures, predicting protein function, and many other proteomics problems. As the number of known protein structures in PDB grows exponentially, the need for accurate automatic domain decomposition methods becomes more essential. In this article, we introduce a bottom‐up algorithm for assigning protein domains using a graph theoretical approach. This algorithm is based on a center‐based clustering approach. For constructing initial clusters, members of an independent dominating set for the graph representation of a protein are considered as the centers. A distance matrix is then defined for these clusters. To obtain final domains, these clusters are merged using the compactness principle of domains and a method similar to the neighbor‐joining algorithm considering some thresholds. The thresholds are computed using a training set consisting of 50 protein chains. The algorithm is implemented using C++ language and is named ProDomAs. To assess the performance of ProDomAs, its results are compared with seven automatic methods, against five publicly available benchmarks. The results show that ProDomAs outperforms other methods applied on the mentioned benchmarks. The performance of ProDomAs is also evaluated against 6342 chains obtained from ASTRAL SCOP 1.71. ProDomAs is freely available at http://www.bioinf.cs.ipm.ir/software/prodomas . Proteins 2014; 82:1937–1946. © 2014 Wiley Periodicals, Inc.     

Berberine Ameliorate Oxidative Stress and Astrogliosis in the Hippocampus of STZ-Induced Diabetic Rats

Diabetes mellitus increases the risk of central nervous system (CNS) disorders such as stroke, seizures, dementia, and cognitive impairment. Berberine, a natural isoquinoline alkaloid, is reported to exhibit beneficial effect in various neurodegenerative and neuropsychiatric disorders. Moreover, astrocytes are proving critical for normal CNS function, and alterations in their activity and impaired oxidative stress could contribute to diabetes-related cognitive dysfunction. Metabolic and oxidative insults often cause rapid changes in glial cells. Key indicators of this response are increased synthesis of glial fibrillary acidic protein (GFAP) as an astrocytic marker. Therefore, we examined the effects of berberine on glial reactivity of hippocampus in streptozotocin (STZ)-induced diabetic rats, using GFAP immunohistochemistry. Lipid peroxidation, superoxide dismutase (SOD) activity, and nitrite levels were assessed as the parameters of oxidative stress. Eight weeks after diabetes induction, we observed increased numbers of GFAP⁺ astrocytes immunostaining associated with increased lipid peroxidation, decreased superoxide dismutase activity, and elevated nitrite levels in the hippocampus of STZ-diabetic rats. In contrast, chronic treatment with berberine (50 and 100 mg/kg p.o. once daily) lowered hyperglycemia, reduced oxidative stress, and prevented the upregulation of GFAP in the brain of diabetic rats. In conclusion, the present study demonstrated that the treatment with berberine resulted in an obvious reduction of oxidative stress and GFAP-immunoreactive astrocytes in the hippocampus of STZ-induced diabetic rats.

Protective effects of Ephedra pachyclada extract on mouse models of carbon tetrachloride- induced chronic and acute liver failure

Inflammation and oxidation are two important factors in the pathogenesis of liver. Ephedra pachyclada (EP) is a traditional medical herb that has anti-inflammatory and anti-oxidant activities. During this study, anti-oxidant activities of the EP extract was measured in vitro by 2,2′- diphenyl-1-picrylhydrazyl (DPPH) and β-Carotene bleaching assays. Then, we examined possible in vivo hepatoprotective effects of EP extract on mouse models of carbon tetrachloride (CCl₄)-induced chronic and acute liver failure. To produce mouse models of chronic and acute liver injuries, male SW1 mice were interaperitoneally injected with 1 ml/kg body weight (bw) CCl₄ biweekly for 42 days and a single dose of 2 ml/kg bw, respectively. In the experimental groups, mouse models were treated with low (140 mg/kg bw) and high (1400 mg/kg bw) doses of the EP extract. Olive oil and water treated mice were considered as controls during model derivation and EP extract treatment respectively. The results showed the antioxidant activity of EP extract and a significant reduction of all parameters of CCl₄-induced liver injury such as relative liver weight, necrosis, fibrosis, inflammation, and serum aspartate transaminase (AST) and alanine aminotransferase (ALT) in mouse models of acute and chronic liver injury treated with EP extract. Therefore, EP induces its hepatoprotective effects probably by suppressing oxidative stress and inhibit inflammation in the liver and is able to protect the liver against CCl₄-induced acute and chronic injuries.     

Antiproliferative Effects and Mechanisms of Liver X Receptor Ligands in Pancreatic Ductal Adenocarcinoma Cells

Pancreatic ductal adenocarcinoma (PDAC) is difficult to detect early and is often resistant to standard chemotherapeutic options, contributing to extremely poor disease outcomes. Members of the nuclear receptor superfamily carry out essential biological functions such as hormone signaling and are successfully targeted in the treatment of endocrine-related malignancies. Liver X receptors (LXRs) are nuclear receptors that regulate cholesterol homeostasis, lipid metabolism, and inflammation, and LXR agonists have been developed to regulate LXR function in these processes. Intriguingly, these compounds also exhibit antiproliferative activity in diverse types of cancer cells. In this study, LXR agonist treatments disrupted proliferation, cell-cycle progression, and colony-formation of PDAC cells. At the molecular level, treatments downregulated expression of proteins involved in cell cycle progression and growth factor signaling. Microarray experiments further revealed changes in expression profiles of multiple gene networks involved in biological processes and pathways essential for cell growth and proliferation following LXR activation. These results establish the antiproliferative effects of LXR agonists and potential mechanisms of action in PDAC cells and provide evidence for their potential application in the prevention and treatment of PDAC.