Multicenter Performance Evaluation of Elecsys Anti-HBc II, Anti-HCV II, HIV combi PT, HBsAg II, and Syphilis Immunoassays.

BACKGROUND: The WHO recommends mandatory serological testing of blood donors for hepatitis B virus, hepatitis C virus (HCV), human immunodeficiency virus (HIV), and syphilis. We evaluated the performance of Elecsys® infectious disease immunoassays against commercially available comparator assays. METHODS: Prospective, routine, anonymized patient or donor samples (n = 8,821) were analyzed at three German sites using Elecsys antihepatitis B core antigen (Anti-HBc II), Anti-HCV II, HIV combi PT, hepatitis B surface antigen (HBsAg II), and Syphilis immunoassays (cobas e 411 analyzer) versus ARCHITECT comparator assays. RESULTS: The Elecsys immunoassays demonstrated comparable sensitivity (≤ 1.54% difference) and equivalent specificity (≤ 0.63% difference) to the respective ARCHITECT comparator assays. Overall sensitivity for the Elecsys and ARCHITECT infectious disease panels was 99.78% vs. 99.40%, respectively, and overall specificity was 99.74% vs. 99.80%, respectively. CONCLUSIONS: The Elecsys infectious disease immunoassays demonstrated high sensitivity and specificity, which were similar to comparator assays, supporting their suitability for routine laboratory practice.

EPIC-TABSAT: analysis tool for targeted bisulfite sequencing experiments and array-based methylation studies.

DNA methylation is one of the major epigenetic modifications and has frequently demonstrated its suitability as diagnostic and prognostic biomarker. In addition to chip and sequencing based epigenome wide methylation profiling methods, targeted bisulfite sequencing (TBS) has been established as a cost-effective approach for routine diagnostics and target validation applications. Yet, an easy-to-use tool for the analysis of TBS data in combination with array-based methylation results has been missing. Consequently, we have developed EPIC-TABSAT, a user-friendly web-based application for the analysis of targeted sequencing data that additionally allows the integration of array-based methylation results. The tool can handle multiple targets as well as multiple sequencing files in parallel and covers the complete data analysis workflow from calculation of quality metrics to methylation calling and interactive result presentation. The graphical user interface offers an unprecedented way to interpret TBS data alone or in combination with array-based methylation studies. Together with the computation of target-specific epialleles it is useful in validation, research, and routine diagnostic environments. EPIC-TABSAT is freely accessible to all users at https://tabsat.ait.ac.at/.

Gene expression profiling identifies pathways involved in seed maturation of Jatropha curcas.

BACKGROUND: Jatropha curcas, a tropical shrub, is a promising biofuel crop, which produces seeds with high content of oil and protein. To better understand the maturation process of J. curcas seeds and to improve its agronomic performance, a two-step approach was performed in six different maturation stages of seeds: 1) generation of the entire transcriptome of J. curcas seeds using 454-Roche sequencing of a cDNA library, 2) comparison of transcriptional expression levels using a custom Agilent 8x60K oligonucleotide microarray. RESULTS: A total of 793,875 high-quality reads were assembled into 19,382 unique full-length contigs, of which 13,507 could be annotated with Gene Ontology (GO) terms. Microarray data analysis identified 9111 probes (out of 57,842 probes), which were differentially expressed between the six maturation stages. The expression results were validated for 75 selected transcripts based on expression levels, predicted function, pathway, and length. Result from cluster analyses showed that transcripts associated with fatty acid, flavonoid, and phenylpropanoid biosynthesis were over-represented in the early stages, while those of lipid storage were over-represented in the late stages. Expression analyses of different maturation stages of J. curcas seed showed that most changes in transcript abundance occurred between the two last stages, suggesting that the timing of metabolic pathways during seed maturation in J. curcas occurs in late stages. The co-expression results showed that the hubs (CB5-D, CDR1, TT8, DFR, HVA22) with the highest number of edges, associated with fatty acid and flavonoid biosynthesis, are showing a decrease in their expression during seed maturation. Furthermore, seed development and hormone pathways are significantly well connected. CONCLUSION: The obtained results revealed differentially expressed sequences (DESs) regulating important pathways related to seed maturation, which could contribute to the understanding of the complex regulatory network during seed maturation with the focus on lipid, flavonoid and phenylpropanoid biosynthesis. This study provides detailed information on transcriptional changes during J. curcas seed maturation and provides a starting point for a genomic survey of seed quality traits. The results highlighted specific genes and processes relevant to the molecular mechanisms involved in Jatropha seed maturation. These data can also be utilized regarding other Euphorbiaceae species.

Oli2go: an automated multiplex oligonucleotide design tool.

The success of widely used oligonucleotide-based experiments, ranging from PCR to microarray, strongly depends on an accurate design. The design process involves a number of steps, which use specific parameters to produce high quality oligonucleotides. Oli2go is an efficient, user friendly, fully automated multiplex oligonucleotide design tool, which performs primer and different hybridization probe designs as well as specificity and cross dimer checks in a single run. The main improvement to existing oligonucleotide design web-tools is that oli2go combines multiple steps in an all-in-one solution, where other web applications only accomplish parts of the whole design workflow. Especially, the oli2go specificity check is not only performed against a single species (e.g. mouse), but against bacteria, viruses, fungi, invertebrates, plants, protozoa, archaea and sequences from whole genome shotgun sequence projects and environmental samples, at once. This allows the design of highly specific oligonucleotides in multiplex applications, which is further assured by performing dimer checks not only on the primers themselves, but in an all-against-all fashion. The software is freely accessible to all users at http://oli2go.ait.ac.at/.

DaMold: A data-mining platform for variant annotation and visualization in molecular diagnostics research.

Next-generation sequencing (NGS) has become a powerful and efficient tool for routine mutation screening in clinical research. As each NGS test yields hundreds of variants, the current challenge is to meaningfully interpret the data and select potential candidates. Analyzing each variant while manually investigating several relevant databases to collect specific information is a cumbersome and time-consuming process, and it requires expertise and familiarity with these databases. Thus, a tool that can seamlessly annotate variants with clinically relevant databases under one common interface would be of great help for variant annotation, cross-referencing, and visualization. This tool would allow variants to be processed in an automated and high-throughput manner and facilitate the investigation of variants in several genome browsers. Several analysis tools are available for raw sequencing-read processing and variant identification, but an automated variant filtering, annotation, cross-referencing, and visualization tool is still lacking. To fulfill these requirements, we developed DaMold, a Web-based, user-friendly tool that can filter and annotate variants and can access and compile information from 37 resources. It is easy to use, provides flexible input options, and accepts variants from NGS and Sanger sequencing as well as hotspots in VCF and BED formats. DaMold is available as an online application at http://damold.platomics.com/index.html, and as a Docker container and virtual machine at https://sourceforge.net/projects/damold/.

ClinQC: a tool for quality control and cleaning of Sanger and NGS data in clinical research.

BACKGROUND: Traditional Sanger sequencing has been used as a gold standard method for genetic testing in clinic to perform single gene test, which has been a cumbersome and expensive method to test several genes in heterogeneous disease such as cancer. With the advent of Next Generation Sequencing technologies, which produce data on unprecedented speed in a cost effective manner have overcome the limitation of Sanger sequencing. Therefore, for the efficient and affordable genetic testing, Next Generation Sequencing has been used as a complementary method with Sanger sequencing for disease causing mutation identification and confirmation in clinical research. However, in order to identify the potential disease causing mutations with great sensitivity and specificity it is essential to ensure high quality sequencing data. Therefore, integrated software tools are lacking which can analyze Sanger and NGS data together and eliminate platform specific sequencing errors, low quality reads and support the analysis of several sample/patients data set in a single run. RESULTS: We have developed ClinQC, a flexible and user-friendly pipeline for format conversion, quality control, trimming and filtering of raw sequencing data generated from Sanger sequencing and three NGS sequencing platforms including Illumina, 454 and Ion Torrent. First, ClinQC convert input read files from their native formats to a common FASTQ format and remove adapters, and PCR primers. Next, it split bar-coded samples, filter duplicates, contamination and low quality sequences and generates a QC report. ClinQC output high quality reads in FASTQ format with Sanger quality encoding, which can be directly used in down-stream analysis. It can analyze hundreds of sample/patients data in a single run and generate unified output files for both Sanger and NGS sequencing data. Our tool is expected to be very useful for quality control and format conversion of Sanger and NGS data to facilitate improved downstream analysis and mutation screening. CONCLUSIONS: ClinQC is a powerful and easy to handle pipeline for quality control and trimming in clinical research. ClinQC is written in Python with multiprocessing capability, run on all major operating systems and is available at https://sourceforge.net/projects/clinqc.

A survey of tools for variant analysis of next-generation genome sequencing data.

Recent advances in genome sequencing technologies provide unprecedented opportunities to characterize individual genomic landscapes and identify mutations relevant for diagnosis and therapy. Specifically, whole-exome sequencing using next-generation sequencing (NGS) technologies is gaining popularity in the human genetics community due to the moderate costs, manageable data amounts and straightforward interpretation of analysis results. While whole-exome and, in the near future, whole-genome sequencing are becoming commodities, data analysis still poses significant challenges and led to the development of a plethora of tools supporting specific parts of the analysis workflow or providing a complete solution. Here, we surveyed 205 tools for whole-genome/whole-exome sequencing data analysis supporting five distinct analytical steps: quality assessment, alignment, variant identification, variant annotation and visualization. We report an overview of the functionality, features and specific requirements of the individual tools. We then selected 32 programs for variant identification, variant annotation and visualization, which were subjected to hands-on evaluation using four data sets: one set of exome data from two patients with a rare disease for testing identification of germline mutations, two cancer data sets for testing variant callers for somatic mutations, copy number variations and structural variations, and one semi-synthetic data set for testing identification of copy number variations. Our comprehensive survey and evaluation of NGS tools provides a valuable guideline for human geneticists working on Mendelian disorders, complex diseases and cancers.

Mutations in ROGDI Cause Kohlschütter-Tönz Syndrome.

Kohlschütter-Tönz syndrome (KTS) is an autosomal-recessive disease characterized by the combination of epilepsy, psychomotor regression, and amelogenesis imperfecta. The molecular basis has not yet been elucidated. Here, we report that KTS is caused by mutations in ROGDI. Using a combination of autozygosity mapping and exome sequencing, we identified a homozygous frameshift deletion, c.229_230del (p.Leu77Alafs(∗)64), in ROGDI in two affected individuals from a consanguineous family. Molecular studies in two additional KTS-affected individuals from two unrelated Austrian and Swiss families revealed homozygosity for nonsense mutation c.286C>T (p.Gln96(∗)) and compound heterozygosity for the splice-site mutations c.531+5G>C and c.532-2A>T in ROGDI, respectively. The latter mutation was also found to be heterozygous in the mother of the Swiss affected individual in whom KTS was reported for the first time in 1974. ROGDI is highly expressed throughout the brain and other organs, but its function is largely unknown. Possible interactions with DISC1, a protein involved in diverse cytoskeletal functions, have been suggested. Our finding that ROGDI mutations cause KTS indicates that the protein product of this gene plays an important role in neuronal development as well as amelogenesis.

SPARQLGraph: a web-based platform for graphically querying biological Semantic Web databases.

BACKGROUND: Semantic Web has established itself as a framework for using and sharing data across applications and database boundaries. Here, we present a web-based platform for querying biological Semantic Web databases in a graphical way. RESULTS: SPARQLGraph offers an intuitive drag & drop query builder, which converts the visual graph into a query and executes it on a public endpoint. The tool integrates several publicly available Semantic Web databases, including the databases of the just recently released EBI RDF platform. Furthermore, it provides several predefined template queries for answering biological questions. Users can easily create and save new query graphs, which can also be shared with other researchers. CONCLUSIONS: This new graphical way of creating queries for biological Semantic Web databases considerably facilitates usability as it removes the requirement of knowing specific query languages and database structures. The system is freely available at http://sparqlgraph.i-med.ac.at.

Personalized Oncology Suite: integrating next-generation sequencing data and whole-slide bioimages.

BACKGROUND: Cancer immunotherapy has recently entered a remarkable renaissance phase with the approval of several agents for treatment. Cancer treatment platforms have demonstrated profound tumor regressions including complete cure in patients with metastatic cancer. Moreover, technological advances in next-generation sequencing (NGS) as well as the development of devices for scanning whole-slide bioimages from tissue sections and image analysis software for quantitation of tumor-infiltrating lymphocytes (TILs) allow, for the first time, the development of personalized cancer immunotherapies that target patient specific mutations. However, there is currently no bioinformatics solution that supports the integration of these heterogeneous datasets. RESULTS: We have developed a bioinformatics platform - Personalized Oncology Suite (POS) - that integrates clinical data, NGS data and whole-slide bioimages from tissue sections. POS is a web-based platform that is scalable, flexible and expandable. The underlying database is based on a data warehouse schema, which is used to integrate information from different sources. POS stores clinical data, genomic data (SNPs and INDELs identified from NGS analysis), and scanned whole-slide images. It features a genome browser as well as access to several instances of the bioimage management application Bisque. POS provides different visualization techniques and offers sophisticated upload and download possibilities. The modular architecture of POS allows the community to easily modify and extend the application. CONCLUSIONS: The web-based integration of clinical, NGS, and imaging data represents a valuable resource for clinical researchers and future application in medical oncology. POS can be used not only in the context of cancer immunology but also in other studies in which NGS data and images of tissue sections are generated. The application is open-source and can be downloaded at http://www.icbi.at/POS.

GAGE-B: an evaluation of genome assemblers for bacterial organisms.

MOTIVATION: A large and rapidly growing number of bacterial organisms have been sequenced by the newest sequencing technologies. Cheaper and faster sequencing technologies make it easy to generate very high coverage of bacterial genomes, but these advances mean that DNA preparation costs can exceed the cost of sequencing for small genomes. The need to contain costs often results in the creation of only a single sequencing library, which in turn introduces new challenges for genome assembly methods. RESULTS: We evaluated the ability of multiple genome assembly programs to assemble bacterial genomes from a single, deep-coverage library. For our comparison, we chose bacterial species spanning a wide range of GC content and measured the contiguity and accuracy of the resulting assemblies. We compared the assemblies produced by this very high-coverage, one-library strategy to the best assemblies created by two-library sequencing, and we found that remarkably good bacterial assemblies are possible with just one library. We also measured the effect of read length and depth of coverage on assembly quality and determined the values that provide the best results with current algorithms. CONTACT: salzberg@jhu.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Differential regulation of LRRC37A2 in gastric cancer by DNA methylation.

Gastric cancer (GC) is one of the leading types of fatal cancer worldwide. Epigenetic manipulation of cancer cells is a useful tool to better understand gene expression regulatory mechanisms and contributes to the discovery of novel biomarkers. Our research group recently reported a list of 83 genes that are potentially modulated by DNA methylation in GC cell lines. Herein, we further explored the regulation of one of these genes, LRRC37A2, in clinical samples. LRRC37A2 expression was evaluated by RT-qPCR, and DNA methylation was studied using next-generation bisulphite sequencing in 36 GC and paired adjacent nonneoplastic tissue samples. We showed that both reduced LRRC37A2 mRNA levels and increased LRRC37A2 exon methylation were associated with undifferentiated and poorly differentiated tumours. Moreover, LRRC37A2 gene expression and methylation levels were inversely correlated at the +45 exon CpG site. We suggest that DNA hypermethylation may contribute to reducing LRRC37A2 expression in undifferentiated and poorly differentiated GC. Therefore, our results show how some genes may be useful to stratify patients who are more likely to benefit from epigenetic therapy.Abbreviations: AR: androgen receptor; 5-AZAdC: 5-aza-2'-deoxycytidine; B2M: beta-2-microglobulin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GC: gastric cancer; GLM: general linear model; LRRC37A2: leucine-rich repeat containing 37 member A2; SD: standard deviation; TFII-I: general transcription factor II-I; TSS: transcription start site; XBP1: X-box binding protein 1.

Evidence of two deeply divergent co-existing mitochondrial genomes in the Tuatara reveals an extremely complex genomic organization.

Animal mitochondrial genomic polymorphism occurs as low-level mitochondrial heteroplasmy and deeply divergent co-existing molecules. The latter is rare, known only in bivalvian mollusks. Here we show two deeply divergent co-existing mt-genomes in a vertebrate through genomic sequencing of the Tuatara (Sphenodon punctatus), the sole-representative of an ancient reptilian Order. The two molecules, revealed using a combination of short-read and long-read sequencing technologies, differ by 10.4% nucleotide divergence. A single long-read covers an entire mt-molecule for both strands. Phylogenetic analyses suggest a 7-8 million-year divergence between genomes. Contrary to earlier reports, all 37 genes typical of animal mitochondria, with drastic gene rearrangements, are confirmed for both mt-genomes. Also unique to vertebrates, concerted evolution drives three near-identical putative Control Region non-coding blocks. Evidence of positive selection at sites linked to metabolically important transmembrane regions of encoded proteins suggests these two mt-genomes may confer an adaptive advantage for an unusually cold-tolerant reptile.

Discovery of Molecular DNA Methylation-Based Biomarkers through Genome-Wide Analysis of Response Patterns to BCG for Bladder Cancer.

BACKGROUND: Bacillus Calmette-Guérin (BCG) immunotherapy, the standard adjuvant intravesical therapy for some intermediate and most high-risk non-muscle invasive bladder cancers (NMIBCs), suffers from a heterogenous response rate. Molecular markers to help guide responses are scarce and currently not used in the clinical setting. METHODS: To identify novel biomarkers and pathways involved in response to BCG immunotherapy, we performed a genome-wide DNA methylation analysis of NMIBCs before BCG therapy. Genome-wide DNA methylation profiles of DNA isolated from tumors of 26 BCG responders and 27 failures were obtained using the Infinium MethylationEPIC BeadChip. RESULTS: Distinct DNA methylation patterns were found by genome-wide analysis in the two groups. Differentially methylated CpG sites were predominantly located in gene promoters and gene bodies associated with bacterial invasion of epithelial cells, chemokine signaling, endocytosis, and focal adhesion. In total, 40 genomic regions with a significant difference in methylation between responders and failures were detected. The differential methylation state of six of these regions, localized in the promoters of the genes GPR158, KLF8, C12orf42, WDR44, FLT1, and CHST11, were internally validated by bisulfite-sequencing. GPR158 promoter hypermethylation was the best predictor of BCG failure with an AUC of 0.809 (p-value < 0.001). CONCLUSIONS: Tumors from BCG responders and BCG failures harbor distinct DNA methylation profiles. Differentially methylated DNA regions were detected in genes related to pathways involved in bacterial invasion of cells or focal adhesion. We identified candidate DNA methylation biomarkers that may help to predict patient prognosis after external validation in larger, well-designed cohorts.

The impact of DNA demethylation on the upregulation of the NRN1 and TNFAIP3 genes associated with advanced gastric cancer.

Gastric cancer (GC) is the third leading cause of cancer-related death worldwide. Very few therapeutic options are currently available in this neoplasia. The use of 5-Aza-2'-deoxycytidine (5-AZAdC) was approved for the treatment of myelodysplastic syndromes, and this drug can treat solid tumours at low doses. Epigenetic manipulation of GC cell lines is a useful tool to better understand gene expression regulatory mechanisms for clinical applications. Therefore, we compared the gene expression profile of 5-AZAdC-treated and untreated GC cell lines by a microarray assay. Among the genes identified in this analysis, we selected NRN1 and TNFAIP3 to be evaluated for gene expression by RT-qPCR and DNA methylation by bisulfite DNA next-generation sequencing in 43 and 52 pairs of GC and adjacent non-neoplastic tissue samples, respectively. We identified 83 candidate genes modulated by DNA methylation in GC cell lines. Increased expression of NRN1 and TNFAIP3 was associated with advanced tumours (P < 0.05). We showed that increased NRN1 and TNFAIP3 expression seems to be regulated by DNA demethylation in GC samples: inverse correlations between the mRNA and DNA methylation levels in the promoter of NRN1 (P < 0.05) and the intron of TNFAIP3 (P < 0.05) were detected. Reduced NRN1 promoter methylation was associated with III/IV TNM stage tumours (P = 0.03) and the presence of Helicobacter pylori infection (P = 0.02). The identification of demethylated activated genes in GC may be useful in clinical practice, stratifying patients who are less likely to benefit from 5-AZAdC-based therapies. KEY MESSAGES: Higher expression of NRN1 and TNFAIP3 is associated with advanced gastric cancer (GC). NRN1 promoter hypomethylation contributes to gene upregulation in advanced GC. TNFAIP3 intronic-specific CpG site demethylation contributes to gene upregulation in GC. These findings may be useful to stratify GC patients who are less likely to benefit from DNA demethylating-based therapies.

QPCR: Application for real-time PCR data management and analysis.

BACKGROUND: Since its introduction quantitative real-time polymerase chain reaction (qPCR) has become the standard method for quantification of gene expression. Its high sensitivity, large dynamic range, and accuracy led to the development of numerous applications with an increasing number of samples to be analyzed. Data analysis consists of a number of steps, which have to be carried out in several different applications. Currently, no single tool is available which incorporates storage, management, and multiple methods covering the complete analysis pipeline. RESULTS: QPCR is a versatile web-based Java application that allows to store, manage, and analyze data from relative quantification qPCR experiments. It comprises a parser to import generated data from qPCR instruments and includes a variety of analysis methods to calculate cycle-threshold and amplification efficiency values. The analysis pipeline includes technical and biological replicate handling, incorporation of sample or gene specific efficiency, normalization using single or multiple reference genes, inter-run calibration, and fold change calculation. Moreover, the application supports assessment of error propagation throughout all analysis steps and allows conducting statistical tests on biological replicates. Results can be visualized in customizable charts and exported for further investigation. CONCLUSION: We have developed a web-based system designed to enhance and facilitate the analysis of qPCR experiments. It covers the complete analysis workflow combining parsing, analysis, and generation of charts into one single application. The system is freely available at http://genome.tugraz.at/QPCR.

PRIMEval: Optimization and screening of multiplex oligonucleotide assays.

The development of multiplex polymerase chain reaction and microarray assays is challenging due to primer dimer formation, unspecific hybridization events, the generation of unspecific by-products, primer depletion, and thus lower amplification efficiencies. We have developed a software workflow with three underlying algorithms that differ in their use case and specificity, allowing the complete in silico evaluation of such assays on user-derived data sets. We experimentally evaluated the method for the prediction of oligonucleotide hybridization events including resulting products and probes, self-dimers, cross-dimers and hairpins at different experimental conditions. The developed method allows explaining the observed artefacts through in silico WGS data and thermodynamic predictions. PRIMEval is available publicly at https://primeval.ait.ac.at.

Autologous induced pluripotent stem cell-derived four-organ-chip.

Microphysiological systems play a pivotal role in progressing toward a global paradigm shift in drug development. Here, we designed a four-organ-chip interconnecting miniaturized human intestine, liver, brain and kidney equivalents. All four organ models were predifferentiated from induced pluripotent stem cells from the same healthy donor and integrated into the microphysiological system. The coculture of the four autologous tissue models in one common medium deprived of tissue specific growth factors was successful over 14-days. Although there were no added growth factors present in the coculture medium, the intestine, liver and neuronal model maintained defined marker expression. Only the renal model was overgrown by coexisting cells and did not further differentiate. This model platform will pave the way for autologous coculture cross-talk assays, disease induction and subsequent drug testing.

The Pattern and Distribution of Induced Mutations in J. curcas Using Reduced Representation Sequencing.

Mutagenesis in combination with Genotyping by Sequencing (GBS) is a powerful tool for introducing variation, studying gene function and identifying causal mutations underlying phenotypes of interest in crop plant genomes. About 400 million paired-end reads were obtained from 82 ethylmethane sulfonate (EMS) induced mutants and 14 wild-type accessions of Jatropha curcas for the detection of Single Nucleotide Polymorphisms (SNPs) and Insertion/Deletions (InDels) by two different approaches (nGBS and ddGBS) on an Illumina HiSeq 2000 sequencer. Using bioinformatics analyses, 1,452 induced SNPs and InDels were identified in coding regions, which were distributed across 995 genes. The predominantly observed mutations were G/C to A/T transitions (64%), while transversions were observed at a lower frequency (36%). Regarding the effect of mutations on gene function, 18% of the mutations were located in intergenic regions. In fact, mutants with the highest number of heterozygous SNPs were found in samples treated with 0.8% EMS for 3 h. Reconstruction of the metabolic pathways showed that in total 16 SNPs were located in six KEGG pathways by nGBS and two pathways by ddGBS. The most highly represented pathways were ether-lipid metabolism and glycerophospholipid metabolism, followed by starch and sucrose metabolism by nGBS and triterpenoid biosynthesis as well as steroid biosynthesis by ddGBS. Furthermore, high genome methylation was observed in J. curcas, which might help to understand the plasticity of the Jatropha genome in response to environmental factors. At last, the results showed that continuously vegetatively propagated tissue is a fast, efficient and accurate method to dissolve chimeras, especially for long-lived plants like J. curcas. Obtained data showed that allelic variations and in silico analyses of gene functions (gene function prediction), which control important traits, could be identified in mutant populations using nGBS and ddGBS. However, the handling of GBS data is more difficult and more challenging than the traditional TILLING strategy in mutated plants, since the Jatropha genome sequence is incomplete, which makes alignment and variant analysis of target sequence reads challenging to perform and interpret. Therefore, providing a complete Jatropha reference genome sequence with high quality should be a priority for any breeding program.

Substantial variation in the hepatitis B surface antigen (HBsAg) in hepatitis B virus (HBV)-positive patients from South Africa: Reliable detection of HBV by the Elecsys HBsAg II assay.

BACKGROUND: It is essential that hepatitis B surface antigen (HBsAg) diagnostic assays reliably detect genetic diversity in the major hydrophilic region (MHR) of HBsAg to avoid false-negative results. Mutations in this domain display marked ethno-geographic variation and may lead to failure to diagnose hepatitis B virus (HBV) infection. OBJECTIVES: Evaluate diagnostic performance of the Elecsys® HBsAg II Qualitative assay in a cohort of South African HBV-positive blood donors. STUDY DESIGN: A total of 179 South African HBsAg- and HBV DNA > 100 IU/mL-positive blood donor samples were included. Samples were sequenced for genetic variation in HBsAg MHR using next-generation ultra-deep sequencing. HBsAg seropositivity was determined using the Roche Elecsys HBsAg II Qualitative assay. Mutation rates were compared between the first (amino acids 124-137) and second (amino acids 139-147) loops of the immunodominant MHR 'a' determinant region. Frequency of occult HBV infection-associated Y100C mutations was also determined. RESULTS: We observed a total of 279 MHR mutations (117 variants) in 102 (57%) samples, of which 91 were located in the 'a' determinant region. The major vaccine-induced escape mutation G145R was observed in two samples. All occult HBV infection-associated Y100C and common diagnostic and vaccine-escape-associated P120T, G145R, K122R, M133L, M133T, Q129H, G130N, and T126S mutations were reliably detected by the assay, which consistently detected the presence of HBsAg in all 179 samples including samples with 11 novel mutations. CONCLUSIONS: Despite substantial variation in HBsAg MHR, the Elecsys HBsAg II Qualitative assay robustly detects HBV infection in this South African cohort.