Combination Of Next-generation Sequencing Research Articles

Characterization of Canna yellow mottle virus in a New Host, Alpinia purpurata, in Hawaii.

Canna yellow mottle virus (CaYMV) is an important badnavirus infecting Canna spp. worldwide. This is the first report of CaYMV in flowering ginger (Alpinia purpurata) in Hawaii, where it is associated with yellow mottling and necrosis of leaves, vein streaking, and stunted plants. We have sequenced CaYMV in A. purpurata (CaYMV-Ap) using a combination of next-generation sequencing and traditional Sanger sequencing techniques. The complete genome of CaYMV-Ap was 7,120 bp with an organization typical of other Badnavirus species. Our results indicated that CaYMV-Ap was present in the episomal form in infected flowering ginger. We determined that this virus disease is prevalent in Hawaii and could potentially have significant economic impact on the marketing of A. purpurata as cut flowers. There is a potential concern that the host range of CaYMV-Ap may expand to include other important tropical plants.

The Vitamin B12-Dependent Photoreceptor AerR Relieves Photosystem Gene Repression by Extending the Interaction of CrtJ with Photosystem Promoters.

ABSTRACTPurple nonsulfur bacteria adapt their physiology to a wide variety of environmental conditions often through the control of transcription. One of the main transcription factors involved in controlling expression of the Rhodobacter capsulatus photosystem is CrtJ, which functions as an aerobic repressor of photosystem genes. Recently, we reported that a vitamin B12 binding antirepressor of CrtJ called AerR is required for anaerobic expression of the photosystem. However, the mechanism whereby AerR regulates CrtJ activity is unclear. In this study, we used a combination of next-generation sequencing and biochemical methods to globally identify genes under control of CrtJ and the role of AerR in controlling this regulation. Our results indicate that CrtJ has a much larger regulon than previously known, with a surprising regulatory function under both aerobic and anaerobic photosynthetic growth conditions. A combination of in vivo chromatin immunoprecipitation-DNA sequencing (ChIP-seq) and ChIP-seq and exonuclease digestion (ChIP-exo) studies and in vitro biochemical studies demonstrate that AerR forms a 1:2 complex with CrtJ (AerR-CrtJ2) and that this complex binds to many promoters under photosynthetic conditions. The results of in vitro and in vivo DNA binding studies indicate that AerR-CrtJ2 anaerobically forms an extended interaction with the bacteriochlorophyll bchC promoter to relieve repression by CrtJ. This is contrasted by aerobic growth conditions where CrtJ alone functions as an aerobic repressor of bchC expression. These results indicate that the DNA binding activity of CrtJ is modified by interacting with AerR in a redox-regulated manner and that this interaction alters CrtJ’s function.

Divergent Relationships between Fecal Microbiota and Metabolome following Distinct Antibiotic-Induced Disruptions.

The intestinal microbiome plays an essential role in regulating many aspects of host physiology, and its disruption through antibiotic exposure has been implicated in the development of a range of serious pathologies. The complex metabolic relationships that exist between members of the intestinal microbiota and the potential redundancy in functional pathways mean that an integrative analysis of changes in both structure and function are needed to understand the impact of antibiotic exposure. We used a combination of next-generation sequencing and nuclear magnetic resonance (NMR) metabolomics to characterize the effects of two clinically important antibiotic treatments, ciprofloxacin and vancomycin-imipenem, on the intestinal microbiomes of female C57BL/6 mice. This assessment was performed longitudinally and encompassed both antibiotic challenge and subsequent microbiome reestablishment. Both antibiotic treatments significantly altered the microbiota and metabolite compositions of fecal pellets during challenge and recovery. Spearman's correlation analysis of microbiota and NMR data revealed that, while some metabolites could be correlated with individual operational taxonomic units (OTUs), frequently multiple OTUs were associated with a significant change in a given metabolite. Furthermore, one metabolite, arginine, can be associated with increases/decreases in different sets of OTUs under differing conditions. Taken together, these findings indicate that reliance on shifts in one data set alone will generate an incomplete picture of the functional effect of antibiotic intervention. A full mechanistic understanding will require knowledge of the baseline microbiota composition, combined with both a comparison and an integration of microbiota, metabolomics, and phenotypic data. IMPORTANCE Despite the fundamental importance of antibiotic therapies to human health, their functional impact on the intestinal microbiome and its subsequent ability to recover are poorly understood. Much research in this area has focused on changes in microbiota composition, despite the interdependency and overlapping functions of many members of the microbial community. These relationships make prediction of the functional impact of microbiota-level changes difficult, while analyses based on the metabolome alone provide relatively little insight into the taxon-level changes that underpin changes in metabolite levels. Here, we used combined microbiota and metabolome profiling to characterize changes associated with clinically important antibiotic combinations with distinct effects on the gut. Correlation analysis of changes in the metabolome and microbiota indicate that a combined approach will be essential for a mechanistic understanding of the functional impact of distinct antibiotic classes.

Microbial community dynamics in Inferno Crater Lake, a thermally fluctuating geothermal spring.

Understanding how microbial communities respond and adjust to ecosystem perturbation is often difficult to interpret due to multiple and often simultaneous variations in observed conditions. In this research, we investigated the microbial community dynamics of Inferno Crater Lake, an acidic geothermal spring in New Zealand with a unique thermal cycle that varies between 30 and 80 °C over a period of 40-60 days. Using a combination of next-generation sequencing, geochemical analysis and quantitative PCR we found that the microbial community composition was predominantly chemolithotrophic and strongly associated with the thermal cycle. At temperatures >65 °C, the microbial community was dominated almost exclusively by sulphur-oxidising archaea (Sulfolobus-like spp.). By contrast, at mesophilic temperatures the community structure was more mixed, comprising both archaea and bacteria but dominated primarily by chemolithotrophic sulphur and hydrogen oxidisers. Multivariate analysis of physicochemical data confirmed that temperature was the only significant variable associated with community turnover. This research contributes to our understanding of microbial community dynamics in variable environments, using a naturally alternating system as a model and extends our limited knowledge of acidophile ecology in geothermal habitats.

Recognizing the tenascin-X deficient type of Ehlers-Danlos syndrome: a cross-sectional study in 17 patients.

The tenascin-X (TNX) deficient type Ehlers-Danlos syndrome (EDS) is similar to the classical type of EDS. Because of the limited awareness among geneticists and the challenge of the molecular analysis of the TNXB gene, the TNX-deficient type EDS is probably to be under diagnosed. We therefore performed an observational, cross-sectional study. History and physical examination were performed. Results of serum TNX measurements were collected and mutation analysis was performed by a combination of next-generation sequencing (NGS), Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). Included were 17 patients of 11 families with autosomal recessive inheritance and childhood onset. All patients had hyperextensible skin without atrophic scarring. Hypermobility of the joints was observed in 16 of 17 patients. Deformities of the hands and feet were observed frequently. TNX serum level was tested and absent in 11 patients (seven families). Genetic testing was performed in all families; 12 different mutations were detected, most of which are suspected to lead to non-sense mRNA mediated decay. In short, patients with the TNX-deficient type EDS typically have generalized joint hypermobility, skin hyperextensibility and easy bruising. In contrast to the classical type, the inheritance pattern is autosomal recessive and atrophic scarring is absent. Molecular analysis of TNXB in a diagnostic setting is challenging.

OR46 Detecting low level clonal somatic mutation in HLA genes using next-generation sequencing in the presence of aplastic anemia

Aim Next-generation sequencing (NGS) has assisted in the full characterization of HLA alleles, allowing in-depth studies of how these genes can play a direct role in the pathophysiology of many diseases outside of the context of transplantation. Here, we sought to determine the degree to which NGS can be used to characterize clonal somatic mutations acquired in HLA genes during the progression of aplastic anemia. Methods Omixon’s Holotype Kit was used to sequence HLA-A, B, C and DRB1 on the Illumina MiSeq targeting an ultra-high depth of coverage of 10,000 across the length of each gene. Omixon’s Twin, GenDx’s NGSengine and a custom pipeline analyzed the HLA allele data. The custom pipeline applies GATK’s best practices and tunes the ploidy parameter to lower the detection threshold of somatic mutations in each HLA allele to 10% of the depth. Results The pathogenesis of aplastic anemia, an autoimmune disease where cytotoxic T lymphocytes (CTL) destroy hematopoietic stem cells (HSC), most likely includes the recognition of a self-antigen in the context of a particular HLA allele by CTLs. Loss of heterozygosity of HLA alleles has been shown to allow HSCs to escape recognition of the CTLs, gaining a selective advantage in the disease state. In one mechanism of immune escape, HSCs acquire loss of function somatic mutations in the HLA allele(s) targeted by CTLs. By sequencing the HLA alleles of aplastic anemia patients, persistent somatic mutations were able to be detected above the background noise level. The frequency of mutations found in this dataset varied, but the lowest frequency mutation found was at 9% depth of coverage compared to the germline HLA allele. Mutations observed in HLA genes include the loss of a single or multiple HLA alleles in an individual by either frameshift mutations with insertions/deletions in exons or point mutations that introduce premature stop codons or mutate the translation start site. Interestingly, we can detect instances of different clones of somatic mutations causing loss of function in the same HLA allele of a single patient. Conclusion The combination of NGS with the Holotype HLA kit and multiple analysis tools, enables detection of clinically significant loss of function somatic mutations present in at least 10% of the depth for the targeted HLA allele in confirmed aplastic anemia cases. D.S. Monos: Scientific/Medical Advisor; Company/Organization; Omixon. Other (Identify); Company/Organization; Royalty from Omixon .

Molecular characterization of an adiponectin receptor homolog in the white leg shrimp, Litopenaeus vannamei.

Adiponectin (AdipoQ) and its receptors (AdipoRs) are strongly related to growth and development of skeletal muscle, as well as glucose and lipid metabolism in vertebrates. Herein we report the identification of the first full-length cDNA encoding an AdipoR homolog (Liv-AdipoR) from the decapod crustacean Litopenaeus vannamei using a combination of next generation sequencing (NGS) technology and bioinformatics analysis. The full-length Liv-AdipoR (1,245 bp) encoded a protein that exhibited the canonical seven transmembrane domains (7TMs) and the inversed topology that characterize members of the progestin and adipoQ receptor (PAQR) family. Based on the obtained sequence information, only a single orthologous AdipoR gene appears to exist in arthropods, whereas two paralogs, AdipoR1 and AdipoR2, have evolved in vertebrates. Transcriptional analysis suggested that the single Liv-AdipoR gene appears to serve the functions of two mammalian AdipoRs. At 72 h after injection of 50 pmol Liv-AdipoR dsRNA (340 bp) into L. vannamei thoracic muscle and deep abdominal muscle, transcription levels of Liv-AdipoR decreased by 93% and 97%, respectively. This confirmed optimal conditions for RNAi of Liv-AdipoR. Knockdown of Liv-AdipoR resulted in significant changes in the plasma levels of ammonia, 3-methylhistine, and ornithine, but not plasma glucose, suggesting that that Liv-AdipoR is important for maintaining muscle fibers. The chronic effect of Liv-AdipoR dsRNA injection was increased mortality. Transcriptomic analysis showed that 804 contigs were upregulated and 212 contigs were downregulated by the knockdown of Liv-AdipoR in deep abdominal muscle. The significantly upregulated genes were categorized as four main functional groups: RNA-editing and transcriptional regulators, molecular chaperones, metabolic regulators, and channel proteins.

A de novo transcriptome of the Malpighian tubules in non-blood-fed and blood-fed Asian tiger mosquitoes Aedes albopictus: insights into diuresis, detoxification, and blood meal processing.

Background. In adult female mosquitoes, the renal (Malpighian) tubules play an important role in the post-prandial diuresis, which removes excess ions and water from the hemolymph of mosquitoes following a blood meal. After the post-prandial diuresis, the roles that Malpighian tubules play in the processing of blood meals are not well described.Methods. We used a combination of next-generation sequencing (paired-end RNA sequencing) and physiological/biochemical assays in adult female Asian tiger mosquitoes (Aedes albopictus) to generate molecular and functional insights into the Malpighian tubules and how they may contribute to blood meal processing (3–24 h after blood ingestion).Results/Discussion. Using RNA sequencing, we sequenced and assembled the first de novo transcriptome of Malpighian tubules from non-blood-fed (NBF) and blood-fed (BF) mosquitoes. We identified a total of 8,232 non-redundant transcripts. The Malpighian tubules of NBF mosquitoes were characterized by the expression of transcripts associated with active transepithelial fluid secretion/diuresis (e.g., ion transporters, water channels, V-type H+-ATPase subunits), xenobiotic detoxification (e.g., cytochrome P450 monoxygenases, glutathione S-transferases, ATP-binding cassette transporters), and purine metabolism (e.g., xanthine dehydrogenase). We also detected the expression of transcripts encoding sodium calcium exchangers, G protein coupled-receptors, and septate junctional proteins not previously described in mosquito Malpighian tubules. Within 24 h after a blood meal, transcripts associated with active transepithelial fluid secretion/diuresis exhibited a general downregulation, whereas those associated with xenobiotic detoxification and purine catabolism exhibited a general upregulation, suggesting a reinvestment of the Malpighian tubules’ molecular resources from diuresis to detoxification. Physiological and biochemical assays were conducted in mosquitoes and isolated Malpighian tubules, respectively, to confirm that the transcriptomic changes were associated with functional consequences. In particular, in vivo diuresis assays demonstrated that adult female mosquitoes have a reduced diuretic capacity within 24 h after a blood meal. Moreover, biochemical assays in isolated Malpighian tubules showed an increase in glutathione S-transferase activity and the accumulation of uric acid (an end product of purine catabolism) within 24 h after a blood meal. Our data provide new insights into the molecular physiology of Malpighian tubules in culicine mosquitoes and reveal potentially important molecular targets for the development of chemical and/or gene-silencing insecticides that would disrupt renal function in mosquitoes.

Abstract B05: Assessment of TP53 mutation status in primary high-grade serous ovarian cancer and cell line models: Comparison between immunohistochemistry and next-generation sequencing.

Abstract Mutations in TP53 have been shown to occur in around 96% of high-grade serous ovarian cancer (HGSOC). Different TP53 mutations can lead to accumulation of mutant p53 in the cell that support gain of function effects, or loss or abrogation of both wild-type and mutant p53. Success of new therapeutic strategies for HGSOC that target mutant p53 will likely depend on detailed knowledge of the type of TP53 mutation present and its influence on mutant p53 levels in the cell. The aim of this study was to identify the spectrum of TP53 mutations in a cohort of primary HGSOCs and compare this with p53 immunostaining patterns. In addition, we sought to identify TP53 mutations in the previously uncharacterised HGSOC cell line models OV167 and OV202, as well as the clear cell adenocarcinoma line OV207. DNA from fresh frozen tumors and cell line samples were processed for next generation sequencing using The Fluidigm Access TP53 Array System run on the Fluidigm Biomark HD™ Real-Time PCR fluidics system (Fluidigm Corporation). Amplicon libraries were pooled and analyses performed in a single massively parallel sequencing run using the MiSeq Sequencing System (Illumina Inc.). Unaligned BAM files were analyzed within the Broad Institute's Genome Analysis Toolkit (GATK) best practice pipeline. The ANNOVAR software tool was used to generate an annotated variants list. Variants were further filtered based on their frequency in the 1000 Genome Database and predicted SIFT scores. Mutations were visualized using the Broad Institute's Integrative Genomics Viewer. All samples in which a mutation was identified underwent Sanger sequencing using primers recommended in the International Agency for Research on Cancer TP53 database. Immunohistochemistry for p53 was performed on formalin fixed paraffin embedded tissue sections using a commercially available mouse monoclonal anti-human antibody (Protein Clone DO-7, cat. #M7001, Dako) on an automated staining platform (Leica BOND-III™ autostainer). The percentage of cells showing positive nuclear staining for p53 was reported. A mutation in TP53 was identified in 94% (68 of 72) of HGSOC. The majority of mutations identified (62%, 42 of 68) were missense, with all except one of these mutations located in the p53 DNA binding domain. Overall, 82% (56 of 68) of mutations occurred within the DNA binding domain. Frameshift, stop or splice mutations (38%, 26 of 68) were identified in exons or flanking intronic sequence between exons 4 – 10. The presence or location of a TP53 mutation did not influence overall survival. Sanger sequencing was unable to reliably detect variants at an allele frequency less than 25%. TP53 mutations were identified in all cell lines at a mutant allele frequency ≥99%. Based on percent of nuclei positive for p53, tumors were immunohistochemically graded as High, Intermediate or Low for p53. All missense mutations scored High for expression of the p53 protein. Considerable variability was observed in p53 staining levels in samples containing stop, frameshift or splice mutations, with samples in all of High, Intermediate and Low categories. Furthermore, a single sample shown to be wild-type for TP53 was graded as High for p53, raising the possibility that p53 had been activated in this tumor via an alternative mechanism. The level of p53 did not affect overall survival. Taken together, these results suggest that caution needs to be exercised when using p53 immunohistochemistry as a surrogate marker for either presence or type of TP53 mutation. A combination of next generation sequencing and p53 immunohistochemistry is required to robustly define both mutation type and effect on mutant p53 levels in the cell. Citation Format: Alexander J. Cole, Trisha Dwight, Ying Zhu, Anthony J. Gill, Kristie-Ann Dickson, Adele Clarkson, Gregory B. Gard, Jayne Maidens, Susan Valmadre, Roderick J. Clifton-Bligh, Deborah J. Marsh. Assessment of TP53 mutation status in primary high-grade serous ovarian cancer and cell line models: Comparison between immunohistochemistry and next-generation sequencing. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B05.

1972 Combination of next generation sequencing and gene chip data to link survival and genotype in breast cancer

1972 Combination of next generation sequencing and gene chip data to link survival and genotype in breast cancer

Molecular profiling of lung adenosquamous carcinoma: hybrid or genuine type?

Lung adenosquamous carcinoma is a particular subtype of non-small cell lung carcinoma that is defined by the coexistence of adenocarcinoma and squamous cell carcinoma components. The aim of this study was to assess the mutational profile in each component of 16 adenosquamous carcinoma samples from a Caucasian population by a combination of next generation sequencing using the cancer hotspot panel as well as the colon and lung cancer panel and FISH. Identified mutations were confirmed by Sanger sequencing of DNA from cancer cells of each component collected by Laser Capture microdissection. Mutations typical for adenocarcinoma as well as squamous cell carcinoma were identified. Driver mutations were predominantly in the trunk suggesting a monoclonal origin of adenosquamous carcinoma. Most remarkably, EGFR mutations and mutations in the PI3K signaling pathway, which accounted for 30% and 25% of tumors respectively, were more prevalent while KRAS mutations were less prevalent than expected for a Caucasian population. Surprisingly, expression of classifier miR-205 was intermediate between that of classical adenocarcinoma and squamous cell carcinoma suggesting that adenosquamous carcinoma is a transitional stage between these tumor types. The high prevalence of therapy-relevant targets opens new options of therapeutic intervention for adenosquamous carcinoma patients.

Prevalence of RAS mutations and individual variation patterns among patients with metastatic colorectal cancer: A pooled analysis of randomised controlled trials

BackgroundThe use of epidermal growth factor receptor inhibitors to treat metastatic colorectal cancer (mCRC) patients requires prior confirmation of tumour wild type (WT) RAS mutation status (exons 2/3/4 for KRAS or NRAS). This retrospective pooled analysis aims to robustly estimate RAS mutation prevalence and individual variation patterns in mCRC patients. MethodIndividual patient data from five randomised, controlled panitumumab studies (three phase III, one phase II and one phase Ib/II) were pooled for this analysis. The phase III studies included mCRC patients independent of RAS mutation status; the phase II and Ib/II studies included mCRC patients with confirmed WT KRAS exon 2 status. Four studies conducted RAS testing using Sanger sequencing; one study used a combination of next-generation sequencing and Sanger sequencing. In order to assign overall RAS status, the mutation status of all exons 2/3/4 KRAS or NRAS was required to be known. ResultsData from 3196 mCRC patients from 36 countries were included in the analysis. The overall unadjusted RAS mutation prevalence in mCRC patients was 55.9% (95% confidence interval (CI): [53.9–57.9%]), with the following distribution observed: KRAS exon 2 (prevalence 42.6% [40.7–44.5%]); KRAS exon 3 (3.8% [2.9–4.9%]); KRAS exon 4 (6.2% [5.0–7.6%]); NRAS exon 2 (2.9% [2.1–3.9%]); NRAS exon 3 (4.2% [3.2–5.4%]); NRAS exon 4 (0.3% [0.1–0.7%]). Differences in RAS mutation prevalence estimates were observed by study (p=0.001), gender (p=0.030), and by country (p=0.028). ConclusionsThis analysis provides robust estimates of overall RAS mutation prevalence and individual variation patterns in mCRC patients.

Prevalence of RAS mutations among patients with metastatic colorectal cancer by country and region in randomized clinical trials: A pooled analysis.

520 Background: Global mCRC treatment guidelines for EGFR inhibitors require prior confirmation of wild-type RAS status (KRAS exons 2, 3, 4 and NRAS 2, 3, 4). The aim of this study was to estimate the prevalence of RAS mutations among mCRC patients by country, demographic characteristics, and clinical risk factors. A secondary aim was to estimate BRAF and KRASexon 2 mutation prevalence. Methods: Data from 5 published Amgen-sponsored randomized clinical trials (RCTs) were merged in a retrospective pooled analysis. There were 3 phase III, 1 phase II, and 1 phase Ib/II studies. For 4 out of 5 RCTs, RAStesting was conducted in a U.S. laboratory (Transgenomic Inc.) using Sanger sequencing on DNA extracted from tumor samples. For the remaining trial, a combination of next-generation sequencing and Sanger sequencing was used. Results: A total of 3,196 patients from 36 countries were included. The overall unadjusted prevalence of RAS mutations among mCRC subjects was 55.9% (95% CI, 53.9%, 57.9%); KRAS exon 2 mutation prevalence was 42.6% (40.7%, 44.5%). The prevalence by exon is given in the table below. BRAFmutation prevalence was 8.1% (6.7%, 9.6%). There were no statistically significant differences in RAS mutation prevalence by gender, age, or clinical factors such as performance status, tumour site, biopsy origin, or metastasis characteristics. Statistically significant differences in RAS mutation prevalence estimates were observed by country and by region with rates for Central West Europe being significantly lower than Eastern Europe (49.4% [44.4%, 54.3%] and 61.5%; [55.3%; 67.4%] respectively). Statistically significant RASmutation prevalence differences were observed between studies which could be due to varying patient characteristics. Conclusions: By merging data from RCTs, the analysis provides robust estimates of RASmutation prevalence. Studies using observational data are needed to confirm these findings. [Table: see text]

A nairovirus isolated from African bats causes haemorrhagic gastroenteritis and severe hepatic disease in mice.

Bats can carry important zoonotic pathogens. Here we use a combination of next-generation sequencing and classical virus isolation methods to identify novel nairoviruses from bats captured from a cave in Zambia. This nairovirus infection is highly prevalent among giant leaf-nosed bats, Hipposideros gigas (detected in samples from 16 individuals out of 38). Whole-genome analysis of three viral isolates (11SB17, 11SB19 and 11SB23) reveals a typical bunyavirus tri-segmented genome. The strains form a single phylogenetic clade that is divergent from other known nairoviruses, and are hereafter designated as Leopards Hill virus (LPHV). When i.p. injected into mice, the 11SB17 strain causes only slight body weight loss, whereas 11SB23 produces acute and lethal disease closely resembling that observed with Crimean–Congo Haemorrhagic Fever virus in humans. We believe that our LPHV mouse model will be useful for research on the pathogenesis of nairoviral haemorrhagic disease.

Exonic mutations in SCN9A (NaV1.7) are found in a minority of patients with erythromelalgia

Background and aims“Gain-of-function” mutations in voltage-gated sodium channel NaV1.7 have been linked to erythromelalgia (EM), characterized by painful hot and red hands and feet. We investigated the proportion of patients with EM that carry a mutation in NaV1.7 or in other pain-related genes and studied possible clinical differences. MethodsIn this study, 48 patients with EM were screened for mutations in a total of 29 candidate genes, including all sodium channel subunits, transient receptor potential channels (TRPA1, TRPV1, TRPM8), neurotrophic factors (NGF, NGFR, BDNF, GDNF, NTRK1 and WNK1) and other known pain-related genes (CACNG2, KCNS1, COMT, P2RX3, TAC1, TACR1), using a combination of next generation sequencing and classical Sanger sequencing. ResultsIn 7/48 patients protein-modifying mutations of NaV1.7 (P187L, I228M, I848T (n=4) and N1245S) were identified. Patients with the I848T mutation could be identified clinically based on early onset and severity of the disease. In contrast, there were no clinical characteristics that differentiated the other patients with NaV1.7 mutation from those patients without. We also found more than twenty rare protein-modifying genetic variants in the genes coding for sodium channels (NaV1.8, NaV1.9, NaV1.6, NaV1.5, NaV2.1, SCN1B, SCN3B), transient receptor potential channel (TRPA1, TRPV1), and other pain-related targets (WNK1 and NGFR). ConclusionsWe conclude that functionally characterized mutations of NaV1.7 (I848T) are present only in a minority of patient with EM. Albeit the majority of patients (27/48) carried rare protein-modifying mutations the vast majority of those will most probably not be causally linked to their disease. ImplicationsThe key question remaining to be solved is the possible role of rare variants of NaV1.8, NaV1.9, or beta-subunits in provoking chronic pain conditions or even EM.

Analysis of the ABCA4 genomic locus in Stargardt disease.

Autosomal recessive Stargardt disease (STGD1, MIM 248200) is caused by mutations in the ABCA4 gene. Complete sequencing of ABCA4 in STGD patients identifies compound heterozygous or homozygous disease-associated alleles in 65-70% of patients and only one mutation in 15-20% of patients. This study was designed to find the missing disease-causing ABCA4 variation by a combination of next-generation sequencing (NGS), array-Comparative Genome Hybridization (aCGH) screening, familial segregation and in silico analyses. The entire 140 kb ABCA4 genomic locus was sequenced in 114 STGD patients with one known ABCA4 exonic mutation revealing, on average, 200 intronic variants per sample. Filtering of these data resulted in 141 candidates for new mutations. Two variants were detected in four samples, two in three samples, and 20 variants in two samples, the remaining 117 new variants were detected only once. Multimodal analysis suggested 12 new likely pathogenic intronic ABCA4 variants, some of which were specific to (isolated) ethnic groups. No copy number variation (large deletions and insertions) was detected in any patient suggesting that it is a very rare event in the ABCA4 locus. Many variants were excluded since they were not conserved in non-human primates, were frequent in African populations and, therefore, represented ancestral, and not disease-associated, variants. The sequence variability in the ABCA4 locus is extensive and the non-coding sequences do not harbor frequent mutations in STGD patients of European-American descent. Defining disease-associated alleles in the ABCA4 locus requires exceptionally well characterized large cohorts and extensive analyses by a combination of various approaches.

Transcriptomic Analysis of Prunus domestica Undergoing Hypersensitive Response to Plum Pox Virus Infection

Plum pox virus (PPV) infects Prunus trees around the globe, posing serious fruit production problems and causing severe economic losses. One variety of Prunus domestica, named ‘Jojo’, develops a hypersensitive response to viral infection. Here we compared infected and non-infected samples using next-generation RNA sequencing to characterize the genetic complexity of the viral population in infected samples and to identify genes involved in development of the resistance response. Analysis of viral reads from the infected samples allowed reconstruction of a PPV-D consensus sequence. De novo reconstruction showed a second viral isolate of the PPV-Rec strain. RNA-seq analysis of PPV-infected ‘Jojo’ trees identified 2,234 and 786 unigenes that were significantly up- or downregulated, respectively (false discovery rate; FDR≤0.01). Expression of genes associated with defense was generally enhanced, while expression of those related to photosynthesis was repressed. Of the total of 3,020 differentially expressed unigenes, 154 were characterized as potential resistance genes, 10 of which were included in the NBS-LRR type. Given their possible role in plant defense, we selected 75 additional unigenes as candidates for further study. The combination of next-generation sequencing and a Prunus variety that develops a hypersensitive response to PPV infection provided an opportunity to study the factors involved in this plant defense mechanism. Transcriptomic analysis presented an overview of the changes that occur during PPV infection as a whole, and identified candidates suitable for further functional characterization.

Massive infection of seabird ticks with bacterial species related to Coxiella burnetii.

Seabird ticks are known reservoirs of bacterial pathogens of medical importance; however, ticks parasitizing tropical seabirds have received less attention than their counterparts from temperate and subpolar regions. Recently, Rickettsia africae was described to infect seabird ticks of the western Indian Ocean and New Caledonia, constituting the only available data on bacterial pathogens associated with tropical seabird tick species. Here, we combined a pyrosequencing-based approach with a classical molecular analysis targeting bacteria of potential medical importance in order to describe the bacterial community in two tropical seabird ticks, Amblyomma loculosum and Carios (Ornithodoros) capensis. We also investigated the patterns of prevalence and host specificity within the biogeographical context of the western Indian Ocean islands. The bacterial community of the two tick species was characterized by a strong dominance of Coxiella and Rickettsia. Our data support a strict Coxiella-host tick specificity, a pattern resembling the one found for Rickettsia spp. in the same two seabird tick species. Both the high prevalence and stringent host tick specificity suggest that these bacteria may be tick symbionts with probable vertical transmission. Detailed studies of the pathogenicity of these bacteria will now be required to determine whether horizontal transmission can occur and to clarify their status as potential human pathogens. More generally, our results show that the combination of next generation sequencing with targeted detection/genotyping approaches proves to be efficient in poorly investigated fields where research can be considered to be starting from scratch.

Next‐Gen phylogeography of rainforest trees: exploring landscape‐level cpDNA variation from whole‐genome sequencing

Standardized phylogeographic studies across codistributed taxa can identify important refugia and biogeographic barriers, and potentially uncover how changes in adaptive constraints through space and time impact on the distribution of genetic diversity. The combination of next-generation sequencing and methodologies that enable uncomplicated analysis of the full chloroplast genome may provide an invaluable resource for such studies. Here, we assess the potential of a shotgun-based method across twelve nonmodel rainforest trees sampled from two evolutionary distinct regions. Whole genomic shotgun sequencing libraries consisting of pooled individuals were used to assemble species-specific chloroplast references (in silicio). For each species, the pooled libraries allowed for the detection of variation within and between data sets (each representing a geographic region). The potential use of nuclear rDNA as an additional marker from the NGS libraries was investigated by mapping reads against available references. We successfully obtained phylogeographically informative sequence data from a range of previously unstudied rainforest trees. Greater levels of diversity were found in northern refugial rainforests than in southern expansion areas. The genetic signatures of varying evolutionary histories were detected, and interesting associative patterns between functional characteristics and genetic diversity were identified. This approach can suit a wide range of landscape-level studies. As the key laboratory-based steps do not require prior species-specific knowledge and can be easily outsourced, the techniques described here are even suitable for researchers without access to wet-laboratory facilities, making evolutionary ecology questions increasingly accessible to the research community.

Using Next-Generation Sequencing and Cross-Species Amplification in the Genus Pseudopungtungia

Nuclear microsatellite markers for Pungtungia herzi were developed using a combination of next-generation sequencing and Sanger sequencing. One hundred primer sets in the flanking region of dinucleotide and trinucleotide repeat motifs were designed and tested for efficiency in polymerase chain reaction amplification. Of these primer sets, 16 new markers (16%) were successfully amplified with unambiguous polymorphic alleles in 16 individuals of Pungtungia herzi. Cross-species amplification with these markers was then examined in two related species, Pseudopungtungia nigra and Pseudopungtungia tenuicorpa. Fifteen and 11 primer pairs resulted in successful amplification in Pseudopungtungia nigra and Pseudopungtungia tenuicorpa, respectively, with various polymorphisms, ranging from one allele (monomorphic) to 11 alleles per marker. These results indicated that developing microsatellite markers for cross-amplification from a species that is abundant and phylogenetically close to the species of interest is a good alternative when tissue samples of an endangered species are insufficient to develop microsatellites.