Combining Next-generation Sequencing Research Articles

Clinical Auditory Phenotypes Associated with GATA3 Gene Mutations in Familial Hypoparathyroidism-deafness-renal Dysplasia Syndrome.

Background:Hypoparathyroidism-deafness-renal dysplasia (HDR) syndrome is an autosomal dominant disorder primarily caused by haploinsufficiency of GATA binding protein 3 (GATA3) gene mutations, and hearing loss is the most frequent phenotypic feature. This study aimed at identifying the causative gene mutation for a three-generation Chinese family with HDR syndrome and analyzing auditory phenotypes in all familial HDR syndrome cases.Methods:Three affected family members underwent otologic examinations, biochemistry tests, and other clinical evaluations. Targeted genes capture combining next-generation sequencing was performed within the family. Sanger sequencing was used to confirm the causative mutation. The auditory phenotypes of all reported familial HDR syndrome cases analyzed were provided.Results:In Chinese family 7121, a heterozygous nonsense mutation c.826C>T (p.R276*) was identified in GATA3. All the three affected members suffered from sensorineural deafness and hypocalcemia; however, renal dysplasia only appeared in the youngest patient. Furthermore, an overview of thirty HDR syndrome families with corresponding GATA3 mutations revealed that hearing impairment occurred earlier in the younger generation in at least nine familial cases (30%) and two thirds of them were found to carry premature stop mutations.Conclusions:This study highlights the phenotypic heterogeneity of HDR and points to a possible genetic anticipation in patients with HDR, which needs to be further investigated.

Endogenous Small-Noncoding RNAs and Potential Functions in Desiccation Tolerance in Physcomitrella Patens

Early land plants like moss Physcomitrella patens have developed remarkable drought tolerance. Phytohormone abscisic acid (ABA) protects seeds during water stress by activating genes through transcription factors such as ABSCISIC ACID INSENSITIVE (ABI3). Small noncoding RNA (sncRNA), including microRNAs (miRNAs) and endogenous small-interfering RNAs (endo-siRNAs), are key gene regulators in eukaryotes, playing critical roles in stress tolerance in plants. Combining next-generation sequencing and computational analysis, we profiled and characterized sncRNA species from two ABI3 deletion mutants and the wild type P. patens that were subject to ABA treatment in dehydration and rehydration stages. Small RNA profiling using deep sequencing helped identify 22 novel miRNAs and 6 genomic loci producing trans-acting siRNAs (ta-siRNAs) including TAS3a to TAS3e and TAS6. Data from degradome profiling showed that ABI3 genes (ABI3a/b/c) are potentially regulated by the plant-specific miR536 and that other ABA-relevant genes are regulated by miRNAs and ta-siRNAs. We also observed broad variations of miRNAs and ta-siRNAs expression across different stages, suggesting that they could potentially influence desiccation tolerance. This study provided evidence on the potential roles of sncRNA in mediating desiccation-responsive pathways in early land plants.

SYNE1 ataxia is a common recessive ataxia with major non-cerebellar features: a large multi-centre study.

Mutations in the synaptic nuclear envelope protein 1 (SYNE1) gene have been reported to cause a relatively pure, slowly progressive cerebellar recessive ataxia mostly identified in Quebec, Canada. Combining next-generation sequencing techniques and deep-phenotyping (clinics, magnetic resonance imaging, positron emission tomography, muscle histology), we here established the frequency, phenotypic spectrum and genetic spectrum of SYNE1 in a screening of 434 non-Canadian index patients from seven centres across Europe. Patients were screened by whole-exome sequencing or targeted panel sequencing, yielding 23 unrelated families with recessive truncating SYNE1 mutations (23/434 = 5.3%). In these families, 35 different mutations were identified, 34 of them not previously linked to human disease. While only 5/26 patients (19%) showed the classical SYNE1 phenotype of mildly progressive pure cerebellar ataxia, 21/26 (81%) exhibited additional complicating features, including motor neuron features in 15/26 (58%). In three patients, respiratory dysfunction was part of an early-onset multisystemic neuromuscular phenotype with mental retardation, leading to premature death at age 36 years in one of them. Positron emission tomography imaging confirmed hypometabolism in extra-cerebellar regions such as the brainstem. Muscle biopsy reliably showed severely reduced or absent SYNE1 staining, indicating its potential use as a non-genetic indicator for underlying SYNE1 mutations. Our findings, which present the largest systematic series of SYNE1 patients and mutations outside Canada, revise the view that SYNE1 ataxia causes mainly a relatively pure cerebellar recessive ataxia and that it is largely limited to Quebec. Instead, complex phenotypes with a wide range of extra-cerebellar neurological and non-neurological dysfunctions are frequent, including in particular motor neuron and brainstem dysfunction. The disease course in this multisystemic neurodegenerative disease can be fatal, including premature death due to respiratory dysfunction. With a relative frequency of ∼5%, SYNE1 is one of the more common recessive ataxias worldwide.

Protective variant for hippocampal atrophy identified by whole exome sequencing.

We used whole-exome sequencing to identify variants other than APOE associated with the rate of hippocampal atrophy in amnestic mild cognitive impairment. An in-silico predicted missense variant in REST (rs3796529) was found exclusively in subjects with slow hippocampal volume loss and validated using unbiased whole-brain analysis and meta-analysis across 5 independent cohorts. REST is a master regulator of neurogenesis and neuronal differentiation that has not been previously implicated in Alzheimer's disease. These findings nominate REST and its functional pathways as protective and illustrate the potential of combining next-generation sequencing with neuroimaging to discover novel disease mechanisms and potential therapeutic targets.

Endogenous small-noncoding RNAs and their roles in chilling response and stress acclimation in Cassava.

BackgroundSmall noncoding RNA (sncRNA), including microRNAs (miRNAs) and endogenous small-interfering RNAs (endo-siRNAs) are key gene regulators in eukaryotes, playing critical roles in plant development and stress tolerance. Trans-acting siRNAs (ta-siRNAs), which are secondary siRNAs triggered by miRNAs, and siRNAs from natural antisense transcripts (nat-siRNAs) are two well-studied classes of endo-siRNAs.ResultsIn order to understand sncRNAs’ roles in plant chilling response and stress acclimation, we performed a comprehensive study of miRNAs and endo-siRNAs in Cassava (Manihot esculenta), a major source of food for the world populations in tropical regions. Combining Next-Generation sequencing and computational and experimental analyses, we profiled and characterized sncRNA species and mRNA genes from the plants that experienced severe and moderate chilling stresses, that underwent further severe chilling stress after chilling acclimation at moderate stress, and that grew under the normal condition. We also included castor bean (Ricinus communis) in our study to understand conservation of sncRNAs. In addition to known miRNAs, we identified 32 (22 and 10) novel miRNAs as well as 47 (26 and 21) putative secondary siRNA-yielding and 8 (7 and 1) nat-siRNA-yielding candidate loci in Cassava and castor bean, respectively. Among the expressed sncRNAs, 114 miRNAs, 12 ta-siRNAs and 2 nat-siRNAs showed significant expression changes under chilling stresses.ConclusionSystematic and computational analysis of microRNAome and experimental validation collectively showed that miRNAs, ta-siRNAs, and possibly nat-siRNAs play important roles in chilling response and chilling acclimation in Cassava by regulating stress-related pathways, e.g. Auxin signal transduction. The conservation of these sncRNA might shed lights on the role of sncRNA-mediated pathways affected by chilling stress and stress acclimation in Euphorbiaceous plants.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-634) contains supplementary material, which is available to authorized users.

Genomic organization and evolution of double minutes/homogeneously staining regions with MYC amplification in human cancer.

The mechanism for generating double minutes chromosomes (dmin) and homogeneously staining regions (hsr) in cancer is still poorly understood. Through an integrated approach combining next-generation sequencing, single nucleotide polymorphism array, fluorescent in situ hybridization and polymerase chain reaction-based techniques, we inferred the fine structure of MYC-containing dmin/hsr amplicons harboring sequences from several different chromosomes in seven tumor cell lines, and characterized an unprecedented number of hsr insertion sites. Local chromosome shattering involving a single-step catastrophic event (chromothripsis) was recently proposed to explain clustered chromosomal rearrangements and genomic amplifications in cancer. Our bioinformatics analyses based on the listed criteria to define chromothripsis led us to exclude it as the driving force underlying amplicon genesis in our samples. Instead, the finding of coexisting heterogeneous amplicons, differing in their complexity and chromosome content, in cell lines derived from the same tumor indicated the occurrence of a multi-step evolutionary process in the genesis of dmin/hsr. Our integrated approach allowed us to gather a complete view of the complex chromosome rearrangements occurring within MYC amplicons, suggesting that more than one model may be invoked to explain the origin of dmin/hsr in cancer. Finally, we identified PVT1 as a target of fusion events, confirming its role as breakpoint hotspot in MYC amplification.

Extended phenotypic spectrum of KIF5A mutations

To establish the phenotypic spectrum of KIF5A mutations and to investigate whether KIF5A mutations cause axonal neuropathy associated with hereditary spastic paraplegia (HSP) or typical Charcot-Marie-Tooth disease type 2 (CMT2). KIF5A sequencing of the motor-domain coding exons was performed in 186 patients with the clinical diagnosis of HSP and in 215 patients with typical CMT2. Another 66 patients with HSP or CMT2 with pyramidal signs were sequenced for all exons of KIF5A by targeted resequencing. One additional patient was genetically diagnosed by whole-exome sequencing. Five KIF5A mutations were identified in 6 unrelated patients: R204W and D232N were novel mutations; R204Q, R280C, and R280H have been previously reported. Three patients had CMT2 as the predominant and presenting phenotype; 2 of them also had pyramidal signs. The other 3 patients presented with HSP but also had significant axonal neuropathy or other additional features. This is currently the largest study investigating KIF5A mutations. By combining next-generation sequencing and conventional sequencing, we confirm that KIF5A mutations can cause variable phenotypes ranging from HSP to CMT2. The identification of mutations in CMT2 broadens the phenotypic spectrum and underlines the importance of KIF5A mutations, which involve degeneration of both the central and peripheral nervous systems and should be tested in HSP and CMT2.

Developing High-Throughput HIV Incidence Assay with Pyrosequencing Platform

Human immunodeficiency virus (HIV) incidence is an important measure for monitoring the epidemic and evaluating the efficacy of intervention and prevention trials. This study developed a high-throughput, single-measure incidence assay by implementing a pyrosequencing platform. We devised a signal-masking bioinformatics pipeline, which yielded a process error rate of 5.8 × 10(-4) per base. The pipeline was then applied to analyze 18,434 envelope gene segments (HXB2 7212 to 7601) obtained from 12 incident and 24 chronic patients who had documented HIV-negative and/or -positive tests. The pyrosequencing data were cross-checked by using the single-genome-amplification (SGA) method to independently obtain 302 sequences from 13 patients. Using two genomic biomarkers that probe for the presence of similar sequences, the pyrosequencing platform correctly classified all 12 incident subjects (100% sensitivity) and 23 of 24 chronic subjects (96% specificity). One misclassified subject's chronic infection was correctly classified by conducting the same analysis with SGA data. The biomarkers were statistically associated across the two platforms, suggesting the assay's reproducibility and robustness. Sampling simulations showed that the biomarkers were tolerant of sequencing errors and template resampling, two factors most likely to affect the accuracy of pyrosequencing results. We observed comparable biomarker scores between AIDS and non-AIDS chronic patients (multivariate analysis of variance [MANOVA], P = 0.12), indicating that the stage of HIV disease itself does not affect the classification scheme. The high-throughput genomic HIV incidence marks a significant step toward determining incidence from a single measure in cross-sectional surveys. Annual HIV incidence, the number of newly infected individuals within a year, is the key measure of monitoring the epidemic's rise and decline. Developing reliable assays differentiating recent from chronic infections has been a long-standing quest in the HIV community. Over the past 15 years, these assays have traditionally measured various HIV-specific antibodies, but recent technological advancements have expanded the diversity of proposed accurate, user-friendly, and financially viable tools. Here we designed a high-throughput genomic HIV incidence assay based on the signature imprinted in the HIV gene sequence population. By combining next-generation sequencing techniques with bioinformatics analysis, we demonstrated that genomic fingerprints are capable of distinguishing recently infected patients from chronically infected patients with high precision. Our high-throughput platform is expected to allow us to process many patients' samples from a single experiment, permitting the assay to be cost-effective for routine surveillance.

Combining next-generation sequencing and online databases for microsatellite development in non-model organisms

Next-generation sequencing (NGS) is revolutionising marker development and the rapidly increasing amount of transcriptomes published across a wide variety of taxa is providing valuable sequence databases for the identification of genetic markers without the need to generate new sequences. Microsatellites are still the most important source of polymorphic markers in ecology and evolution. Motivated by our long-term interest in the adaptive radiation of a non-model species complex of whitefishes (Coregonus spp.), in this study, we focus on microsatellite characterisation and multiplex optimisation using transcriptome sequences generated by Illumina® and Roche-454, as well as online databases of Expressed Sequence Tags (EST) for the study of whitefish evolution and demographic history. We identified and optimised 40 polymorphic loci in multiplex PCR reactions and validated the robustness of our analyses by testing several population genetics and phylogeographic predictions using 494 fish from five lakes and 2 distinct ecotypes.

Taiwan schizophrenia linkage study: Lessons learned from endophenotype‐based genome‐wide linkage scans and perspective

Taiwan Schizophrenia Linkage Study (TSLS) was initiated with a linkage strategy for locating multiple genes, each of small to moderate effect, and aimed to recruit a large enough sample of pairs of affected siblings and their families ascertained from a multisite study. With a sample of 607 families successfully recruited, a total of 2,242 individuals (1,207 affected and 1,035 unaffected) from 557 families were genotyped using 386 microsatellite markers spaced at an average of 9-cM intervals. Here the author reviews the establishment of TSLS and initial signal derived from linkage scan using the diagnosis of schizophrenia. Based on the limited success of the initial linkage analysis, a sufficient-component causal model is proposed to incorporate endophenotypes and genes for schizophrenia. Four types of candidate endophenotype measured in TSLS, including schizotypal personality, Continuous Performance Test, Wisconsin Card Sorting Test, and niacin skin flush test, are briefly described. The author discusses different strategies of linkage analysis incorporating these endophenotypes, including quantitative trait loci (QTL) linkage analysis, clustering-derived subgroups, ordered subset analysis (OSA), and latent classes for linkage scan. Then the author summarizes the linkage signals generated from seven studies of endophenotype-based linkage analysis using TSLS, including QTL scan of neurocognitive performance, QTL scan of niacin skin flush, the family cluster of attention deficit and execution deficit, OSA by schizophrenia-schizotypy factors, nested OSA by age at onset and neurocognitive performance, and the latent class of deficit schizophrenia for linkage analysis. The perspective of combining next-generation sequencing with linkage analysis of families is also discussed.

Combining Next-Generation Sequencing and Immune Assays: A Novel Method for Identification of Antigen-Specific T Cells

In this study, we combined a novel sequencing method, which can identify individual clonotypes based on their unique T cell receptor (TCR) rearrangement, with existing immune assays to characterize antigen-specific T cell responses. We validated this approach using three types of assays routinely used to measure antigen-specific responses: pentamers which enable identification of T cells bearing specific TCRs, activation marker expression following antigen stimulation and antigen-induced proliferation to identify cytomegalovirus (CMV) specific clonotypes. In one individual, 8 clonotypes were identified using a pentamer reagent derived from the CMV pp65 protein. The same 8 clonotypes were also identified following sequencing of cells that upregulated an activation marker following incubation with an identical peptide derived from pp65. These 8 and an additional 8 clonotypes were identified using a more sensitive CFSE-based proliferation assay. We found clear sequence homology among some of the clonotypes identified, and the CDR3 region in one clonotype was identical to a previously published pp65-specific clonotype sequence. Many of these CMV-specific clonotypes were present at frequencies below 10−5 which are undetectable using standard flow-cytometric methods. These studies suggest that an immune response is comprised of a diverse set of clones, many of which are present at very low frequencies. Thus, the combination of immune assays and sequencing depicts the richness and diversity of an immune response at a level that is not possible using standard immune assays alone. The methods articulated in this work provide an enhanced understanding of T cell-mediated immune responses at the clonal level.

Rapid, economical single‐nucleotide polymorphism and microsatellite discovery based on de novo assembly of a reduced representation genome in a non‐model organism: a case study of Atlantic cod Gadus morhua

By combining next-generation sequencing technology (454) and reduced representation library (RRL) construction, the rapid and economical isolation of over 25 000 potential single-nucleotide polymorphisms (SNP) and >6000 putative microsatellite loci from c. 2% of the genome of the non-model teleost, Atlantic cod Gadus morhua from the Celtic Sea, south of Ireland, was demonstrated. A small-scale validation of markers indicated that 80% (11 of 14) of SNP loci and 40% (6 of 15) of the microsatellite loci could be amplified and showed variability. The results clearly show that small-scale next-generation sequencing of RRL genomes is an economical and rapid approach for simultaneous SNP and microsatellite discovery that is applicable to any species. The low cost and relatively small investment in time allows for positive exploitation of ascertainment bias to design markers applicable to specific populations and study questions.

Combining Next-Generation Sequencing and Microarray Technology into a Transcriptomics Approach for the Non-Model Organism Chironomus riparius

Whole-transcriptome gene-expression analyses are commonly performed in species that have a sequenced genome and for which microarrays are commercially available. To do such analyses in species with no or limited genome data, i.e. non-model organisms, necessary transcriptomics resources, i.e. an annotated transcriptome and a validated gene-expression microarray, must first be developed. The aim of the present study was to establish an advanced approach for developing transcriptomics resources for non-model organisms by combining next-generation sequencing (NGS) and microarray technology. We applied our approach to the non-biting midge Chironomus riparius, an ecologically relevant species that is widely used in sediment ecotoxicity testing. We sampled extensively covering all C. riparius developmental stages as well as toxicant exposed larvae and obtained from a normalized cDNA library 1.5 M NGS reads totalling 501 Mbp. Using the NGS data we developed transcriptomics resources in several steps. First, we designed 844 k probes directly on the NGS reads, as well as 76 k probes targeting expressed sequence tags of related species. These probes were tested for their affinity to C. riparius DNA and mRNA, by performing two biological experiments with a 1 M probe-selection microarray that contained the entire probe-library. Subsequently, the 1.5 M NGS reads were assembled into 23,709 isotigs and 135,082 singletons, which were associated to ∼55 k, respectively, ∼61 k gene ontology terms and which corresponded together to 22,593 unique protein accessions. An algorithm was developed that took the assembly and the probe affinities to DNA and mRNA into account, what resulted in 59 k highly-reliable probes that targeted uniquely 95% of the isotigs and 18% of the singletons. Concluding, our approach allowed the development of high-quality transcriptomics resources for C. riparius, and is applicable to any non-model organism. It is expected, that these resources will advance ecotoxicity testing with C. riparius as whole-transcriptome gene-expression analysis are now possible with this species.

Computational prediction of furin cleavage sites by a hybrid method and understanding mechanism underlying diseases

Furin cleaves diverse types of protein precursors in the secretory pathway. The substrates for furin cleavage possess a specific 20-residue recognition sequence motif. In this report, based on the functional characterisation of the 20-residue sequence motif, we developed a furin cleavage site prediction tool, PiTou, using a hybrid method composed of a hidden Markov model and biological knowledge-based cumulative probability score functions. PiTou can accurately predict the presence and location of furin cleavage sites in protein sequences with high sensitivity (96.9%) and high specificity (97.3%). PiTou's prediction scores are biological meaningful and reflect binding strength and solvent accessibility of furin substrates. A prediction result is interpreted within cellular contexts: subcellular localisation, cellular function and interference by other dynamic protein modifications. Combining next-generation sequencing, PiTou can help with elucidating the molecular mechanism of furin cleavage-associated human diseases. PiTou has been made freely available at the associated website.

RNA-based assessment of diversity and composition of active archaeal communities in the German Bight.

Archaea play an important role in various biogeochemical cycles. They are known extremophiles inhabiting environments such as thermal springs or hydrothermal vents. Recent studies have revealed a significant abundance of Archaea in moderate environments, for example, temperate sea water. Nevertheless, the composition and ecosystem function of these marine archaeal communities is largely unknown. To assess diversity and composition of active archaeal communities in the German Bight, seven marine water samples were taken and studied by RNA-based analysis of ribosomal 16S rRNA. For this purpose, total RNA was extracted from the samples and converted to cDNA. Archaeal community structures were investigated by pyrosequencing-based analysis of 16S rRNA amplicons generated from cDNA. To our knowledge, this is the first study combining next-generation sequencing and metatranscriptomics to study archaeal communities in marine habitats. The pyrosequencing-derived dataset comprised 62,045 archaeal 16S rRNA sequences. We identified Halobacteria as the predominant archaeal group across all samples with increased abundance in algal blooms. Thermoplasmatales (Euryarchaeota) and the Marine Group I (Thaumarchaeota) were identified in minor abundances. It is indicated that archaeal community patterns were influenced by environmental conditions.

Analysis of the coding genome of diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common form of human lymphoma. While a number of structural alterations have been associated with the pathogenesis of this malignancy, the full spectrum of genetic lesions that are present in the DLBCL genome, and therefore the identity of dysregulated cellular pathways, remains unknown. By combining next-generation sequencing and copy number analysis, we show that the DLBCL coding genome contains on average more than 30 clonally represented gene alterations/case. This analysis also revealed mutations in genes not previously implicated in DLBCL pathogenesis, including those regulating chromatin methylation (MLL2, 24% of cases) and immune recognition by T cells. These results provide initial data on the complexity of the DLBCL coding genome and identify novel dysregulated pathways underlying its pathogenesis.

Analysis of the chronic lymphocytic leukemia coding genome: role of NOTCH1 mutational activation

The pathogenesis of chronic lymphocytic leukemia (CLL), the most common leukemia in adults, is still largely unknown. The full spectrum of genetic lesions that are present in the CLL genome, and therefore the number and identity of dysregulated cellular pathways, have not been identified. By combining next-generation sequencing and copy number analysis, we show here that the typical CLL coding genome contains <20 clonally represented gene alterations/case, including predominantly nonsilent mutations, and fewer copy number aberrations. These analyses led to the discovery of several genes not previously known to be altered in CLL. Although most of these genes were affected at low frequency in an expanded CLL screening cohort, mutational activation of NOTCH1, observed in 8.3% of CLL at diagnosis, was detected at significantly higher frequency during disease progression toward Richter transformation (31.0%), as well as in chemorefractory CLL (20.8%). Consistent with the association of NOTCH1 mutations with clinically aggressive forms of the disease, NOTCH1 activation at CLL diagnosis emerged as an independent predictor of poor survival. These results provide initial data on the complexity of the CLL coding genome and identify a dysregulated pathway of diagnostic and therapeutic relevance.