Illumina Genome Analyzer Research Articles

RNA-Seq of Human Breast Ductal Carcinoma In Situ Models Reveals Aldehyde Dehydrogenase Isoform 5A1 as a Novel Potential Target

Breast ductal carcinoma in situ (DCIS) is being found in great numbers of women due to the widespread use of mammography. To increase knowledge of DCIS, we determined the expression changes that are common among three DCIS models (MCF10.DCIS, SUM102 and SUM225) compared to the MCF10A model of non-tumorigenic mammary epithelial cells in three dimensional (3D) overlay culture with reconstituted basement membrane (rBM). Extracted mRNA was subjected to 76 cycles of deep sequencing (RNA-Seq) using Illumina Genome Analyzer GAIIx. Analysis of RNA-Seq results showed 295 consistently differentially expressed transcripts in the DCIS models. These differentially expressed genes encode proteins that are associated with a number of signaling pathways such as integrin, fibroblast growth factor and TGFβ signaling, show association with cell-cell signaling, cell-cell adhesion and cell proliferation, and have a notable bias toward localization in the extracellular and plasma membrane compartments. RNA-Seq data was validated by quantitative real-time PCR of selected differentially expressed genes. Aldehyde dehydrogenase 5A1 (ALDH5A1) which is an enzyme that is involved in mitochondrial glutamate metabolism, was over-expressed in all three DCIS models at both the mRNA and protein levels. Disulfiram and valproic acid are known to inhibit ALDH5A1 and are safe for chronic use in humans for other disorders. Both of these drugs significantly inhibited net proliferation of the DCIS 3D rBM overlay models, but had minimal effect on MCF10A 3D rBM overlay models. These results suggest that ALDH5A1 may play an important role in DCIS and potentially serve as a novel molecular therapeutic target.

205 RNA-SEq PROFILING OF BOVINE PRE-IMPLANTATION EMBRYOS

In order to provide a comprehensive view of the transcriptome changes during the earliest stages of bovine development, we sequenced the total RNA content of bovine oocytes, 4-cell, 8-cell, and 16-cell embryos and the inner cell mass and trophoblast envelope of expanded blastocysts on the Illumina Genome Analyzer IIx. For each experiment pools of in vitro matured oocytes from the German Simmental cows were fertilized by sperm of a single bull, and 10 oocytes or embryos per developmental stage were collected to generate total RNA pools used for sequencing. Synthesis of cDNA was initiated directly in the cell lysate in order to avoid any losses during RNA preparation and was random primed in order to capture all RNA species. Amplified cDNA and unstranded sequencing libraries were prepared using kits from Nugen (Ovation RNA-Seq, Nugen, San Carlos, CA, USA). Biological replicates were generated by inseminating the oocytes with sperm from the distant breeds Jersey (n = 3) and Brahman (n = 3). This cross-breeding design allowed tracking of single sequencing reads back to the maternal or paternal genome, where breed-specific SNP are present in the expressed transcripts. The analysis of this dataset resulted in monitoring of zygotic genome activation and parent-specific expression for single transcripts, a catalogue of splicing isoforms, novel transcripts, and non-coding RNAs and differentially expressed genes between the single developmental stages. Using the program DESEqn, 2784 genes showed differential expression between any of the stages at a false discovery rate of 1%. Specifically, we found 200 genes differentially expressed between immature and matured oocytes, 209 genes between matured oocytes and 4-cell embryos, 580 genes between the 4-cell and 8-cell stage, 567 genes between the 8-cell and 16-cell stage, 987 genes between the 16-cell stage and the inner cell mass, and 1569 genes between the 16-cell and the trophoblast. Functional analysis revealed stage-specific functions of the differentially expressed genes. In summary, by fully exploiting the single-nucleotide resolution of the RNA-Seq method, this dataset provides an invaluable resource for the study of zygotic genome activation, imprinting, transcript annotation, and gene expression in the earliest developmental stages of bovine embryos.

Accurate Diagnostics for Bovine tuberculosis Based on High-Throughput Sequencing

BackgroundBovine tuberculosis (bTB) is an enduring contagious disease of cattle that has caused substantial losses to the global livestock industry. Despite large-scale eradication efforts, bTB continues to persist. Current bTB tests rely on the measurement of immune responses in vivo (skin tests), and in vitro (bovine interferon-γ release assay). Recent developments are characterized by interrogating the expression of an increasing number of genes that participate in the immune response. Currently used assays have the disadvantages of limited sensitivity and specificity, which may lead to incomplete eradication of bTB. Moreover, bTB that reemerges from wild disease reservoirs requires early and reliable diagnostics to prevent further spread. In this work, we use high-throughput sequencing of the peripheral blood mononuclear cells (PBMCs) transcriptome to identify an extensive panel of genes that participate in the immune response. We also investigate the possibility of developing a reliable bTB classification framework based on RNA-Seq reads.Methodology/Principal FindingsPooled PBMC mRNA samples from unaffected calves as well as from those with disease progression of 1 and 2 months were sequenced using the Illumina Genome Analyzer II. More than 90 million reads were splice-aligned against the reference genome, and deposited to the database for further expression analysis and visualization. Using this database, we identified 2,312 genes that were differentially expressed in response to bTB infection (p<10−8). We achieved a bTB infected status classification accuracy of more than 99% with split-sample validation on newly designed and learned mixtures of expression profiles.Conclusions/SignificanceWe demonstrated that bTB can be accurately diagnosed at the early stages of disease progression based on RNA-Seq high-throughput sequencing. The inclusion of multiple genes in the diagnostic panel, combined with the superior sensitivity and broader dynamic range of RNA-Seq, has the potential to improve the accuracy of bTB diagnostics. The computational pipeline used for the project is available from http://code.google.com/p/bovine-tb-prediction.

Exome Sequencing Identifies Recurring DNMT3A Mutations in Adult ETP-ALL

AbstractAbstract 1377 Introduction:Early T-cell precursor (ETP) ALL accounting for 10% of all T-ALL cases is of special interest because of its proposed origin from early thymic progenitors with multilineage differentiation potential. ETP-ALL is associated with a poorer outcome in pediatric and adult patients. On the molecular level, ETP-ALL is characterized by a specific immunophenotype (CD1-, CD5weak, CD8-, co-expression of stem cell and/or myeloid antigens) and distinct molecular features (expression of stem cell genes, high frequency of FLT3 mutations with absence of NOTCH1 mutations). Whereas a highly heterogeneous genetic pattern was revealed by whole genome sequencing in pediatric patients, the genetic background of adult ETP-ALL remains largely unknown. Here we investigated genetic alterations in adult ETP-ALL by whole exome sequencing and subsequently analyzed specific target genes. Patients and methods:We performed whole exome sequencing of five paired (diagnosis/remission) adult ETP-ALL patients enrolled in German Acute Lymphoblastic Leukemia Multicenter Study Group (GMALL) trials. Using exon capturing from genomic DNA, followed by 76-bp paired-end sequencing on an Illumina Genome Analyzer IIx platform, we generated at least 5 Gb of exome sequence from each ETP-ALL and remission samples. Somatic mutations were identified by comparing the ETP-ALL with the remission exome sequence, excluding all annotated polymorphisms (dbSNP130), non-coding positions and positions with evidence of a variant in the corresponding remission samples. Candidate variants were confirmed by capillary sequencing of genomic DNA. The DNMT3A mutations status was analyzed by Sanger sequencing of exons 11–23 in additional 68 adult ETP-ALL (55 male, 13 female, median age: 38 years) as well as the mutation status of the polycomb repressor complex (PRC) genes EZH2 and SUZ12. For 52 of 68 patients clinical follow-up data were available. Results:Using whole exome sequencing we found a total of 56 non-synonymous somatic mutations or indels in the five ETP-ALL patients (range: 6 to 16 per patient). Eleven mutations/indels affected cancer genes. DNMT3A (2/5) and FAT3 (2/5) were recurrently mutated in the five patients. The DNA-methyl-transferase DNMT3A is a frequent mutational target in acute myeloid leukemia (AML; 20%), whereas FAT3 (FAT, tumor suppressor homolog 3) mutations were recently reported in ovarian carcinoma (TCGA, Nature 2011). Novel mutations identified in adult ETP-ALL involved genes in epigenetic regulation (e.g. MLL2, MLL3, BMI1), and in genes previously reported to be mutated in ETP-ALL (e.g. in JAK1, ETV6, NOTCH1, DNM2).By Sanger sequencing, we screened for DNMT3A mutations in a larger cohort of adult ETP-ALL. DNMT3A mutations were present in 11 of the 68 (16%) patients, a mutation rate similar to AML. Amino acid R882 (exon 23), the most frequently mutated amino acid in AML, was mutated in five ETP-ALL. The remaining six mutations occurred in single spots, with one exception in the ZNF or the MTF domain. Patients with a DNMT3A mutation were significantly older (median: 63 vs 37 years, P=0.016). No correlation was found between DNMT3A and FLT3 mutations (27% in DNMT3A mut pts. vs. 37% in DNMT3A wt pts., P=0.41) or NOTCH1 mutations (10% in DNMT3A mut pts. vs. 16% in DNMT3A wt pts., P=0.47).In addition, we investigated genetic alterations in epigenetic regulators including members of the polycomb repressor complex (PRC). Mutations were seen in EZH2 in 4/68 (6%), SUZ12 in 1/68 (1%) and SH2B3 in 4/69 (6%) of ETP-ALL. Interestingly, patients with at least one mutation in an epigenetic regulator gene (DNMT3A, SUZ12, SH2B3, MLL2, or EZH2) showed a trend towards an inferior survival (one-year-survival: 50% vs. 85%, P=0.08). Conclusion:Adult ETP-ALL patients display a heterogenous spectrum of mutations, particularly affecting genes involved in epigenetic regulation. The spectrum is different to pediatric patients with a lower rate of polycomb repressor complex and a higher rate of DNMT3A mutations. The higher rate of DNMT3A mutations in older patients might point to a different pathogenesis compared to pediatric ETP-ALL. Like in AML, DNMT3A mutations in adult ETP-ALL show a similar frequency, within the same hot spots and are correlated with an adverse prognostic value, underscoring the myeloid character of ETP-ALL. Thus, these data may provide a rationale to use epigenetic therapy in ETP-ALL. Disclosures:Krebs:Illumina: Honoraria. Greif:Illumina: Honoraria.

Integrated Analysis of Whole-Exome Sequencing and Micrornas Expression in Blast Crisis Transformation of Chronic Myeloid Leukemia

AbstractAbstract 3727 Objective.The molecular events leading to the evolution of BCR/ABL+ chronic myeloid leukemia (CML) from the chronic phase (CP) to the advanced phase (blast crisis-BP) are poorly understood. The aggressiveness of BP and the poor over-all survival of BP patients needs deep investigation of the biological basis of blastic transformation. We here present the results obtained from the analysis of paired (CP)/(BP) CML samples from patients that underwent progression after standard therapy. The access to matched CP/BP samples will render this data highly valuable. Methods.We performed whole-exome sequencing analysis using high-throughput technologies (Illumina Genome Analyzer IIx) from genomic DNA of four paired samples. The cross-match between BP and CP exomes was performed with dedicated in-house C#software. Gross chromosomal rearrangements were evaluated using CEQer(Comparative-Exonic-Quantification-Analyzer) software from whole-exome sequencing data. We also evaluated microRNA(miRNA) differential expression extracting RNA from five paired samples, using Nanostring nCounter miRNA expression assay. Differentially-expressed miRNAs with a p-value<0.001 were considered significant. Putative targets of significantly deregulated miRNAs were generated using miRGen software analysis(http://www.diana.pcbi.upenn.edu/miRGen.html). Results.By comparing exome-sequences of four paired CP (used as a control) and BP samples we found a total of 8 single nucleotide somatic mutations. Among the 8 variants identified, 4 of them ranked >1 in the GenRanker cancer scoring system (http://cbio.mskcc.org/tcga-generanker). We show here that, unexpectedly, blast crisis samples have a limited number of acquired mutations compared to chronic phases (average=2 mutations/patient) with patient number two and four displaying the lowest and the higher frequencies, respectively (patient n.2=0 mutations, patient n.4=4 mutations). CEQer analysis of whole exome data showed that 3/4 patients present gross chromosomal rearrangements of at least 2 chromosomes (bulky alteration of chromosome7 present in 2/4 patients); critical regulators of cell cycle control (e.g. CDKN2A and p53) have also been shown to be deleted in patient number 1 and 4, respectively. The individual mutations and rearrangements identified will be presented at the meeting.Differential expression of miRNAs showed that miR-106a, miR-17, miR-20a and miR-20b were significantly down-regulated while miR-148a was significantly up-regulated in all the blast crisis compared to chronic phase samples. In-silico analysis of the putative deregulated targets revealed a strong enrichment of genes involved in molecular mechanisms of cancer, with the 20% of these genes involved in cell cycle regulation. The integrated analysis of these informative data will help to understand the molecular mechanisms responsible for blast crisis progression. Disclosures:No relevant conflicts of interest to declare.

Quality control of next‐generation sequencing library through an integrative digital microfluidic platform

We have developed an automated quality control (QC) platform for next-generation sequencing (NGS) library characterization by integrating a droplet-based digital microfluidic (DMF) system with a capillary-based reagent delivery unit and a quantitative CE module. Using an in-plane capillary-DMF interface, a prepared sample droplet was actuated into position between the ground electrode and the inlet of the separation capillary to complete the circuit for an electrokinetic injection. Using a DNA ladder as an internal standard, the CE module with a compact LIF detector was capable of detecting dsDNA in the range of 5-100 pg/μL, suitable for the amount of DNA required by the Illumina Genome Analyzer sequencing platform. This DMF-CE platform consumes tenfold less sample volume than the current Agilent BioAnalyzer QC technique, preserving precious sample while providing necessary sensitivity and accuracy for optimal sequencing performance. The ability of this microfluidic system to validate NGS library preparation was demonstrated by examining the effects of limited-cycle PCR amplification on the size distribution and the yield of Illumina-compatible libraries, demonstrating that as few as ten cycles of PCR bias the size distribution of the library toward undesirable larger fragments.

SmRNAome profiling to identify conserved and novel microRNAs in Stevia rebaudiana Bertoni

BackgroundMicroRNAs (miRNAs) constitute a family of small RNA (sRNA) population that regulates the gene expression and plays an important role in plant development, metabolism, signal transduction and stress response. Extensive studies on miRNAs have been performed in different plants such as Arabidopsis thaliana, Oryza sativa etc. and volume of the miRNA database, mirBASE, has been increasing on day to day basis. Stevia rebaudiana Bertoni is an important perennial herb which accumulates high concentrations of diterpene steviol glycosides which contributes to its high indexed sweetening property with no calorific value. Several studies have been carried out for understanding molecular mechanism involved in biosynthesis of these glycosides, however, information about miRNAs has been lacking in S. rebaudiana. Deep sequencing of small RNAs combined with transcriptomic data is a powerful tool for identifying conserved and novel miRNAs irrespective of availability of genome sequence data.ResultsTo identify miRNAs in S. rebaudiana, sRNA library was constructed and sequenced using Illumina genome analyzer II. A total of 30,472,534 reads representing 2,509,190 distinct sequences were obtained from sRNA library. Based on sequence similarity, we identified 100 miRNAs belonging to 34 highly conserved families. Also, we identified 12 novel miRNAs whose precursors were potentially generated from stevia EST and nucleotide sequences. All novel sequences have not been earlier described in other plant species. Putative target genes were predicted for most conserved and novel miRNAs. The predicted targets are mainly mRNA encoding enzymes regulating essential plant metabolic and signaling pathways.ConclusionsThis study led to the identification of 34 highly conserved miRNA families and 12 novel potential miRNAs indicating that specific miRNAs exist in stevia species. Our results provided information on stevia miRNAs and their targets building a foundation for future studies to understand their roles in key stevia traits.

Prevalence of single nucleotide polymorphism among 27 diverse alfalfa genotypes as assessed by transcriptome sequencing.

BackgroundAlfalfa, a perennial, outcrossing species, is a widely planted forage legume producing highly nutritious biomass. Currently, improvement of cultivated alfalfa mainly relies on recurrent phenotypic selection. Marker assisted breeding strategies can enhance alfalfa improvement efforts, particularly if many genome-wide markers are available. Transcriptome sequencing enables efficient high-throughput discovery of single nucleotide polymorphism (SNP) markers for a complex polyploid species.ResultThe transcriptomes of 27 alfalfa genotypes, including elite breeding genotypes, parents of mapping populations, and unimproved wild genotypes, were sequenced using an Illumina Genome Analyzer IIx. De novo assembly of quality-filtered 72-bp reads generated 25,183 contigs with a total length of 26.8 Mbp and an average length of 1,065 bp, with an average read depth of 55.9-fold for each genotype. Overall, 21,954 (87.2%) of the 25,183 contigs represented 14,878 unique protein accessions. Gene ontology (GO) analysis suggested that a broad diversity of genes was represented in the resulting sequences. The realignment of individual reads to the contigs enabled the detection of 872,384 SNPs and 31,760 InDels. High resolution melting (HRM) analysis was used to validate 91% of 192 putative SNPs identified by sequencing. Both allelic variants at about 95% of SNP sites identified among five wild, unimproved genotypes are still present in cultivated alfalfa, and all four US breeding programs also contain a high proportion of these SNPs. Thus, little evidence exists among this dataset for loss of significant DNA sequence diversity from either domestication or breeding of alfalfa. Structure analysis indicated that individuals from the subspecies falcata, the diploid subspecies caerulea, and the tetraploid subspecies sativa (cultivated tetraploid alfalfa) were clearly separated.ConclusionWe used transcriptome sequencing to discover large numbers of SNPs segregating in elite breeding populations of alfalfa. Little loss of SNP diversity was evident between unimproved and elite alfalfa germplasm. The EST and SNP markers generated from this study are publicly available at the Legume Information System ( http://medsa.comparative-legumes.org/) and can contribute to future alfalfa research and breeding applications.

Local Assemblies of Paired-End Reduced Representation Libraries Sequenced with the Illumina Genome Analyzer in Maize

The use of next-generation DNA sequencing technologies has greatly facilitated reference-guided variant detection in complex plant genomes. However, complications may arise when regions adjacent to a read of interest are used for marker assay development, or when reference sequences are incomplete, as short reads alone may not be long enough to ascertain their uniqueness. Here, the possibility of generating longer sequences in discrete regions of the large and complex genome of maize is demonstrated, using a modified version of a paired-end RAD library construction strategy. Reads are generated from DNA fragments first digested with a methylation-sensitive restriction endonuclease, sheared, enriched with biotin and a selective PCR amplification step, and then sequenced at both ends. Sequences are locally assembled into contigs by subgrouping pairs based on the identity of the read anchored by the restriction site. This strategy applied to two maize inbred lines (B14 and B73) generated 183,609 and 129,018 contigs, respectively, out of which at least 76% were >200 bps in length. A subset of putative single nucleotide polymorphisms from contigs aligning to the B73 reference genome with at least one mismatch was resequenced, and 90% of those in B14 were confirmed, indicating that this method is a potent approach for variant detection and marker development in species with complex genomes or lacking extensive reference sequences.

SNP Discovery with EST and NextGen Sequencing in Switchgrass (Panicum virgatum L.)

Although yield trials for switchgrass (Panicum virgatum L.), a potentially high value biofuel feedstock crop, are currently underway throughout North America, the genetic tools for crop improvement in this species are still in the early stages of development. Identification of high-density molecular markers, such as single nucleotide polymorphisms (SNPs), that are amenable to high-throughput genotyping approaches, is the first step in a quantitative genetics study of this model biofuel crop species. We generated and sequenced expressed sequence tag (EST) libraries from thirteen diverse switchgrass cultivars representing both upland and lowland ecotypes, as well as tetraploid and octoploid genomes. We followed this with reduced genomic library preparation and massively parallel sequencing of the same samples using the Illumina Genome Analyzer technology platform. EST libraries were used to generate unigene clusters and establish a gene-space reference sequence, thus providing a framework for assembly of the short sequence reads. SNPs were identified utilizing these scaffolds. We used a custom software program for alignment and SNP detection and identified over 149,000 SNPs across the 13 short-read sequencing libraries (SRSLs). Approximately 25,000 additional SNPs were identified from the entire EST collection available for the species. This sequencing effort generated data that are suitable for marker development and for estimation of population genetic parameters, such as nucleotide diversity and linkage disequilibrium. Based on these data, we assessed the feasibility of genome wide association mapping and genomic selection applications in switchgrass. Overall, the SNP markers discovered in this study will help facilitate quantitative genetics experiments and greatly enhance breeding efforts that target improvement of key biofuel traits and development of new switchgrass cultivars.

A defect in iron uptake enhances the susceptibility of Cryptococcus neoformans to azole antifungal drugs

The high-affinity reductive iron uptake system that includes a ferroxidase (Cfo1) and an iron permease (Cft1) is critical for the pathogenesis of Cryptococcus neoformans. In addition, a mutant lacking CFO1 or CFT1 not only has reduced iron uptake but also displays a markedly increased susceptibility to azole antifungal drugs. Altered antifungal susceptibility of the mutants was of particular interest because the iron uptake system has been proposed as an alternative target for antifungal treatment. In this study, we used transcriptome analysis to begin exploring the molecular mechanisms of altered antifungal susceptibility in a cfo1 mutant. The wild-type strain and the cfo1 mutant were cultured with or without the azole antifungal drug fluconazole and their transcriptomes were compared following sequencing with Illumina Genome Analyzer IIx (GAIIx) technology. As expected, treatment of both strains with fluconazole caused elevated expression of genes in the ergosterol biosynthetic pathway that includes the target enzyme Erg11. Additionally, genes differentially expressed in the cfo1 mutant were involved in iron uptake and homeostasis, mitochondrial functions and respiration. The cfo1 mutant also displayed phenotypes consistent with these changes including a reduced ratio of NAD+/NADH and down-regulation of Fe–S cluster synthesis. Moreover, combination treatment of the wild-type strain with fluconazole and the respiration inhibitor diphenyleneiodonium dramatically increased susceptibility to fluconazole. This result supports the hypothesis that down-regulation of genes required for respiration contributed to the altered fluconazole susceptibility of the cfo1 mutant. Overall, our data suggest that iron uptake and homeostasis play a key role in antifungal susceptibility and could be used as novel targets for combination treatment of cryptococcosis. Indeed, we found that iron chelation in combination with fluconazole treatment synergistically inhibited the growth of C. neoformans.

Transcriptome profile of a bovine respiratory disease pathogen: Mannheimia haemolytica PHL213

BackgroundComputational methods for structural gene annotation have propelled gene discovery but face certain drawbacks with regards to prokaryotic genome annotation. Identification of transcriptional start sites, demarcating overlapping gene boundaries, and identifying regulatory elements such as small RNA are not accurate using these approaches. In this study, we re-visit the structural annotation of Mannheimia haemolytica PHL213, a bovine respiratory disease pathogen. M. haemolytica is one of the causative agents of bovine respiratory disease that results in about $3 billion annual losses to the cattle industry. We used RNA-Seq and analyzed the data using freely-available computational methods and resources. The aim was to identify previously unannotated regions of the genome using RNA-Seq based expression profile to complement the existing annotation of this pathogen.ResultsUsing the Illumina Genome Analyzer, we generated 9,055,826 reads (average length ~76 bp) and aligned them to the reference genome using Bowtie. The transcribed regions were analyzed using SAMTOOLS and custom Perl scripts in conjunction with BLAST searches and available gene annotation information. The single nucleotide resolution map enabled the identification of 14 novel protein coding regions as well as 44 potential novel sRNA. The basal transcription profile revealed that 2,506 of the 2,837 annotated regions were expressed in vitro, at 95.25% coverage, representing all broad functional gene categories in the genome. The expression profile also helped identify 518 potential operon structures involving 1,086 co-expressed pairs. We also identified 11 proteins with mutated/alternate start codons.ConclusionsThe application of RNA-Seq based transcriptome profiling to structural gene annotation helped correct existing annotation errors and identify potential novel protein coding regions and sRNA. We used computational tools to predict regulatory elements such as promoters and terminators associated with the novel expressed regions for further characterization of these novel functional elements. Our study complements the existing structural annotation of Mannheimia haemolytica PHL213 based on experimental evidence. Given the role of sRNA in virulence gene regulation and stress response, potential novel sRNA described in this study can form the framework for future studies to determine the role of sRNA, if any, in M. haemolytica pathogenesis.

The genome of flax (Linum usitatissimum) assembled de novo from short shotgun sequence reads

Flax (Linum usitatissimum) is an ancient crop that is widely cultivated as a source of fiber, oil and medicinally relevant compounds. To accelerate crop improvement, we performed whole-genome shotgun sequencing of the nuclear genome of flax. Seven paired-end libraries ranging in size from 300 bp to 10 kb were sequenced using an Illumina genome analyzer. A de novo assembly, comprised exclusively of deep-coverage (approximately 94× raw, approximately 69× filtered) short-sequence reads (44-100 bp), produced a set of scaffolds with N(50) =694 kb, including contigs with N(50)=20.1 kb. The contig assembly contained 302 Mb of non-redundant sequence representing an estimated 81% genome coverage. Up to 96% of published flax ESTs aligned to the whole-genome shotgun scaffolds. However, comparisons with independently sequenced BACs and fosmids showed some mis-assembly of regions at the genome scale. A total of 43384 protein-coding genes were predicted in the whole-genome shotgun assembly, and up to 93% of published flax ESTs, and 86% of A. thaliana genes aligned to these predicted genes, indicating excellent coverage and accuracy at the gene level. Analysis of the synonymous substitution rates (K(s) ) observed within duplicate gene pairs was consistent with a recent (5-9 MYA) whole-genome duplication in flax. Within the predicted proteome, we observed enrichment of many conserved domains (Pfam-A) that may contribute to the unique properties of this crop, including agglutinin proteins. Together these results show that de novo assembly, based solely on whole-genome shotgun short-sequence reads, is an efficient means of obtaining nearly complete genome sequence information for some plant species.

De Novo Sequencing and Transcriptome Analysis of the Central Nervous System of Mollusc Lymnaea stagnalis by Deep RNA Sequencing

The pond snail Lymnaea stagnalis is among several mollusc species that have been well investigated due to the simplicity of their nervous systems and large identifiable neurons. Nonetheless, despite the continued attention given to the physiological characteristics of its nervous system, the genetic information of the Lymnaea central nervous system (CNS) has not yet been fully explored. The absence of genetic information is a large disadvantage for transcriptome sequencing because it makes transcriptome assembly difficult. We here performed transcriptome sequencing for Lymnaea CNS using an Illumina Genome Analyzer IIx platform and obtained 81.9 M of 100 base pair (bp) single end reads. For de novo assembly, five programs were used: ABySS, Velvet, OASES, Trinity and Rnnotator. Based on a comparison of the assemblies, we chose the Rnnotator dataset for the following blast searches and gene ontology analyses. The present dataset, 116,355 contigs of Lymnaea transcriptome shotgun assembly (TSA), contained longer sequences and was much larger compared to the previously reported Lymnaea expression sequence tag (EST) established by classical Sanger sequencing. The TSA sequences were subjected to blast analyses against several protein databases and Aplysia EST data. The results demonstrated that about 20,000 sequences had significant similarity to the reported sequences using a cutoff value of 1e-6, and showed the lack of molluscan sequences in the public databases. The richness of the present TSA data allowed us to identify a large number of new transcripts in Lymnaea and molluscan species.

Large‐scale transcriptome characterization and mass discovery of SNPs in globe artichoke and its related taxa

Cynara cardunculus (2n = 2× = 34) is a member of the Asteraceae family that contributes significantly to the agricultural economy of the Mediterranean basin. The species includes two cultivated varieties, globe artichoke and cardoon, which are grown mainly for food. Cynara cardunculus is an orphan crop species whose genome/transcriptome has been relatively unexplored, especially in comparison to other Asteraceae crops. Hence, there is a significant need to improve its genomic resources through the identification of novel genes and sequence-based markers, to design new breeding schemes aimed at increasing quality and crop productivity. We report the outcome of cDNA sequencing and assembly for eleven accessions of C. cardunculus. Sequencing of three mapping parental genotypes using Roche 454-Titanium technology generated 1.7 × 10⁶ reads, which were assembled into 38,726 reference transcripts covering 32 Mbp. Putative enzyme-encoding genes were annotated using the KEGG-database. Transcription factors and candidate resistance genes were surveyed as well. Paired-end sequencing was done for cDNA libraries of eight other representative C. cardunculus accessions on an Illumina Genome Analyzer IIx, generating 46 × 10⁶ reads. Alignment of the IGA and 454 reads to reference transcripts led to the identification of 195,400 SNPs with a Bayesian probability exceeding 95%; a validation rate of 90% was obtained by Sanger-sequencing of a subset of contigs. These results demonstrate that the integration of data from different NGS platforms enables large-scale transcriptome characterization, along with massive SNP discovery. This information will contribute to the dissection of key agricultural traits in C. cardunculus and facilitate the implementation of marker-assisted selection programs.

Self-organizing Approach for the Human Gut Meta-genome

We extend the self-organizing approach for annotation of a bacterial genome to analyzing the raw sequencing data of the human gut metagenome without sequence assembling. The original approach divides the genomic sequence of a bacterium into non-overlapping segments of equal length and assigns to each segment one of seven ‘phases’, among which one is for the noncoding regions, three for the direct coding regions to indicate the three possible codon positions of the segment starting site, and three for the reverse coding regions. The noncoding phase and the six coding phases are described by two frequency tables of the 64 triplet types or ‘codon usages’. A set of codon usages can be used to update the phase assignment and vice versa. After an initialization of phase assignment or codon usage tables, an iteration leads to a convergent phase assignment to give an annotation of the genome. In the extension of the approach to a metagenome, we consider a mixture model of a number of categories of genomes. The Illumina Genome Analyzer sequencing data of the total DNA from faecal samples are then examined to understand the diversity of the human gut microbiome.

Slim-Filter: an interactive windows-based application for illumina genome analyzer data assessment and manipulation

BackgroundThe emergence of Next Generation Sequencing technologies has made it possible for individual investigators to generate gigabases of sequencing data per week. Effective analysis and manipulation of these data is limited due to large file sizes, so even simple tasks such as data filtration and quality assessment have to be performed in several steps. This requires (potentially problematic) interaction between the investigator and a bioinformatics/computational service provider. Furthermore, such services are often performed using specialized computational facilities.ResultsWe present a Windows-based application, Slim-Filter designed to interactively examine the statistical properties of sequencing reads produced by Illumina Genome Analyzer and to perform a broad spectrum of data manipulation tasks including: filtration of low quality and low complexity reads; filtration of reads containing undesired subsequences (such as parts of adapters and PCR primers used during the sample and sequencing libraries preparation steps); excluding duplicated reads (while keeping each read’s copy number information in a specialized data format); and sorting reads by copy numbers allowing for easy access and manual editing of the resulting files. Slim-Filter is organized as a sequence of windows summarizing the statistical properties of the reads. Each data manipulation step has roll-back abilities, allowing for return to previous steps of the data analysis process. Slim-Filter is written in C++ and is compatible with fasta, fastq, and specialized AS file formats presented in this manuscript. Setup files and a user’s manual are available for download at the supplementary web site ( https://www.bioinfo.uh.edu/Slim_Filter/).ConclusionThe presented Windows-based application has been developed with the goal of providing individual investigators with integrated sequencing reads analysis, curation, and manipulation capabilities.

Next‐generation sequencing for mitochondrial diseases: A wide diagnostic spectrum

The current diagnostic approach for mitochondrial disorders requires invasive procedures such as muscle biopsy and multiple biochemical testing but the results are often inconclusive. Clinical sequencing tests are available only for a limited number of genes. Recently, massively parallel sequencing has become a powerful tool for testing genetically heterogeneous conditions such as mitochondrial disorders. Targeted next-generation sequencing was performed on 26 patients with known or suspected mitochondrial disorders using in-solution capture for the exons of 908 known and candidate nuclear genes and an Illumina genome analyzer. None of the 18 patients with various abnormal respiratory chain complex (RCC) activities had molecular defects in either subunits or assembly factors of mitochondrial RCC enzymes except a reference control sample with known mutations in SURF1. Instead, several variants in known pathogenic genes including CPT2, POLG, PDSS1, UBE3A, SDHD, and a few potentially pathogenic variants in candidate genes such as MTO1 or SCL7A13 were identified. Sequencing only nuclear genes for RCC subunits and assembly factors may not provide the diagnostic answers for suspected patients with mitochondrial disorders. The present findings indicate that the diagnostic spectrum of mitochondrial disorders is much broader than previously thought, which could potentially lead to misdiagnosis and/or inappropriate treatment. Overall analytic sensitivity and precision appear acceptable for clinical testing. Despite the limitations in finding mutations in all patients, the present findings underscore the considerable clinical benefits of targeted next-generation sequencing and serve as a prototype for extending the clinical evaluation in this clinically heterogeneous patient group.

Characterization of the rainbow trout egg microRNA transcriptome.

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNA molecules that regulate post-transcriptional expression of target genes and play important roles in animal development. The objectives of this study were to characterize the egg miRNA transcriptome and identify novel egg-predominant miRNAs in rainbow trout. Small RNAs isolated from mature unfertilized rainbow trout eggs were subjected to deep sequencing using an Illumina Genome Analyzer. The massive sequencing produced 24,621,741 quality reads, among which, 266 known miRNAs were identified and 230 putatively novel miRNAs were predicted. The most abundantly known miRNAs are let-7 and miR-21, accounting for 24.06% and 18.71% of the known miRNAs, respectively. Other known miRNAs which are abundantly present in eggs include miR-24, miR-202, miR-148, miR-30, miR-10, miR-146, miR-25, and miR-143. Real time PCR analysis using cDNAs derived from 10 tissues validated 87 out of 90 selected putative miRNAs and identified three novel miRNAs predominantly expressed in rainbow trout eggs. Each of these novel egg-predominant miRNAs is predicted to target a significant number of genes, most of which are significantly down-regulated in naturally ovulated rainbow trout eggs based on analysis of publicly available microarray data sets. Quantitative real time PCR analysis also demonstrated low expression of a selected number of target genes in eggs relative to liver and muscle tissues. This study represents the first complete survey of miRNAs in fish eggs and provides a starting point for future studies aimed at understanding the roles of miRNAs in controlling egg quality and early embryogenesis in rainbow trout.

Enrichment of genomic DNA for polymorphism detection in a non‐model highly polyploid crop plant

Large polyploid genomes of non-model species remain challenging targets for DNA polymorphism discovery despite the increasing throughput and continued reductions in cost of sequencing with new technologies. For these species especially, there remains a requirement to enrich genomic DNA to discover polymorphisms in regions of interest because of large genome size and to provide the sequence depth to enable estimation of copy number. Various methods of enriching DNA have been utilised, but some recent methods enable the efficient sampling of large regions (e.g. the exome). We have utilised one of these methods, solution-based hybridization (Agilent SureSelect), to capture regions of the genome of two sugarcane genotypes (one Saccharum officinarum and one Saccharum hybrid) based mainly on gene sequences from the close relative Sorghum bicolor. The capture probes span approximately 5.8 megabases (Mb). The enrichment over whole-genome shotgun sequencing was 10-11-fold for the two genotypes tested. This level of enrichment has important consequences for detecting single nucleotide polymorphisms (SNPs) from a single lane of Illumina (Genome Analyzer) sequence reads. The detection of polymorphisms was enabled by the depth of sequence at or near probe sites and enabled the detection of 270 000-280 000 SNPs within each genotype from a single lane of sequence using stringent detection parameters. The SNPs were present in 13 000-16 000 targeted genes, which would enable mapping of a large number of these chosen genes. SNP validation from 454 sequencing and between-genotype confirmations gave an 87%-91% validation rate.