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The olive fruit fly, Bactrocera oleae, is the most important pest for the olive fruit but lacks adequate transcriptomic characterization that could aid in molecular control approaches. We apply nanopore long-read RNA-seq with internal RNA standards allowing absolute transcript quantification to analyze transcription dynamics during early embryo development for the first time in this organism. Sequencing on the MinION platform generated over 31 million reads. Over 50% of the expressed genes had at least one read covering its entire length validating our full-length approach. We generated a de novo transcriptome assembly and identified 1768 new genes and a total of 79,810 isoforms; a fourfold increase in transcriptome diversity compared to the current NCBI predicted transcriptome. Absolute transcript quantification per embryo allowed an insight into the dramatic re-organization of maternal transcripts. We further identified Zelda as a possible regulator of early zygotic genome activation in B. oleae and provide further insights into the maternal-to-zygotic transition. These data show the utility of long-read RNA in improving characterization of non-model organisms that lack a fully annotated genome, provide potential targets for sterile insect technic approaches, and provide the first insight into the transcriptome landscape of the developing olive fruit fly embryo.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The olive fruit fly or olive fly (Bactrocera oleae) is the most important pest of cultivated olive trees. Like all insects the olive fly undergoes complete metamorphosis. However, the transcription dynamics that occur during early embryonic development have not been explored, while detailed transcriptomic analysis in the absence of a fully annotated genome is challenging. We collected olive fly embryos at hourly intervals for the first 6 hours of development and performed full-length cDNA-Seq using a purpose designed SMARTer cDNA synthesis protocol followed by sequencing on the MinION (Oxford Nanopore Technologies). We generated 31 million total reads across the timepoints (median yield 4.2 million per timepoint). The reads showed 98 % alignment rate to the olive fly genome and 91 % alignment rate to the NBCI predicted B. oleae gene models. Over 50 % of the expressed genes had at least one read covering its entire length validating our full-length RNA-Seq procedure. Expression of 68 % of the predicted B. oleae genes was detected in the first six hours of development. We generated a de novo transcriptome assembly of the olive fly and identified 3553 novel genes and a total of 79,810 transcripts; a fourfold increase in transcriptome diversity compared to the NCBI predicted transcriptome. On a global scale, the first six hours of embryo development were characterized by dramatic transcriptome changes with the total number of transcripts per embryo dropping to half from the first hour to the second hour of embryo development. Clustering of genes based on temporal co-expression followed by gene-set enrichment analysiss of genes expressed in the first six hours of embryo development showed that genes involved in transcription and translation, macro-molecule biosynthesis, and neurodevelopment were highly enriched. These data provide the first insight into the transcriptome landscape of the developing olive fly embryo. The data also reveal transcript signatures of sex development. Overall, full-length sequencing of the cDNA molecules permitted a detailed characterization of the isoform complexity and the transcriptional dynamics of the first embryonic stages of the B. oleae.

Newly released Rapid Low Input by PCR Barcoding Sequencing Kit (SQK-RLB001); minor adjustments indicated by (*).

RNA sequencing using next-generation sequencing (NGS, RNA-Seq) technologies is currently the standard approach for gene expression profiling, particularly for large-scale high-throughput studies. NGS technologies comprise short-read RNA-Seq (dominated by Illumina) and long-read RNA-Seq technologies provided by Pacific Bioscience (PacBio) and Oxford Nanopore Technologies (ONT). Although short-read sequencing technologies are the most widely used, long-read technologies are increasingly becoming the standard approach for de novo transcriptome assembly and isoform expression quantification due to the complex nature of the transcriptome which consists of variable lengths of transcripts and multiple alternatively spliced isoforms for most genes. In this chapter, we describe experimental procedures for library preparation, sequencing, and associated data analysis approaches for PacBio and ONT with a major focus on full length cDNA synthesis, de novo transcriptome assembly, and isoform quantification.

The transcriptome encompasses a range of species including messenger RNA, and other noncoding RNA such as rRNA, tRNA, and short and long noncoding RNAs. Due to the huge role played by mRNA in development and disease, several methods have been developed to sequence and characterize mRNA, with RNA sequencing (RNA-Seq) emerging as the current method of choice particularly for large high-throughput studies. Short-read RNA-Seq which involves sequencing of short cDNA fragments and computationally assembling them to reconstruct the transcriptome, or aligning them to a reference is the most widely used approach. However, due to inherent limitations of this approach in de novo transcriptome assembly and isoform quantification, long-read RNA-Seq approaches, which also happen to be single molecule sequencing approaches, are increasingly becoming the standard for de novo transcriptome assembly and isoform quantification. In this chapter, we review the technical aspects of the current methods of RNA-Seq, both short and long-read approaches, and data analysis methods available. We discuss recent advances in single-cell RNA-Seq and direct RNA-Seq approaches, which perhaps will dominate the future of RNA-Seq.

RNA sequencing using next-generation sequencing (NGS, RNA-Seq) technologies is currently the standard approach for gene expression profiling, particularly for large-scale high-throughput studies. NGS technologies comprise short-read RNA-Seq (dominated by Illumina) and long-read RNA-Seq technologies provided by Pacific Bioscience (PacBio) and Oxford Nanopore Technologies (ONT). Although short-read sequencing technologies are the most widely used, long-read technologies are increasingly becoming the standard approach for de novo transcriptome assembly and isoform expression quantification due to the complex nature of the transcriptome which consists of variable lengths of transcripts and multiple alternatively spliced isoforms for most genes. In this chapter, we describe experimental procedures for library preparation, sequencing, and associated data analysis approaches for PacBio and ONT with a major focus on full length cDNA synthesis, de novo transcriptome assembly, and isoform quantification.

The transcriptome encompasses a range of species including messenger RNA, and other noncoding RNA such as rRNA, tRNA, and short and long noncoding RNAs. Due to the huge role played by mRNA in development and disease, several methods have been developed to sequence and characterize mRNA, with RNA sequencing (RNA-Seq) emerging as the current method of choice particularly for large high-throughput studies. Short-read RNA-Seq which involves sequencing of short cDNA fragments and computationally assembling them to reconstruct the transcriptome, or aligning them to a reference is the most widely used approach. However, due to inherent limitations of this approach in de novo transcriptome assembly and isoform quantification, long-read RNA-Seq approaches, which also happen to be single molecule sequencing approaches, are increasingly becoming the standard for de novo transcriptome assembly and isoform quantification. In this chapter, we review the technical aspects of the current methods of RNA-Seq, both short and long-read approaches, and data analysis methods available. We discuss recent advances in single-cell RNA-Seq and direct RNA-Seq approaches, which perhaps will dominate the future of RNA-Seq.

Background Genetic studies have provided ample evidence of the influence of non-coding DNA polymorphisms on trait variance, particularly those occurring within transcription factor binding sites. Protein binding microarrays and other platforms that can map these sites with great precision have enhanced our understanding of how a single nucleotide polymorphism can alter binding potential within an in vitro setting, allowing for greater predictive capability of its effect on a transcription factor binding site. Results We have used protein binding microarrays and electrophoretic mobility shift assay-sequencing (EMSA-Seq), a deep sequencing based method we developed to analyze nine distinct human NF-κB dimers. This family of transcription factors is one of the most extensively studied, but our understanding of its DNA binding preferences has been limited to the originally described consensus motif, GGRRNNYYCC. We highlight differences between NF-κB family members and also put under the spotlight non-canonical motifs that have so far received little attention. We utilize our data to interpret the binding of transcription factors between individuals across 1,405 genomic regions laden with single nucleotide polymorphisms. We also associated binding correlations made using our data with risk alleles of disease and demonstrate its utility as a tool for functional studies of single nucleotide polymorphisms in regulatory regions. Conclusions NF-κB dimers bind specifically to non-canonical motifs and these can be found within genomic regions in which a canonical motif is not evident. Binding affinity data generated with these different motifs can be used in conjunction with data from chromatin immunoprecipitation-sequencing (ChIP-Seq) to enable allele-specific analyses of expression and transcription factor-DNA interactions on a genome-wide scale., Wellcome Trust (London, England) (grant 075491/Z/04), European Commission (Seventh Framework Programme FP7/2007-2013: Model-In (222008)), European Commission (Seventh Framework Programme FP7 ITN Network INTEGER (214902)), Medical Research Council (Canada) (MRC project grant G0700818)

To assess the performance of the Oxford Nanopore Technologies MinION sequencing platform, cDNAs from the External RNA Controls Consortium (ERCC) RNA Spike-In mix were sequenced. This mix mimics mammalian mRNA species and consists of 92 polyadenylated transcripts with known concentration. cDNA libraries were generated using a template switching protocol to facilitate the direct comparison between different sequencing platforms. The MinION performance was assessed for its ability to sequence the cDNAs directly with good accuracy in terms of abundance and full length. The abundance of the ERCC cDNA molecules sequenced by MinION agreed with their expected concentration. No length or GC content bias was observed. The majority of cDNAs were sequenced as full length. Additionally, a complex cDNA population derived from a human HEK-293 cell line was sequenced on an Illumina HiSeq 2500, PacBio RS II and ONT MinION platforms. We observed that there was a good agreement in the measured cDNA abundance between PacBio RS II and ONT MinION (rpearson = 0.82, isoforms with length more than 700bp) and between Illumina HiSeq 2500 and ONT MinION (rpearson = 0.75). This indicates that the ONT MinION can sequence quantitatively both long and short full length cDNA molecules.

Hypoxia-inducible factor (HIF) regulates the major transcriptional cascade central to the response of all mammalian cells to alterations in oxygen tension. Expression arrays indicate that many hundreds of genes are regulated by this pathway, controlling diverse processes that in turn orchestrate both oxygen delivery and utilization. However, the extent to which HIF exerts direct versus indirect control over gene expression together with the factors dictating the range of HIF-regulated genes remains unclear. Using chromatin immunoprecipitation linked to high throughput sequencing, we identify HIF-binding sites across the genome, independently of gene architecture. Using gene set enrichment analysis, we demonstrate robust associations with the regulation of gene expression by HIF, indicating that these sites operate over long genomic intervals. Analysis of HIF-binding motifs demonstrates sequence preferences outside of the core RCGTG-binding motif but does not reveal any additional absolute sequence requirements. Across the entire genome, only a small proportion of these potential binding sites are bound by HIF, although occupancy of potential sites was enhanced approximately 20-fold at normoxic DNAse1 hypersensitivity sites (irrespective of distance from promoters), suggesting that epigenetic regulation of chromatin may have an important role in defining the response to hypoxia.

Summary Interferon Regulatory Factor 5 (IRF5) plays a major role in setting up an inflammatory macrophage phenotype, but the molecular basis of its transcriptional activity is not fully understood. In this study, we conduct a comprehensive genome-wide analysis of IRF5 recruitment in macrophages stimulated with bacterial lipopolysaccharide and discover that IRF5 binds to regulatory elements of highly transcribed genes. Analysis of protein:DNA microarrays demonstrates that IRF5 recognizes the canonical IRF-binding (interferon-stimulated response element [ISRE]) motif in vitro. However, IRF5 binding in vivo appears to rely on its interactions with other proteins. IRF5 binds to a noncanonical composite PU.1:ISRE motif, and its recruitment is aided by RelA. Global gene expression analysis in macrophages deficient in IRF5 and RelA highlights the direct role of the RelA:IRF5 cistrome in regulation of a subset of key inflammatory genes. We map the RelA:IRF5 interaction domain and suggest that interfering with it would offer selective targeting of macrophage inflammatory activities., Graphical Abstract, Highlights • Genome-wide function of IRF5 in LPS-stimulated macrophages was analyzed • IRF5 cistrome overlaps with RelA cistrome at multiple loci • IRF5 targets regulatory elements of highly inducible inflammatory genes • IRF5 recruitment to key inflammatory loci is assisted by RelA, Saliba et al. show that the genome-wide binding of IRF5 and RelA in LPS-stimulated macrophages overlaps at multiple loci. IRF5 and RelA target regulatory elements of highly inducible inflammatory genes. IRF5 is recruited to a composite PU.1:ISRE motif, and its binding is aided by NF-κb. This study systematically defines the mechanism of IRF5 and RelA transcriptional regulation of a key subset of inflammatory genes and highlights IRF5:RelA interaction interface as a target for specific interventions.

The Transforming Growth Factor-β (TGF-β) signaling pathway is one of the major pathways essential for normal embryonic development and tissue homeostasis, with anti-tumor but also pro-metastatic properties in cancer. This pathway directly regulates several target genes that mediate its downstream functions, however very few microRNAs (miRNAs) have been identified as targets. miRNAs are modulators of gene expression with essential roles in development and a clear association with diseases including cancer. Little is known about the transcriptional regulation of the primary transcripts (pri-miRNA, pri-miR) from which several mature miRNAs are often derived. Here we present the identification of miRNAs regulated by TGF-β signaling in mouse embryonic stem (ES) cells and early embryos. We used an inducible ES cell system to maintain high levels of the TGF-β activated/phosphorylated Smad2/3 effectors, which are the transcription factors of the pathway, and a specific inhibitor that blocks their activation. By performing short RNA deep-sequencing after 12 hours Smad2/3 activation and after 16 hours inhibition, we generated a database of responsive miRNAs. Promoter/enhancer analysis of a subset of these miRNAs revealed that the transcription of pri-miR-181c/d and the pri-miR-341∼3072 cluster were found to depend on activated Smad2/3. Several of these miRNAs are expressed in early mouse embryos, when the pathway is known to play an essential role. Treatment of embryos with TGF-β inhibitor caused a reduction of their levels confirming that they are targets of this pathway in vivo. Furthermore, we showed that pri-miR-341∼3072 transcription also depends on FoxH1, a known Smad2/3 transcription partner during early development. Together, our data show that miRNAs are regulated directly by the TGF-β/Smad2/3 pathway in ES cells and early embryos. As somatic abnormalities in functions known to be regulated by the TGF-β/Smad2/3 pathway underlie tumor suppression and metastasis, this research also provides a resource for miRNAs involved in cancer.

Hypoxia is a common feature of most solid tumors and is associated with poor prognosis in breast cancer. In the current study, we investigated the hypoxia transcription landscape of an ER positive breast cancer cell line [MCF7] using high throughput strand-specific total RNA sequencing (tssRNA-seq). MCF7 cells were grown under hypoxia and normoxia (24 hours, 1% O2), and we found about 7.3% of annotated genome got significantly differential expressed in hypoxic stress, which includes 79% (1293) up regulated and 21% (344) down regulated transcripts (P value of 1.5). Analysis revealed deregulation of a range of non coding transcripts including piwiRNA (0.36%), miRNA (0.098%), sn/snoRNA (0.028%), tRNA (4.5%), lncRNA (6.5%) under hypoxia (P value of 1.5). Many non-poly A transcripts and natural anti-sense transcripts (n=71) were detected and differently expressed in hypoxia. We characterized two significant up regulated natural anti-sense transcripts in hypoxia and found that these transcripts are long (>200bp), lack of protein coding frames, localized in nucleus and found highly expressed (range fold >5-15) in different hypoxic breast cancer cell lines. To define which hypoxic altered transcripts are dependent on, hypoxia-inducible factor (HIF), we suppressed HIF-alpha subunits in hypoxic MFC-7 cells using short interfering RNA and subjected them to mRNA-seq. Results revealed that HIF-1, HIF-2 and combined HIF-1/2 knockdown resulted in significant down-regulation of 179, 84 and 149 transcripts, respectively, which were up regulated in hypoxic MCF-7 cells. Despite detection of previously reported HIF dependent transcripts, we detected a substantial number of HIF dependent transcripts (n=64) that were not reported before as hypoxia regulated genes. To our understanding, this is first study to show hypoxia dependent natural anti-sense transcripts and genome-wide HIF dependent transcripts analysis at single base pair resolution in hypoxic tumors. These finding will open new avenues in our efforts to understand the hypoxia induced transcriptional response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3946. doi:1538-7445.AM2012-3946

Transcriptional responses to hypoxia are central to the pathogenesis of many types of cancer. Today, pan-genome analyses of hypoxia have focused on protein-coding genes, however, the role of non-coding RNAs, in particular long non-coding RNAs (lncRNA) is not well characterised. We undertook an integrated pan-genomic analysis of the transcriptional responses to hypoxia in MCF7 breast cancer cells, employing total RNA-seq together with ChIP-seq for the hypoxia-inducible transcription factor (HIF) and for epigenetic marks of transcriptional activation (RNApol2 and histone H3K4me3). Analyses revealed that all classes of RNA are significantly regulated by hypoxia. We found significant numbers of lncRNAs are up-regulated in hypoxia and these are associated with epigenetic marks of increased transcription and HIF binding. We describe a number of hypoxia regulated non-annotated RNA species, including several that are antisense to hypoxia regulated protein-coding RNAs. The most hypoxia up-regulated lncRNA was NEAT1. The role of NEAT1 in cancer has not been previously studied. We demonstrate that NEAT1 induction is common in breast cancer cell lines and xenograft models. Finally, selected hypoxia regulated lncRNAs are analysed in a large cohort of breast cancers (n=2000) and found to be associated with poor clinical outcome and clinicopathological features. Our findings extend knowledge of the hypoxic transcriptional response into the spectrum of non-coding transcripts. These HIF-regulated non-coding transcripts have the potential to act as biomarkers for breast cancer as well as potential novel therapeutic targets.