Unique Molecular Identifiers reveal a novel sequencing artefact with implications for RNA-Seq based gene expression analysis.

Attaching Unique Molecular Identifiers (UMI) to RNA molecules in the first step of sequencing library preparation establishes a distinct identity for each input molecule. This makes it possible to eliminate the effects of PCR amplification bias, which is particularly important where many PCR cycles are required, for example, in single cell studies. After PCR, molecules sharing a UMI are assumed to be derived from the same input molecule. In our single cell RNA-Seq studies of Physcomitrella patens, we discovered that reads sharing a UMI, and therefore presumed to be derived from the same mRNA molecule, frequently map to different, but closely spaced locations. This behaviour occurs in all such libraries that we have produced, and in multiple other UMI-containing RNA-Seq data sets in the public domain. This apparent paradox, that reads of identical origin map to distinct genomic coordinates may be partially explained by PCR stutter, which is often seen in low-entropy templates and those containing simple tandem repeats. In the absence of UMI this artefact is undetectable. We show that the common assumption that sequence reads having different mapping coordinates are derived from different starting molecules does not hold. Unless taken into account, this artefact is likely to result in over-estimation of certain transcript abundances, depending on the counting method employed.

A Whole Genome Assembly of the Horn Fly, Haematobia irritans, and Prediction of Genes with Roles in Metabolism and Sex Determination.

Haematobia irritans, commonly known as the horn fly, is a globally distributed blood-feeding pest of cattle that is responsible for significant economic losses to cattle producers. Chemical insecticides are the primary means for controlling this pest but problems with insecticide resistance have become common in the horn fly. To provide a foundation for identification of genomic loci for insecticide resistance and for discovery of new control technology, we report the sequencing, assembly, and annotation of the horn fly genome. The assembled genome is 1.14 Gb, comprising 76,616 scaffolds with N50 scaffold length of 23 Kb. Using RNA-Seq data, we have predicted 34,413 gene models of which 19,185 have been assigned functional annotations. Comparative genomics analysis with the Dipteran flies Musca domestica L., Drosophila melanogaster, and Lucilia cuprina, show that the horn fly is most closely related to M. domestica, sharing 8,748 orthologous clusters followed by D. melanogaster and L. cuprina, sharing 7,582 and 7,490 orthologous clusters respectively. We also identified a gene locus for the sodium channel protein in which mutations have been previously reported that confers target site resistance to the most common class of pesticides used in fly control. Additionally, we identified 276 genomic loci encoding members of metabolic enzyme gene families such as cytochrome P450s, esterases and glutathione S-transferases, and several genes orthologous to sex determination pathway genes in other Dipteran species.

Draft Genome Sequence of a Gordonia sp. Isolated from the Soil of a Red Alder Plant.

A Gordonia species was cultured from soil of a red alder (Alnus rubra) plant. Here we present the assembled and annotated genome sequence to aid investigations into the potential of this organism as a symbiont and comparative studies of the genus Gordonia.

A survey of the sorghum transcriptome using single-molecule long reads.

Alternative splicing and alternative polyadenylation (APA) of pre-mRNAs greatly contribute to transcriptome diversity, coding capacity of a genome and gene regulatory mechanisms in eukaryotes. Second-generation sequencing technologies have been extensively used to analyse transcriptomes. However, a major limitation of short-read data is that it is difficult to accurately predict full-length splice isoforms. Here we sequenced the sorghum transcriptome using Pacific Biosciences single-molecule real-time long-read isoform sequencing and developed a pipeline called TAPIS (Transcriptome Analysis Pipeline for Isoform Sequencing) to identify full-length splice isoforms and APA sites. Our analysis reveals transcriptome-wide full-length isoforms at an unprecedented scale with over 11,000 novel splice isoforms. Additionally, we uncover APA of ∼11,000 expressed genes and more than 2,100 novel genes. These results greatly enhance sorghum gene annotations and aid in studying gene regulation in this important bioenergy crop. The TAPIS pipeline will serve as a useful tool to analyse Iso-Seq data from any organism.

Impacts on Sirtuin Function and Bioavailability of the Dietary Bioactive Compound Dihydrocoumarin.

The plant secondary metabolite and common food additive dihydrocoumarin (DHC) is an inhibitor of the Sirtuin family of NAD+-dependent deacetylases. Sirtuins are key regulators of epigenetic processes that maintain silent chromatin in yeast and have been linked to gene expression, metabolism, apoptosis, tumorogenesis and age-related processes in multiple organisms, including humans. Here we report that exposure to the polyphenol DHC led to defects in several Sirtuin-regulated processes in budding yeast including the establishment and maintenance of Sir2p-dependent silencing by causing disassembly of silent chromatin, Hst1p-dependent repression of meiotic-specific genes during the mitotic cell cycle. As both transient and prolonged exposure to environmental and dietary factors have the potential to lead to heritable alterations in epigenetic states and to modulate additional Sirtuin-dependent phenotypes, we examined the bioavailability and digestive stability of DHC using an in vivo rat model and in vitro digestive simulator. Our analyses revealed that DHC was unstable during digestion and could be converted to melilotic acid (MA), which also caused epigenetic defects, albeit less efficiently. Upon ingestion, DHC was observed primarily in intestinal tissues, but did not accumulate over time and was readily cleared from the animals. MA displayed a wider tissue distribution and, in contrast to DHC, was also detected in the blood plasma, interstitial fluid, and urine, implying that the conversion of DHC to the less bioactive compound, MA, occurred efficiently in vivo.

Repertoire of virus-derived small RNAs produced by mosquito and mammalian cells in response to dengue virus infection.

RNA interference (RNAi) is the major defense of many arthropods against arthropod-borne RNA viruses (arboviruses), but the role of RNAi in vertebrate immunity to arboviruses is not clear. RNA viruses can trigger RNAi in vertebrate cells, but the vertebrate interferon response may obscure this interaction. We quantified virus-derived small RNAs (vRNAs) generated by mosquito (U4.4) cells and interferon-deficient (Vero) and interferon-competent (HuH-7) mammalian cells infected with a single isolate of mosquito-borne dengue virus. Mosquito cells produced significantly more vRNAs than mammalian cells, and mosquito cell vRNAs were derived from both the positive- and negative-sense dengue genomes whereas mammalian cell vRNAs were derived primarily from positive-sense genome. Mosquito cell vRNAs were predominantly 21 nucleotides in length whereas mammalian cell vRNAs were between 12 and 36 nucleotides with a modest peak at 24 nucleotides. Hot-spots, regions of the virus genome that generated a disproportionate number of vRNAs, overlapped among the cell lines.

Proliferating cell nuclear antigen (PCNA) is required for cell cycle-regulated silent chromatin on replicated and nonreplicated genes.

In Saccharomyces cerevisiae, silent chromatin is formed at HMR upon the passage through S phase, yet neither the initiation of DNA replication at silencers nor the passage of a replication fork through HMR is required for silencing. Paradoxically, mutations in the DNA replication processivity factor, POL30, disrupt silencing despite this lack of requirement for DNA replication in the establishment of silencing. We tested whether pol30 mutants could establish silencing at either replicated or non-replicated HMR loci during S phase and found that pol30 mutants were defective in establishing silencing at HMR regardless of its replication status. Although previous studies tie the silencing defect of pol30 mutants to the chromatin assembly factors Asf1p and CAF-1, we found pol30 mutants did not exhibit a gross defect in packaging HMR into chromatin. Rather, the pol30 mutants exhibited defects in histone modifications linked to ASF1 and CAF-1-dependent pathways, including SAS-I- and Rtt109p-dependent acetylation events at H4-K16 and H3-K9 (plus H3-K56; Miller, A., Yang, B., Foster, T., and Kirchmaier, A. L. (2008) Genetics 179, 793-809). Additional experiments using FLIM-FRET revealed that Pol30p interacted with SAS-I and Rtt109p in the nuclei of living cells. However, these interactions were disrupted in pol30 mutants with defects linked to ASF1- and CAF-1-dependent pathways. Together, these results imply that Pol30p affects epigenetic processes by influencing the composition of chromosomal histone modifications.

Inhibition of histone deacetylases antagonized FGF2 and IL-1beta effects on MMP expression in human articular chondrocytes.

Fibroblast growth factor-2 (FGF2) and interleukin-1beta (IL-1beta) stimulate the expression of matrix metalloproteinases (MMPs) in articular chondrocytes, which may contribute to cartilage degradation and development of osteoarthritis. Histone deacetylases (HDACs) have recently been implicated in the regulation of MMP gene expression. To investigate the functional involvement of HDACs in the signaling pathway of FGF2 and IL-1beta, we examined the effects of HDAC inhibition on activities of FGF2 or IL-1beta on gene expression of MMP-1, MMP-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS5), collagen type II, and aggrecan. Human articular chondrocyte cultures were treated with FGF2 or IL-1beta in the presence or absence of HDAC inhibitor (trichostatin A, TSA). Gene expression levels after treatments were assessed using quantitative real time PCR. Results showed that FGF2 and IL-1beta both increased MMP-1 and -13 expression, while IL-1beta also increased MMP-3 mRNA levels. These effects were attenuated in the presence of TSA in a dose dependent manner. In contrast to the effects on MMPs, FGF2 decreased mRNA levels of ADAMTS-5, which was not affected by HDAC inhibition. FGF2, IL-1beta, and TSA inhibited expression of aggrecan, while TSA also decreased mRNA levels of collagen type II. These findings showed that HDAC inhibition antagonized FGF2 and IL-1beta induced MMP expression. Combination of FGF2 and the HDAC inhibitor decreases both anabolic and catabolic genes, which may slow the cartilage turnover and be beneficial for maintaining cartilage integrity.