Matthew P Davis

Kraken: a set of tools for quality control and analysis of high-throughput sequence data.

New sequencing technologies pose significant challenges in terms of data complexity and magnitude. It is essential that efficient software is developed with performance that scales with this growth in sequence information. Here we present a comprehensive and integrated set of tools for the analysis of data from large scale sequencing experiments. It supports adapter detection and removal, demultiplexing of barcodes, paired-end data, a range of read architectures and the efficient removal of sequence redundancy. Sequences can be trimmed and filtered based on length, quality and complexity. Quality control plots track sequence length, composition and summary statistics with respect to genomic annotation. Several use cases have been integrated into a single streamlined pipeline, including both mRNA and small RNA sequencing experiments. This pipeline interfaces with existing tools for genomic mapping and differential expression analysis.

Extracellular vesicles from neural stem cells transfer IFN-γ via Ifngr1 to activate Stat1 signaling in target cells.

The idea that stem cell therapies work only via cell replacement is challenged by the observation of consistent intercellular molecule exchange between the graft and the host. Here we defined a mechanism of cellular signaling by which neural stem/precursor cells (NPCs) communicate with the microenvironment via extracellular vesicles (EVs), and we elucidated its molecular signature and function. We observed cytokine-regulated pathways that sort proteins and mRNAs into EVs. We described induction of interferon gamma (IFN-γ) pathway in NPCs exposed to proinflammatory cytokines that is mirrored in EVs. We showed that IFN-γ bound to EVs through Ifngr1 activates Stat1 in target cells. Finally, we demonstrated that endogenous Stat1 and Ifngr1 in target cells are indispensable to sustain the activation of Stat1 signaling by EV-associated IFN-γ/Ifngr1 complexes. Our study identifies a mechanism of cellular signaling regulated by EV-associated IFN-γ/Ifngr1 complexes, which grafted stem cells may use to communicate with the host immune system.

MiR-25 regulates Wwp2 and Fbxw7 and promotes reprogramming of mouse fibroblast cells to iPSCs

Background miRNAs are a class of small non-coding RNAs that regulate gene expression and have critical functions in various biological processes. Hundreds of miRNAs have been identified in mammalian genomes but only a small number of them have been functionally characterized. Recent studies also demonstrate that some miRNAs have important roles in reprogramming somatic cells to induced pluripotent stem cells (iPSCs). Methods We screened 52 miRNAs cloned in a piggybac (PB) vector for their roles in reprogramming of mouse embryonic fibroblast cells to iPSCs. To identify targets of miRNAs, we made Dgcr8-deficient embryonic stem (ES) cells and introduced miRNA mimics to these cells, which lack miRNA biogenesis. The direct target genes of miRNA were identified through global gene expression analysis and target validation. Results and conclusion We found that over-expressing miR-25 or introducing miR-25 mimics enhanced production of iPSCs. We identified a number of miR-25 candidate gene targets. Of particular interest were two ubiquitin ligases, Wwp2 and Fbxw7, which have been proposed to regulate Oct4, c-Myc and Klf5, respectively. Our findings thus highlight the complex interplay between miRNAs and transcription factors involved in reprogramming, stem cell self-renewal and maintenance of pluripotency.

Large-scale analysis of microRNA expression, epi-transcriptomic features and biogenesis.

Abstract MicroRNAs are important genetic regulators in both animals and plants. They have a range of functions spanning development, differentiation, growth, metabolism and disease. The advent of next-generation sequencing technologies has made it a relatively straightforward task to detect these molecules and their relative expression via sequencing. There are a large number of published studies with deposited datasets. However, there are currently few resources that capitalize on these data to better understand the features, distribution and biogenesis of miRNAs. Herein, we focus on Human and Mouse for which the majority of data are available. We reanalyse sequencing data from 461 samples into a coordinated catalog of microRNA expression. We use this to perform large-scale analyses of miRNA function and biogenesis. These analyses include global expression comparison, co-expression of miRNA clusters and the prediction of miRNA strand-specificity and underlying constraints. Additionally, we report for the first time a global analysis of miRNA epi-transcriptomic modifications and assess their prevalence across tissues, samples and families. Finally, we report a list of potentially mis-annotated miRNAs in miRBase based on their aggregated modification profiles. The results have been collated into a comprehensive online repository of miRNA expression and features such as modifications and RNA editing events, which is available at: http://wwwdev.ebi.ac.uk/enright-dev/miratlas. We believe these findings will further contribute to our understanding of miRNA function in animals and benefit the miRNA community in general.

Lack of correlation between predicted and actual off-target effects of short-interfering RNAs targeting the human papillomavirus type 16 E7 oncogene.

Background:When designing therapeutic short-interfering RNAs (siRNAs), off-target effects (OTEs) are usually predicted by computational quantification of messenger RNAs (mRNAs) that contain matches to the siRNA seed sequence in their 3′ UTRs. It is assumed that the higher the number of predicted transcriptional OTEs, the greater the size of the actual OTE signature and the more detrimental the phenotypic consequences in target-negative cells.Methods:We tested this general assumption by investigating the OTEs of potential therapeutic siRNAs targeting the human papillomavirus (HPV) type-16 E7 oncogene. We studied HPV-negative squamous epithelial cells, from normal cervix (NCx) and skin (HaCaT), which would be vulnerable to ‘bystander’ OTEs following transfection in vivo.Results:We observed no correlation between the number of computationally predicted OTEs and the actual number of seed-dependent OTEs (P=0.76). On average only 20.5% of actual transcriptional OTEs were seed-dependent (i.e., predicted). The unpredicted OTEs included stimulation of innate immune pathways, as well as indirect (downstream) effects of other OTEs, which affected important cancer-associated pathways. Although most significant OTEs observed were seen in both NCx and HaCaT cells, only 0–5.9% of differentially expressed genes overlapped between the two cell types.Conclusion:These data do not support the assumption that actual OTEs correlate well with predicted OTEs.

A high-resolution mRNA expression time course of embryonic development in zebrafish

We have produced an mRNA expression time course of zebrafish development across 18 time points from 1 cell to 5 days post-fertilisation sampling individual and pools of embryos. Using poly(A) pulldown stranded RNA-seq and a 3′ end transcript counting method we characterise temporal expression profiles of 23,642 genes. We identify temporal and functional transcript co-variance that associates 5024 unnamed genes with distinct developmental time points. Specifically, a class of over 100 previously uncharacterised zinc finger domain containing genes, located on the long arm of chromosome 4, is expressed in a sharp peak during zygotic genome activation. In addition, the data reveal new genes and transcripts, differential use of exons and previously unidentified 3′ ends across development, new primary microRNAs and temporal divergence of gene paralogues generated in the teleost genome duplication. To make this dataset a useful baseline reference, the data can be browsed and downloaded at Expression Atlas and Ensembl.

Large-Scale Identification of MicroRNA Targets in Murine Dgcr8-Deficient Embryonic Stem Cell Lines

Small RNAs such as microRNAs play important roles in embryonic stem cell maintenance and differentiation. A broad range of microRNAs is expressed in embryonic stem cells while only a fraction of their targets have been identified. We have performed large-scale identification of embryonic stem cell microRNA targets using a murine embryonic stem cell line deficient in the expression of Dgcr8. These cells are heavily depleted for microRNAs, allowing us to reintroduce specific microRNA duplexes and identify refined target sets. We used deep sequencing of small RNAs, mRNA expression profiling and bioinformatics analysis of microRNA seed matches in 3′ UTRs to identify target transcripts. Consequently, we have identified a network of microRNAs that converge on the regulation of several important cellular pathways. Additionally, our experiments have revealed a novel candidate for Dgcr8-independent microRNA genesis and highlighted the challenges currently facing miRNA annotation.

Transposon-driven transcription is a conserved feature of vertebrate spermatogenesis and transcript evolution

Spermatogenesis is associated with major and unique changes to chromosomes and chromatin. Here, we sought to understand the impact of these changes on spermatogenic transcriptomes. We show that long terminal repeats (LTRs) of specific mouse endogenous retroviruses (ERVs) drive the expression of many long non‐coding transcripts (lncRNA). This process occurs post‐mitotically predominantly in spermatocytes and round spermatids. We demonstrate that this transposon‐driven lncRNA expression is a conserved feature of vertebrate spermatogenesis. We propose that transposon promoters are a mechanism by which the genome can explore novel transcriptional substrates, increasing evolutionary plasticity and allowing for the genesis of novel coding and non‐coding genes. Accordingly, we show that a small fraction of these novel ERV‐driven transcripts encode short open reading frames that produce detectable peptides. Finally, we find that distinct ERV elements from the same subfamilies act as differentially activated promoters in a tissue‐specific context. In summary, we demonstrate that LTRs can act as tissue‐specific promoters and contribute to post‐mitotic spermatogenic transcriptome diversity.

Mirnovo: genome-free prediction of microRNAs from small RNA sequencing data and single-cells using decision forests.

Abstract The discovery of microRNAs (miRNAs) remains an important problem, particularly given the growth of high-throughput sequencing, cell sorting and single cell biology. While a large number of miRNAs have already been annotated, there may well be large numbers of miRNAs that are expressed in very particular cell types and remain elusive. Sequencing allows us to quickly and accurately identify the expression of known miRNAs from small RNA-Seq data. The biogenesis of miRNAs leads to very specific characteristics observed in their sequences. In brief, miRNAs usually have a well-defined 5′ end and a more flexible 3′ end with the possibility of 3′ tailing events, such as uridylation. Previous approaches to the prediction of novel miRNAs usually involve the analysis of structural features of miRNA precursor hairpin sequences obtained from genome sequence. We surmised that it may be possible to identify miRNAs by using these biogenesis features observed directly from sequenced reads, solely or in addition to structural analysis from genome data. To this end, we have developed mirnovo, a machine learning based algorithm, which is able to identify known and novel miRNAs in animals and plants directly from small RNA-Seq data, with or without a reference genome. This method performs comparably to existing tools, however is simpler to use with reduced run time. Its performance and accuracy has been tested on multiple datasets, including species with poorly assembled genomes, RNaseIII (Drosha and/or Dicer) deficient samples and single cells (at both embryonic and adult stage).

Extracellular vesicles from neural stem cells transfer the IFN-γ/IFNGR1 complex to activate Stat1-dependent signalling in target cells

important tool because of their multipotent capacity. The cannabinoid system is a complex network of ligands, receptors and enzymes. There is robust evidence of the wide spectrum of actions that cannabinoids exert on mammalian cells; their receptors regulate numerous physiological processes. Information on their role in BM-MSCs is very limited. The aim of the study was to evaluate the expression of cannabinoid receptors in BM-MSCs, and to analyze the effects of cannabinoids WIN 55,212-2 (WIN) and cannabidiol (CBD) on immunomodulatory and neuroprotective properties of these cells. BM-MSCs were isolated from healthy donors. CB1 and CB2 receptor proteins were determined by confocal microscopy and flow cytometry. Cannabinoid toxicity on BM-MSCs was assessed by MTT. Non-toxic concentrations of cannabinoids were added to the cells to assess their ability for adipogenic, osteogenic and chondrogenic transformation, colony forming unit (CFU) potential, antiproliferative effect on T cells, as well as their capacity to modulate cytokines and neurotrophic factors. Differentiation was analyzed by confocal microscopy; CFU and proliferation were determined by crystal violet count and MTT assays, respectively; neurotrophic factors and cytokine production were measured by ELISA. Our study showed that both CB1 and CB2 receptors had a very low expression in BM-MSCs surface. However, the expression of both proteins was greatly increased when their intracellular distribution was analyzed. Besides, the incubation of BM-MSC in the presence of cannabinoids showed an augmented osteogenic differentiation with CBD, whereas WIN had no effect. CB1 and CB2 receptors are membrane-associated G-protein coupled receptors. It has been assumed that their presence in BM-MSCs is either low or absent; here we show for the first time that they can be detected when cells undergo membrane permeabilization. WIN is a cannabinoid agonist acting mostly via CB2, and CBD is an antagonist of the orphan receptor GPR55, independent of CB1 and CB2. In bone, cells express CB1, CB2, GPR55 and TRPV1, indicating a deep involvement of the cannabinoid system in bone metabolism. Recently, it has been shown that CBD mediates its bone remodeling action via GPR55. Our results showing increased osteogenic transformation of BM-MSCs with CBD but not with WIN, suggest that this effect may be mediated through the GPR55 receptor.