Ultrafast and memory-efficient alignment of short DNA sequences to the human genome

Bowtie: a new ultrafast memory-efficient tool for the alignment of short DNA sequence reads to large genomes

1012-101 Vascular Smooth Muscle-Directed Adenovlral Vectors

Gene transfer to the vascular wall utilizing locally-delivered recombinant adenoviral vectors has shown promise as a novel technique for therapeutic as well as experimental modulation of vascular wall gene expression. Infusion of such vectors using porous balloon catheters (PBC) has previously been demonstrated to result in transduction of extravascular cells at the delivery site, as well as substantial systemic transduction as a consequence of release of vector into the circulation. Introduction of a vascular-directed promoter into the adenoviral vector should thus contribute to targeting the expression of genes to the vascular wall, while reducing peri-vascular and systemic expression. In order to test the feasibility of utilizing the vascular smooth muscle α-actin (SMA) promoter to confer tissue specificity upon a recombinant adenoviral vector, we constructed an adenovirus (AvLacZ5) employing a 1.1 kilobase region of the murine SMA promoter to direct the expression of the nuclear-targeted beta-galactosidase (lacZ) gene and evaluated gene transduction by this vector, in comparison with a vector differing only by the presence of the RSV-LTR promoter. Several cell types were used as targets, including bovine aortic smooth muscle cells (BASMC). human pulmonary epithelial carcinoma cells (A549 cells), and transformed human embryonic kidney epithelial cells which are competent to replicate these adenoviral vectors (293 cells). The vector incorporating the SMA promoter demonstrated substantial selectivity for vascular smooth muscle gene expression, with typical transductions carried out in parallel under identical conditions manifesting 90–95% lacZ-expressing BASMC, 0.3% lacZ-positive A549 cells, and 4% positive 293 cells. Conversely, parallel transductions with the vector employing the RSV promoter typically resulted in 95–99% lac-expressing 293 cells at vector concentrations yielding only 5–10% positive BASMC. These data support cell lineage-specificity of AvLacZ5 at the level of promoter function rather than due to intrinsic cellular differences in capacity for adenovirally-mediated transduction. However, it is notable that a limited subpopulation of 293 cells clearly are able to direct sufficient transcription from the SMA promoter sequences chosen to yield detectable lacZ expression; the molecular basis for this heterogeneity of expression remains to be determined. Adenoviral vectors utilizing these promoter sequences may render vascular-restricted gene transfer feasible when used in conjunction with mechanical devices providing a component of spatial localization

Improving RNA-Seq expression estimates by correcting for fragment bias

The biochemistry of RNA-Seq library preparation results in cDNA fragments that are not uniformly distributed within the transcripts they represent. This non-uniformity must be accounted for when estimating expression levels, and we show how to perform the needed corrections using a likelihood based approach. We find improvements in expression estimates as measured by correlation with independently performed qRT-PCR and show that correction of bias leads to improved replicability of results across libraries and sequencing technologies

TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions

TopHat is a popular spliced aligner for RNA-sequence (RNA-seq) experiments. In this paper, we describe TopHat2, which incorporates many significant enhancements to TopHat. TopHat2 can align reads of various lengths produced by the latest sequencing technologies, while allowing for variable-length indels with respect to the reference genome. In addition to de novo spliced alignment, TopHat2 can align reads across fusion breaks, which can occur after genomic translocations. TopHat2 combines the ability to identify novel splice sites with direct mapping to known transcripts, producing sensitive and accurate alignments, even for highly repetitive genomes or in the presence of pseudogenes. TopHat2 is available at http://ccb.jhu.edu/software/tophat

Thyroid hormone regulates distinct paths to maturation in pigment cell lineages

Thyroid hormone (TH) regulates diverse developmental events and can drive disparate cellular outcomes. In zebrafish, TH has opposite effects on neural crest derived pigment cells of the adult stripe pattern, limiting melanophore population expansion, yet increasing yellow/orange xanthophore numbers. To learn how TH elicits seemingly opposite responses in cells having a common embryological origin, we analyzed individual transcriptomes from thousands of neural crest-derived cells, reconstructed developmental trajectories, identified pigment cell-lineage specific responses to TH, and assessed roles for TH receptors. We show that TH promotes maturation of both cell types but in distinct ways. In melanophores, TH drives terminal differentiation, limiting final cell numbers. In xanthophores, TH promotes accumulation of orange carotenoids, making the cells visible. TH receptors act primarily to repress these programs when TH is limiting. Our findings show how a single endocrine factor integrates very different cellular activities during the generation of adult form

RNase-mediated protein footprint sequencing reveals protein-binding sites throughout the human transcriptome

Although numerous approaches have been developed to map RNA-binding sites of individual RNA-binding proteins (RBPs), few methods exist that allow assessment of global RBP–RNA interactions. Here, we describe PIP-seq, a universal, high-throughput, ribonuclease-mediated protein footprint sequencing approach that reveals RNA-protein interaction sites throughout a transcriptome of interest. We apply PIP-seq to the HeLa transcriptome and compare binding sites found using different cross-linkers and ribonucleases. From this analysis, we identify numerous putative RBP-binding motifs, reveal novel insights into co-binding by RBPs, and uncover a significant enrichment for disease-associated polymorphisms within RBP interaction sites

Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks

Recent advances in high-throughput cDNA sequencing (RNA-seq) can reveal new genes and splice variants and quantify expression genome-wide in a single assay. The volume and complexity of data from RNA-seq experiments necessitate scalable, fast and mathematically principled analysis software. TopHat and Cufflinks are free, open-source software tools for gene discovery and comprehensive expression analysis of high-throughput mRNA sequencing (RNA-seq) data. Together, they allow biologists to identify new genes and new splice variants of known ones, as well as compare gene and transcript expression under two or more conditions. This protocol describes in detail how to use TopHat and Cufflinks to perform such analyses. It also covers several accessory tools and utilities that aid in managing data, including CummeRbund, a tool for visualizing RNA-seq analysis results. Although the procedure assumes basic informatics skills, these tools assume little to no background with RNA-seq analysis and are meant for novices and experts alike. The protocol begins with raw sequencing reads and produces a transcriptome assembly, lists of differentially expressed and regulated genes and transcripts, and publication-quality visualizations of analysis results. The protocol's execution time depends on the volume of transcriptome sequencing data and available computing resources but takes less than 1 d of computer time for typical experiments and ~1 h of hands-on time

Modulation of NF-κB-dependent gene transcription using programmable DNA minor groove binders

Nuclear factor κB (NF-κB) is a transcription factor that regulates various aspects of immune response, cell death, and differentiation as well as cancer. In this study we introduce the Py-Im polyamide 1 that binds preferentially to the sequences 5′-WGGWWW-3′ and 5′GGGWWW-3′. The compound is capable of binding to κB sites and reducing the expression of various NF-κB–driven genes including IL6 and IL8 by qRT-PCR. Chromatin immunoprecipitation experiments demonstrate a reduction of p65 occupancy within the proximal promoters of those genes. Genome-wide expression analysis by RNA-seq compares the DNA-binding polyamide with the well-characterized NF-κB inhibitor PS1145, identifies overlaps and differences in affected gene groups, and shows that both affect comparable numbers of TNF-α–inducible genes. Inhibition of NF-κB DNA binding via direct displacement of the transcription factor is a potential alternative to the existing antagonists

RNA editing signature during myeloid leukemia cell differentiation

Adenosine deaminases acting on RNA (ADARs) are key proteins for hematopoietic stem cell self-renewal and for survival of differentiating progenitor cells. However, their specific role in myeloid cell maturation has been poorly investigated. Here we show that ADAR1 is present at basal level in the primary myeloid leukemia cells obtained from patients at diagnosis as well as in myeloid U-937 and THP1 cell lines and its expression correlates with the editing levels. Upon phorbol-myristate acetate or Vitamin D3/granulocyte macrophage colony-stimulating factor (GM-CSF)-driven differentiation, both ADAR1 and ADAR2 enzymes are upregulated, with a concomitant global increase of A-to-I RNA editing. ADAR1 silencing caused an editing decrease at specific ADAR1 target genes, without, however, interfering with cell differentiation or with ADAR2 activity. Remarkably, ADAR2 is absent in the undifferentiated cell stage, due to its elimination through the ubiquitin–proteasome pathway, being strongly upregulated at the end of the differentiation process. Of note, peripheral blood monocytes display editing events at the selected targets similar to those found in differentiated cell lines. Taken together, the data indicate that ADAR enzymes play important and distinct roles in myeloid cells

IDBA-tran: a more robust de novo de Bruijn graph assembler for transcriptomes with uneven expression levels

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