Comparing de novo assemblers for 454 transcriptome data

<p>Abstract</p> <p>Background</p> <p>Roche 454 pyrosequencing has become a method of choice for generating transcriptome data from non-model organisms. Once the tens to hundreds of thousands of short (250-450 base) reads have been produced, it is important to correctly assemble these to estimate the sequence of all the transcripts. Most transcriptome assembly projects use only one program for assembling 454 pyrosequencing reads, but there is no evidence that the programs used to date are optimal. We have carried out a systematic comparison of five assemblers (CAP3, MIRA, Newbler, SeqMan and CLC) to establish best practices for transcriptome assemblies, using a new dataset from the parasitic nematode <it>Litomosoides sigmodontis</it>.</p> <p>Results</p> <p>Although no single assembler performed best on all our criteria, Newbler 2.5 gave longer contigs, better alignments to some reference sequences, and was fast and easy to use. SeqMan assemblies performed best on the criterion of recapitulating known transcripts, and had more novel sequence than the other assemblers, but generated an excess of small, redundant contigs. The remaining assemblers all performed almost as well, with the exception of Newbler 2.3 (the version currently used by most assembly projects), which generated assemblies that had significantly lower total length. As different assemblers use different underlying algorithms to generate contigs, we also explored merging of assemblies and found that the merged datasets not only aligned better to reference sequences than individual assemblies, but were also more consistent in the number and size of contigs.</p> <p>Conclusions</p> <p>Transcriptome assemblies are smaller than genome assemblies and thus should be more computationally tractable, but are often harder because individual contigs can have highly variable read coverage. Comparing single assemblers, Newbler 2.5 performed best on our trial data set, but other assemblers were closely comparable. Combining differently optimal assemblies from different programs however gave a more credible final product, and this strategy is recommended.</p

The maternal and early embryonic transcriptome of the milkweed bug Oncopeltus fasciatus

<p>Abstract</p> <p>Background</p> <p>Most evolutionary developmental biology ("evo-devo") studies of emerging model organisms focus on small numbers of candidate genes cloned individually using degenerate PCR. However, newly available sequencing technologies such as 454 pyrosequencing have recently begun to allow for massive gene discovery in animals without sequenced genomes. Within insects, although large volumes of sequence data are available for holometabolous insects, developmental studies of basally branching hemimetabolous insects typically suffer from low rates of gene discovery.</p> <p>Results</p> <p>We used 454 pyrosequencing to sequence over 500 million bases of cDNA from the ovaries and embryos of the milkweed bug <it>Oncopeltus fasciatus</it>, which lacks a sequenced genome. This indirectly developing insect occupies an important phylogenetic position, branching basal to Diptera (including fruit flies) and Hymenoptera (including honeybees), and is an experimentally tractable model for short-germ development. 2,087,410 reads from both normalized and non-normalized cDNA assembled into 21,097 sequences (isotigs) and 112,531 singletons. The assembled sequences fell into 16,617 unique gene models, and included predictions of splicing isoforms, which we examined experimentally. Discovery of new genes plateaued after assembly of ~1.5 million reads, suggesting that we have sequenced nearly all transcripts present in the cDNA sampled. Many transcripts have been assembled at close to full length, and there is a net gain of sequence data for over half of the pre-existing <it>O. fasciatus </it>accessions for developmental genes in GenBank. We identified 10,775 unique genes, including members of all major conserved metazoan signaling pathways and genes involved in several major categories of early developmental processes. We also specifically address the effects of cDNA normalization on gene discovery in <it>de novo </it>transcriptome analyses.</p> <p>Conclusions</p> <p>Our sequencing, assembly and annotation framework provide a simple and effective way to achieve high-throughput gene discovery for organisms lacking a sequenced genome. These data will have applications to the study of the evolution of arthropod genes and genetic pathways, and to the wider evolution, development and genomics communities working with emerging model organisms.</p> <p>[The sequence data from this study have been submitted to GenBank under study accession number SRP002610 (<url>http://www.ncbi.nlm.nih.gov/sra?term=SRP002610</url>). Custom scripts generated are available at <url>http://www.extavourlab.com/protocols/index.html</url>. Seven Additional files are available.]</p

SalmoNet, an integrated network of ten Salmonella enterica strains reveals common and distinct pathways to host adaptation

Salmonella enterica is a prominent bacterial pathogen with implications on human and animal health. Salmonella serovars could be classified as gastro-intestinal or extra-intestinal. Genome-wide comparisons revealed that extra-intestinal strains are closer relatives of gastro-intestinal strains than to each other indicating a parallel evolution of this trait. Given the complexity of the differences, a systems-level comparison could reveal key mechanisms enabling extra-intestinal serovars to cause systemic infections. Accordingly, in this work, we introduce a unique resource, SalmoNet, which combines manual curation, high-throughput data and computational predictions to provide an integrated network for Salmonella at the metabolic, transcriptional regulatory and protein-protein interaction levels. SalmoNet provides the networks separately for five gastro-intestinal and five extra-intestinal strains. As a multi-layered, multi-strain database containing experimental data, SalmoNet is the first dedicated network resource for Salmonella. It comprehensively contains interactions between proteins encoded in Salmonella pathogenicity islands, as well as regulatory mechanisms of metabolic processes with the option to zoom-in and analyze the interactions at specific loci in more detail. Application of SalmoNet is not limited to strain comparisons as it also provides a Salmonella resource for biochemical network modeling, host-pathogen interaction studies, drug discovery, experimental validation of novel interactions, uncovering new pathological mechanisms from emergent properties and epidemiological studies. SalmoNet is available at http://salmonet.org

Rib biomechanical properties exhibit diagnostic potential for accurate ageing in forensic investigations

Age estimation remains one of the most challenging tasks in forensic practice when establishing a biological profile of unknown skeletonised remains. Morphological methods based on developmental markers of bones can provide accurate age estimates at a young age, but become highly unreliable for ages over 35 when all developmental markers disappear. This study explores the changes in the biomechanical properties of bone tissue and matrix, which continue to change with age even after skeletal maturity, and their potential value for age estimation. As a proof of concept we investigated the relationship of 28 variables at the macroscopic and microscopic level in rib autopsy samples from 24 individuals. Stepwise regression analysis produced a number of equations one of which with seven variables showed an R2=0.949; a mean residual error of 2.13 yrs ±0.4 (SD) and a maximum residual error value of 2.88 yrs. For forensic purposes, by using only bench top machines in tests which can be carried out within 36 hrs, a set of just 3 variables produced an equation with an R2=0.902 a mean residual error of 3.38 yrs ±2.6 (SD) and a maximum observed residual error 9.26yrs. This method outstrips all existing age-at-death methods based on ribs, thus providing a novel lab based accurate tool in the forensic investigation of human remains. The present application is optimised for fresh (uncompromised by taphonomic conditions) remains, but the potential of the principle and method is vast once the trends of the biomechanical variables are established for other environmental conditions and circumstances

Overexpression of caveolin-1 and -2 in cell lines and in human samples of inflammatory breast cancer

Inflammatory breast cancer (IBC) is the most aggressive form of locally advanced breast cancer (LABC). The IBC phenotype is characterized by an infiltrative growth pattern, increased (lymph)angiogenesis and the propensity to invade dermal lymphatics. In pancreatic cancer, interactions between caveolin-1 and RhoC GTPase, a key molecule in causing the IBC phenotype, regulate tumour cell motility and invasion. In this study we sought to investigate the role of caveolin-1 and -2 in IBC cell lines and in human IBC samples.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44235/1/10549_2005_Article_9002.pd

Opposing actions of TGFbeta1 and FGF2 on growth, differentiation and extracellular matrix accumulation in prostatic stromal cells.

TGFbeta 1 and FGF2 are autocrine growth factors in prostatic stroma and are elevated in benign prostatic hyperplasia (BPH), a disease characterized by enlargement of the stromal compartment of the prostate. TGFbeta1 has a biphasic effect on proliferation of prostatic stromal cells, inducing proliferation at low doses (< 1 ng/ml), but inhibiting growth above 1 ng/ml. This study investigated the role of TGFP 1 and FGF2 on growth factor bioavailability and extracellular matrix (ECM) accumulation synthesis in cultured prostatic stromal cells. Real-Time-PCR showed that TGFbeta1 expression is auto-inductive, whereas FGF2 is auto-repressive. FGF2 also induced TGFbeta1 secretion in the absence of increased TGFbeta1 mRNA expression. TGFbeta1 and FGF2 have opposing actions on Type 1 collagen expression, a finding confirmed by Western blotting. The bioavailability of TGFbeta1 regulated by FGF2 may represent part of a negative feedback mechanism controlling stromal growth, differentiation and ECM. Dysregulation of this pathway in favour of TGFbeta1 bioactivity may exacerbate BPH