Incorporating alignment uncertainty into Felsenstein's phylogenetic bootstrap to improve its reliability.

MOTIVATION: Most evolutionary analyses are based on pre-estimated multiple sequence alignment. Wong et al. established the existence of an uncertainty induced by multiple sequence alignment when reconstructing phylogenies. They were able to show that in many cases different aligners produce different phylogenies, with no simple objective criterion sufficient to distinguish among these alternatives. RESULTS: We demonstrate that incorporating MSA induced uncertainty into bootstrap sampling can significantly increase correlation between clade correctness and its corresponding bootstrap value. Our procedure involves concatenating several alternative multiple sequence alignments of the same sequences, produced using different commonly used aligners. We then draw bootstrap replicates while favoring columns of the more unique aligner among the concatenated aligners. We named this concatenation and bootstrapping method, Weighted Partial Super Bootstrap (wpSBOOT). We show on three simulated datasets of 16, 32 and 64 tips that our method improves the predictive power of bootstrap values. We also used as a benchmark an empirical collection of 853 one to one orthologous genes from seven yeast species and found wpSBOOT to significantly improve discrimination capacity between topologically correct and incorrect trees. Bootstrap values of wpSBOOT are comparable to similar readouts estimated using a single method. However, for reduced trees by 50 and 95% bootstrap thresholds, wpSBOOT comes out the lowest Type I error (less FP). AVAILABILITY AND IMPLEMENTATION: The automated generation of replicates has been implemented in the T-Coffee package, which is available as open source freeware available from www.tcoffee.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Generalized Bootstrap Supports for Phylogenetic Analyses of Protein Sequences Incorporating Alignment Uncertainty.

Phylogenetic reconstructions are essential in genomics data analyses and depend on accurate multiple sequence alignment (MSA) models. We show that all currently available large-scale progressive multiple alignment methods are numerically unstable when dealing with amino-acid sequences. They produce significantly different output when changing sequence input order. We used the HOMFAM protein sequences dataset to show that on datasets larger than 100 sequences, this instability affects on average 21.5% of the aligned residues. The resulting Maximum Likelihood (ML) trees estimated from these MSAs are equally unstable with over 38% of the branches being sensitive to the sequence input order. We established that about two-thirds of this uncertainty stems from the unordered nature of children nodes within the guide trees used to estimate MSAs. To quantify this uncertainty we developed unistrap, a novel approach that estimates the combined effect of alignment uncertainty and site sampling on phylogenetic tree branch supports. Compared with the regular bootstrap procedure, unistrap provides branch support estimates that take into account a larger fraction of the parameters impacting tree instability when processing datasets containing a large number of sequences.

PSI/TM-Coffee: a web server for fast and accurate multiple sequence alignments of regular and transmembrane proteins using homology extension on reduced databases.

The PSI/TM-Coffee web server performs multiple sequence alignment (MSA) of proteins by combining homology extension with a consistency based alignment approach. Homology extension is performed with Position Specific Iterative (PSI) BLAST searches against a choice of redundant and non-redundant databases. The main novelty of this server is to allow databases of reduced complexity to rapidly perform homology extension. This server also gives the possibility to use transmembrane proteins (TMPs) reference databases to allow even faster homology extension on this important category of proteins. Aside from an MSA, the server also outputs topological prediction of TMPs using the HMMTOP algorithm. Previous benchmarking of the method has shown this approach outperforms the most accurate alignment methods such as MSAProbs, Kalign, PROMALS, MAFFT, ProbCons and PRALINE™. The web server is available at http://tcoffee.crg.cat/tmcoffee.

Rfam 12.0: updates to the RNA families database.

The Rfam database (available at http://rfam.xfam.org) is a collection of non-coding RNA families represented by manually curated sequence alignments, consensus secondary structures and annotation gathered from corresponding Wikipedia, taxonomy and ontology resources. In this article, we detail updates and improvements to the Rfam data and website for the Rfam 12.0 release. We describe the upgrade of our search pipeline to use Infernal 1.1 and demonstrate its improved homology detection ability by comparison with the previous version. The new pipeline is easier for users to apply to their own data sets, and we illustrate its ability to annotate RNAs in genomic and metagenomic data sets of various sizes. Rfam has been expanded to include 260 new families, including the well-studied large subunit ribosomal RNA family, and for the first time includes information on short sequence- and structure-based RNA motifs present within families.

HCVIVdb: The hepatitis-C IRES variation database.

BACKGROUND: Sequence variability in the hepatitis C virus (HCV) genome has led to the development and classification of six genotypes and a number of subtypes. The HCV 5' untranslated region mainly comprises an internal ribosomal entry site (IRES) responsible for cap-independent synthesis of the viral polyprotein and is conserved among all HCV genotypes. DESCRIPTION: Considering the possible high impact of variations in HCV IRES on viral protein production and thus virus replication, we decided to collect the available data on known nucleotide variants in the HCV IRES and their impact on IRES function in translation initiation. The HCV IRES variation database (HCVIVdb) is a collection of naturally occurring and engineered mutation entries for the HCV IRES. Each entry contains contextual information pertaining to the entry such as the HCV genotypic background and links to the original publication. Where available, quantitative data on the IRES efficiency in translation have been collated along with details on the reporter system used to generate the data. Data are displayed both in a tabular and graphical formats and allow direct comparison of results from different experiments. Together the data provide a central resource for researchers in the IRES and hepatitis C-oriented fields. CONCLUSION: The collation of over 1900 mutations enables systematic analysis of the HCV IRES. The database is mainly dedicated to detailed comparative and functional analysis of all the HCV IRES domains, which can further lead to the development of site-specific drug designs and provide a guide for future experiments. HCVIVdb is available at http://www.hcvivdb.org .

[Nextflow, an efficient tool to improve computation numerical stability in genomic analysis]. / Nextflow : un outil efficace pour l'amélioration de la stabilité numérique des calculs en analyse génomique.

Reproducing routine bioinformatics analysis is challenging owing to a combination of factors hard to control for. Nextflow is a flow management framework that uses container technology to insure efficient deployment and reproducibility of computational analysis pipelines. Third party pipelines can be ported into Nextflow with minimum re-coding. We used RNA-Seq quantification, genome annotation and phylogeny reconstruction examples to show how two seemingly irreproducible analyzes can be made stable across platforms when ported into Nextflow.

Biophysical characterization of ovine forestomach extracellular matrix biomaterials.

Ovine forestomach matrix (OFM) is a native and functional decellularized extracellular matrix biomaterial that supports cell adhesion and proliferation and is remodeled during the course of tissue regeneration. Small angle X-ray scattering demonstrated that OFM retains a native collagen architecture (d spacing = 63.5 ± 0.2 nm, orientation index = 20°). The biophysical properties of OFM were further defined using ball-burst, uniaxial and suture retention testing, as well as a quantification of aqueous permeability. OFM biomaterial was relatively strong (yield stress = 10.15 ± 1.81 MPa) and elastic (modulus = 0.044 ± 0.009 GPa). Lamination was used to generate new OFM-based biomaterials with a range of biophysical properties. The resultant multi-ply OFM biomaterials had suitable biophysical characteristics for clinical applications where the grafted biomaterial is under load.

Quantification of in vitro and in vivo angiogenesis stimulated by ovine forestomach matrix biomaterial.

Ovine forestomach matrix (OFM) biomaterial acts as a biomimetic of native extracellular matrix (ECM) by providing structural and functional cues to orchestrate cell activity during tissue regeneration. The ordered collagen matrix of the biomaterial is supplemented with secondary ECM-associated macromolecules that function in cell adhesion, migration and communication. As angiogenesis and vasculogenesis are critical processes during tissue regeneration we sought to quantify the angiogenic properties of the OFM biomaterial. In vitro studies demonstrated that soluble OFM components stimulated human umbilical vein endothelial cell (HUVEC) migration and increased vascular sprouting from an aorta. Blood vessel density and branch points increased in response to OFM in an ex ovo chicken chorioallantoic membrane (CAM) assay. The OFM biomaterial was shown to undergo remodeling in a porcine full-thickness excisional model and gave rise to significantly more blood vessels than wounds treated with small intestinal submucosa decellularized ECM or untreated wounds.

A functional extracellular matrix biomaterial derived from ovine forestomach.

Extracellular matrix (ECM) based biomaterials have an established place as medical devices for wound healing and tissue regeneration. In the search for biomaterials we have identified ovine forestomach matrix (OFM), a thick, large format ECM which is biochemically diverse and biologically functional. OFM was purified using an osmotic process that was shown to reduce the cellularity of the ECM and aid tissue delamination. OFM produced using this technique was shown to retain residual basement membrane components, as evidence by the presence of laminin and collagen IV. The collagenous microarchitecture of OFM retained many components of native ECM including fibronectin, glycosaminoglycans, elastin and fibroblast growth factor basic. OFM was non-toxic to mammalian cells and supported fibroblast and keratinocyte migration, differentiation and infiltration. OFM is a culturally acceptable alternative to current collagen-based biomaterials and has immediate clinical applications in wound healing and tissue regeneration.