The OGCleaner: filtering false-positive homology clusters.

Detecting homologous sequences in organisms is an essential step in protein structure and function prediction, gene annotation and phylogenetic tree construction. Heuristic methods are often employed for quality control of putative homology clusters. These heuristics, however, usually only apply to pairwise sequence comparison and do not examine clusters as a whole. We present the Orthology Group Cleaner (the OGCleaner), a tool designed for filtering putative orthology groups as homology or non-homology clusters by considering all sequences in a cluster. The OGCleaner relies on high-quality orthologous groups identified in OrthoDB to train machine learning algorithms that are able to distinguish between true-positive and false-positive homology groups. This package aims to improve the quality of phylogenetic tree construction especially in instances of lower-quality transcriptome assemblies. AVAILABILITY AND IMPLEMENTATION: https://github.com/byucsl/ogcleaner CONTACT: sfujimoto@gmail.comSupplementary information: Supplementary data are available at Bioinformatics online.

Opsins have evolved under the permanent heterozygote model: insights from phylotranscriptomics of Odonata.

Gene duplication plays a central role in adaptation to novel environments by providing new genetic material for functional divergence and evolution of biological complexity. Several evolutionary models have been proposed for gene duplication to explain how new gene copies are preserved by natural selection, but these models have rarely been tested using empirical data. Opsin proteins, when combined with a chromophore, form a photopigment that is responsible for the absorption of light, the first step in the phototransduction cascade. Adaptive gene duplications have occurred many times within the animal opsins' gene family, leading to novel wavelength sensitivities. Consequently, opsins are an attractive choice for the study of gene duplication evolutionary models. Odonata (dragonflies and damselflies) have the largest opsin repertoire of any insect currently known. Additionally, there is tremendous variation in opsin copy number between species, particularly in the long-wavelength-sensitive (LWS) class. Using comprehensive phylotranscriptomic and statistical approaches, we tested various evolutionary models of gene duplication. Our results suggest that both the blue-sensitive (BS) and LWS opsin classes were subjected to strong positive selection that greatly weakens after multiple duplication events, a pattern that is consistent with the permanent heterozygote model. Due to the immense interspecific variation and duplicability potential of opsin genes among odonates, they represent a unique model system to test hypotheses regarding opsin gene duplication and diversification at the molecular level.

A novel approach for multi-SNP GWAS and its application in Alzheimer's disease.

BACKGROUND: Genome-wide association studies (GWAS) have effectively identified genetic factors for many diseases. Many diseases, including Alzheimer's disease (AD), have epistatic causes, requiring more sophisticated analyses to identify groups of variants which together affect phenotype. RESULTS: Based on the GWAS statistical model, we developed a multi-SNP GWAS analysis to identify pairs of variants whose common occurrence signaled the Alzheimer's disease phenotype. CONCLUSIONS: Despite not having sufficient data to demonstrate significance, our preliminary experimentation identified a high correlation between GRIA3 and HLA-DRB5 (an AD gene). GRIA3 has not been previously reported in association with AD, but is known to play a role in learning and memory.

Detecting false positive sequence homology: a machine learning approach.

BACKGROUND: Accurate detection of homologous relationships of biological sequences (DNA or amino acid) amongst organisms is an important and often difficult task that is essential to various evolutionary studies, ranging from building phylogenies to predicting functional gene annotations. There are many existing heuristic tools, most commonly based on bidirectional BLAST searches that are used to identify homologous genes and combine them into two fundamentally distinct classes: orthologs and paralogs. Due to only using heuristic filtering based on significance score cutoffs and having no cluster post-processing tools available, these methods can often produce multiple clusters constituting unrelated (non-homologous) sequences. Therefore sequencing data extracted from incomplete genome/transcriptome assemblies originated from low coverage sequencing or produced by de novo processes without a reference genome are susceptible to high false positive rates of homology detection. RESULTS: In this paper we develop biologically informative features that can be extracted from multiple sequence alignments of putative homologous genes (orthologs and paralogs) and further utilized in context of guided experimentation to verify false positive outcomes. We demonstrate that our machine learning method trained on both known homology clusters obtained from OrthoDB and randomly generated sequence alignments (non-homologs), successfully determines apparent false positives inferred by heuristic algorithms especially among proteomes recovered from low-coverage RNA-seq data. Almost ~42 % and ~25 % of predicted putative homologies by InParanoid and HaMStR respectively were classified as false positives on experimental data set. CONCLUSIONS: Our process increases the quality of output from other clustering algorithms by providing a novel post-processing method that is both fast and efficient at removing low quality clusters of putative homologous genes recovered by heuristic-based approaches.

ScaffoldScaffolder: solving contig orientation via bidirected to directed graph reduction.

MOTIVATION: The contig orientation problem, which we formally define as the MAX-DIR problem, has at times been addressed cursorily and at times using various heuristics. In setting forth a linear-time reduction from the MAX-CUT problem to the MAX-DIR problem, we prove the latter is NP-complete. We compare the relative performance of a novel greedy approach with several other heuristic solutions. RESULTS: Our results suggest that our greedy heuristic algorithm not only works well but also outperforms the other algorithms due to the nature of scaffold graphs. Our results also demonstrate a novel method for identifying inverted repeats and inversion variants, both of which contradict the basic single-orientation assumption. Such inversions have previously been noted as being difficult to detect and are directly involved in the genetic mechanisms of several diseases. AVAILABILITY AND IMPLEMENTATION: http://bioresearch.byu.edu/scaffoldscaffolder. CONTACT: paulmbodily@gmail.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Heterozygous genome assembly via binary classification of homologous sequence.

BACKGROUND: Genome assemblers to date have predominantly targeted haploid reference reconstruction from homozygous data. When applied to diploid genome assembly, these assemblers perform poorly, owing to the violation of assumptions during both the contigging and scaffolding phases. Effective tools to overcome these problems are in growing demand. Increasing parameter stringency during contigging is an effective solution to obtaining haplotype-specific contigs; however, effective algorithms for scaffolding such contigs are lacking. METHODS: We present a stand-alone scaffolding algorithm, ScaffoldScaffolder, designed specifically for scaffolding diploid genomes. The algorithm identifies homologous sequences as found in "bubble" structures in scaffold graphs. Machine learning classification is used to then classify sequences in partial bubbles as homologous or non-homologous sequences prior to reconstructing haplotype-specific scaffolds. We define four new metrics for assessing diploid scaffolding accuracy: contig sequencing depth, contig homogeneity, phase group homogeneity, and heterogeneity between phase groups. RESULTS: We demonstrate the viability of using bubbles to identify heterozygous homologous contigs, which we term homolotigs. We show that machine learning classification trained on these homolotig pairs can be used effectively for identifying homologous sequences elsewhere in the data with high precision (assuming error-free reads). CONCLUSION: More work is required to comparatively analyze this approach on real data with various parameters and classifiers against other diploid genome assembly methods. However, the initial results of ScaffoldScaffolder supply validity to the idea of employing machine learning in the difficult task of diploid genome assembly. Software is available at http://bioresearch.byu.edu/scaffoldscaffolder.

Effects of error-correction of heterozygous next-generation sequencing data.

BACKGROUND: Error correction is an important step in increasing the quality of next-generation sequencing data for downstream analysis and use. Polymorphic datasets are a challenge for many bioinformatic software packages that are designed for or assume homozygosity of an input dataset. This assumption ignores the true genomic composition of many organisms that are diploid or polyploid. In this survey, two different error correction packages, Quake and ECHO, are examined to see how they perform on next-generation sequence data from heterozygous genomes. RESULTS: Quake and ECHO perform well and were able to correct many errors found within the data. However, errors that occur at heterozygous positions had unique trends. Errors at these positions were sometimes corrected incorrectly, introducing errors into the dataset with the possibility of creating a chimeric read. Quake was much less likely to create chimeric reads. Quake's read trimming removed a large portion of the original data and often left reads with few heterozygous markers. ECHO resulted in more chimeric reads and introduced more errors than Quake but preserved heterozygous markers. CONCLUSIONS: These findings suggest that Quake and ECHO both have strengths and weaknesses when applied to heterozygous data. With the increased interest in haplotype specific analysis, new tools that are designed to be haplotype-aware are necessary that do not have the weaknesses of Quake and ECHO.

Probabilistic alignment leads to improved accuracy and read coverage for bisulfite sequencing data.

BACKGROUND: DNA methylation has been linked to many important biological phenomena. Researchers have recently begun to sequence bisulfite treated DNA to determine its pattern of methylation. However, sequencing reads from bisulfite-converted DNA can vary significantly from the reference genome because of incomplete bisulfite conversion, genome variation, sequencing errors, and poor quality bases. Therefore, it is often difficult to align reads to the correct locations in the reference genome. Furthermore, bisulfite sequencing experiments have the additional complexity of having to estimate the DNA methylation levels within the sample. RESULTS: Here, we present a highly accurate probabilistic algorithm, which is an extension of the Genomic Next-generation Universal MAPper to accommodate bisulfite sequencing data (GNUMAP-bs), that addresses the computational problems associated with aligning bisulfite sequencing data to a reference genome. GNUMAP-bs integrates uncertainty from read and mapping qualities to help resolve the difference between poor quality bases and the ambiguity inherent in bisulfite conversion. We tested GNUMAP-bs and other commonly-used bisulfite alignment methods using both simulated and real bisulfite reads and found that GNUMAP-bs and other dynamic programming methods were more accurate than the more heuristic methods. CONCLUSIONS: The GNUMAP-bs aligner is a highly accurate alignment approach for processing the data from bisulfite sequencing experiments. The GNUMAP-bs algorithm is freely available for download at: http://dna.cs.byu.edu/gnumap. The software runs on multiple threads and multiple processors to increase the alignment speed.

Probabilistic inference and ranking of gene regulatory pathways as a shortest-path problem.

BACKGROUND: Since the advent of microarray technology, numerous methods have been devised to infer gene regulatory relationships from gene expression data. Many approaches that infer entire regulatory networks. This produces results that are rich in information and yet so complex that they are often of limited usefulness for researchers. One alternative unit of regulatory interactions is a linear path between genes. Linear paths are more comprehensible than networks and still contain important information. Such paths can be extracted from inferred regulatory networks or inferred directly. Since criteria for inferring networks generally differs from criteria for inferring paths, indirect and direct inference of paths may achieve different results. RESULTS: This paper explores a strategy to infer linear pathways by converting the path inference problem into a shortest-path problem. The edge weights used are the negative log-transformed probabilities of directness derived from the posterior joint distributions of pairwise mutual information between gene expression levels. Directness is inferred using the data processing inequality. The method was designed with two goals. One is to achieve better accuracy in path inference than extraction of paths from inferred networks. The other is to facilitate priorization of interactions for laboratory validation. A method is proposed for achieving this by ranking paths according to the joint probability of directness of each path's edges. The algorithm is evaluated using simulated expression data and is compared to extraction of shortest paths from networks inferred by two alternative methods, ARACNe and a minimum spanning tree algorithm. CONCLUSIONS: Direct path inference appears to achieve accuracy competitive with that obtained by extracting paths from networks inferred by the other methods. Preliminary exploration of the use of joint edge probabilities to rank paths is largely inconclusive. Suggestions for a better framework for such comparisons are discussed.

Pathoscope: species identification and strain attribution with unassembled sequencing data.

Emerging next-generation sequencing technologies have revolutionized the collection of genomic data for applications in bioforensics, biosurveillance, and for use in clinical settings. However, to make the most of these new data, new methodology needs to be developed that can accommodate large volumes of genetic data in a computationally efficient manner. We present a statistical framework to analyze raw next-generation sequence reads from purified or mixed environmental or targeted infected tissue samples for rapid species identification and strain attribution against a robust database of known biological agents. Our method, Pathoscope, capitalizes on a Bayesian statistical framework that accommodates information on sequence quality, mapping quality, and provides posterior probabilities of matches to a known database of target genomes. Importantly, our approach also incorporates the possibility that multiple species can be present in the sample and considers cases when the sample species/strain is not in the reference database. Furthermore, our approach can accurately discriminate between very closely related strains of the same species with very little coverage of the genome and without the need for multiple alignment steps, extensive homology searches, or genome assembly--which are time-consuming and labor-intensive steps. We demonstrate the utility of our approach on genomic data from purified and in silico "environmental" samples from known bacterial agents impacting human health for accuracy assessment and comparison with other approaches.

Rhizobial plasmids that cause impaired symbiotic nitrogen fixation and enhanced host invasion.

The genetic rules that dictate legume-rhizobium compatibility have been investigated for decades, but the causes of incompatibility occurring at late stages of the nodulation process are not well understood. An evaluation of naturally diverse legume (genus Medicago) and rhizobium (genus Sinorhizobium) isolates has revealed numerous instances in which Sinorhizobium strains induce and occupy nodules that are only minimally beneficial to certain Medicago hosts. Using these ineffective strain-host pairs, we identified gain-of-compatibility (GOC) rhizobial variants. We show that GOC variants arise by loss of specific large accessory plasmids, which we call HR plasmids due to their effect on symbiotic host range. Transfer of HR plasmids to a symbiotically effective rhizobium strain can convert it to incompatibility, indicating that HR plasmids can act autonomously in diverse strain backgrounds. We provide evidence that HR plasmids may encode machinery for their horizontal transfer. On hosts in which HR plasmids impair N fixation, the plasmids also enhance competitiveness for nodule occupancy, showing that naturally occurring, transferrable accessory genes can convert beneficial rhizobia to a more exploitative lifestyle. This observation raises important questions about agricultural management, the ecological stability of mutualisms, and the genetic factors that distinguish beneficial symbionts from parasites.

Targeted amplicon sequencing (TAS): a scalable next-gen approach to multilocus, multitaxa phylogenetics.

Next-gen sequencing technologies have revolutionized data collection in genetic studies and advanced genome biology to novel frontiers. However, to date, next-gen technologies have been used principally for whole genome sequencing and transcriptome sequencing. Yet many questions in population genetics and systematics rely on sequencing specific genes of known function or diversity levels. Here, we describe a targeted amplicon sequencing (TAS) approach capitalizing on next-gen capacity to sequence large numbers of targeted gene regions from a large number of samples. Our TAS approach is easily scalable, simple in execution, neither time-nor labor-intensive, relatively inexpensive, and can be applied to a broad diversity of organisms and/or genes. Our TAS approach includes a bioinformatic application, BarcodeCrucher, to take raw next-gen sequence reads and perform quality control checks and convert the data into FASTA format organized by gene and sample, ready for phylogenetic analyses. We demonstrate our approach by sequencing targeted genes of known phylogenetic utility to estimate a phylogeny for the Pancrustacea. We generated data from 44 taxa using 68 different 10-bp multiplexing identifiers. The overall quality of data produced was robust and was informative for phylogeny estimation. The potential for this method to produce copious amounts of data from a single 454 plate (e.g., 325 taxa for 24 loci) significantly reduces sequencing expenses incurred from traditional Sanger sequencing. We further discuss the advantages and disadvantages of this method, while offering suggestions to enhance the approach.

Parallel Mapping Approaches for GNUMAP.

Mapping short next-generation reads to reference genomes is an important element in SNP calling and expression studies. A major limitation to large-scale whole-genome mapping is the large memory requirements for the algorithm and the long run-time necessary for accurate studies. Several parallel implementations have been performed to distribute memory on different processors and to equally share the processing requirements. These approaches are compared with respect to their memory footprint, load balancing, and accuracy. When using MPI with multi-threading, linear speedup can be achieved for up to 256 processors.

The GNUMAP algorithm: unbiased probabilistic mapping of oligonucleotides from next-generation sequencing.

MOTIVATION: The advent of next-generation sequencing technologies has increased the accuracy and quantity of sequence data, opening the door to greater opportunities in genomic research. RESULTS: In this article, we present GNUMAP (Genomic Next-generation Universal MAPper), a program capable of overcoming two major obstacles in the mapping of reads from next-generation sequencing runs. First, we have created an algorithm that probabilistically maps reads to repeat regions in the genome on a quantitative basis. Second, we have developed a probabilistic Needleman-Wunsch algorithm which utilizes _prb.txt and _int.txt files produced in the Solexa/Illumina pipeline to improve the mapping accuracy for lower quality reads and increase the amount of usable data produced in a given experiment. AVAILABILITY: The source code for the software can be downloaded from http://dna.cs.byu.edu/gnumap.

Pharmacogenomics: analysing SNPs in the CYP2D6 gene using amino acid properties.

The CYP2D6 gene is responsible for metabolising a large portion of the commonly prescribed drugs. Because of its importance, various approaches have been taken to analyse CYP2D6 and Single Nucleotide Polymorphisms (SNPs) throughout its sequence. This study introduces a novel method to analyse the effects of SNPs on encoded protein complexes by focusing on the biochemical properties of each non-synonymous substitution using the program TreeSAAP. Our results show four SNPs in CYP2D6 that exhibit radical changes in amino acid properties which may cause a lack of functionality in the CYP2D6 gene and contribute to a person's inability to metabolise specific drugs.

Phylogenies scores for exhaustive searches and parsimony scores searches.

Fundamental to Multiple Sequence Alignment (MSA) algorithms is modelling insertions and deletions (gaps). The most prevalent model is to use Gap Open Penalties (GOP) and Gap Extension Penalties (GEP). While GOP and GEP are well understood conceptually, their effects on MSA and consequently on phylogeny scores are not as well understood. We use exhaustive phylogeny searching to explore the effects of varying the GOP and GEP for three nuclear ribosomal data sets. Particular attention is given to optimal maximum likelihood and parsimony phylogeny scores for various alignments of a range of GOP and GEP and their respective distribution of phylogeny scores.

Taking the first steps towards a standard for reporting on phylogenies: Minimum Information About a Phylogenetic Analysis (MIAPA).

In the eight years since phylogenomics was introduced as the intersection of genomics and phylogenetics, the field has provided fundamental insights into gene function, genome history and organismal relationships. The utility of phylogenomics is growing with the increase in the number and diversity of taxa for which whole genome and large transcriptome sequence sets are being generated. We assert that the synergy between genomic and phylogenetic perspectives in comparative biology would be enhanced by the development and refinement of minimal reporting standards for phylogenetic analyses. Encouraged by the development of the Minimum Information About a Microarray Experiment (MIAME) standard, we propose a similar roadmap for the development of a Minimal Information About a Phylogenetic Analysis (MIAPA) standard. Key in the successful development and implementation of such a standard will be broad participation by developers of phylogenetic analysis software, phylogenetic database developers, practitioners of phylogenomics, and journal editors.