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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2014

Formalization of Genome Interval Relations

In order to take full advantage of next generation genomics data, I need informatics methods to be based on agreed upon formally specified standards that can be implemented easily in a uniform fashion without ambiguity. These standards should be encoded as logical formulae, so that provably correct and efficient decision procedures can be used for query answering and validation. In this paper I present the core of such a standard for sequence data: a collection of definitions of relations that hold between genomic intervals, and an alegbra for performing operations upon these intervals. I show how these relations can be used to extend formalize concepts in the Sequence Ontology (SO).

Bioinformatics↗

bioRxiv2014

Reconstructing subclonal composition and evolution from whole genome sequencing of tumors

Tumors often contain multiple, genetically distinct subpopulations of cancerous cells. These so-called subclonal populations are defined by distinct somatic mutations that include point mutations such as single nucleotide variants and small indels - collectively called simple somatic mutations (SSMs) - as well as larger structural changes that result in copy number variations (CNVs). In some cases, the genotype and prevalence of these subpopulations can be reconstructed based on high-throughput, short-read sequencing of DNA in one or more tumor samples. To date, no automated SSM-based subclonal reconstructions have been attempted on WGS data; and CNV-based reconstructions are limited to tumors with two or fewer cancerous subclonal populations and with a small number of CNVs.\n\nWe describe a new automated method, PhyloWGS, that can be applied to WGS data from one or more tumor samples to perform subclonal reconstruction based on both CNVs and SSMs. PhyloWGS successfully recovers the composition of mixtures of a highly rearranged TGCA cell line when a CNV-based method fails. On WGS data with average read depth of 40 from five time-series chronic lymphocytic leukemia samples, PhyloWGS recovers the same tumor phylogeny previously reconstructed using deep targeted resequencing. To further explore the limits of WGS-based subclonal reconstruction, we ran PhyloWGS on simulated data: PhyloWGS can reliably reconstruct as many as three cancerous subpopulations based on 30-50x coverage WGS data from a single tumor sample with 10s to 1000s of SSMs per subpopulation. At least five cancerous subpopulations can be reconstructed if provided with read depths of 200 or more.\n\nPhyloWGS is the first automated method that can be applied to WGS tumor data that accurately reconstructs the frequency, genotype and phylogeny of the subclonal populations based on both SSMs and CNVs. It also provides a principled, automated approach to combining overlapping SSM and CNV data. By demonstrating the utility of PhyloWGS on medium depth WGS data, including from examples with highly rearranged chromosomes, we have greatly expanded the range of tumors for which subclonal reconstruction is possible.

Bioinformatics↗

bioRxiv2014

Recurrent alternative splicing isoform switches in tumor samples provide novel signatures of cancer

Cancer genomics has been instrumental to determine the genetic alterations that are predictive of various tumor conditions. However, the majority of these alterations occur at low frequencies, motivating the need to expand the catalogue of cancer signatures. Alternative pre-mRNA splicing alterations, which bear major importance for the understanding of cancer, have not been exhaustively studied yet in the context of recent cancer genome projects. In this article we analyze RNA sequencing data for more than 4000 samples from The Cancer Genome Atlas (TCGA) project, including paired normal samples, to detect recurrent alternative splicing isoform switches in 9 different cancer types. We first investigate whether alternative splicing isoform changes are predictive of tumors by applying a rank-based algorithm based on the reversal of the relative expression of transcript isoforms. We find that consistent alternative splicing isoform changes can separate with high accuracy tumor and normal samples, as well as some cancer subtypes. We then searched for those changes that occur in the most abundant isoform, i.e isoform switches, and are therefore more likely to have a functional impact. In total we detected 244 isoform switches, which are associated to functional pathways that are frequently altered in cancer and also separate tumor and normal samples accurately. We further assessed whether these isoform changes are associated to somatic mutations. Surprisingly, only a few cases appear to have association, including the putative tumor suppressor FBLN2 and the tumor driver MYH11, which show association of an isoform switch to mutations and indels on the alternatively spliced exon. However, the number of observed mutations is in general not sufficient to explain the frequency of the found isoform switches, suggesting that recurrent isoform switching in cancer is mostly independent of somatic mutations. In summary, we present an effective approach to detect novel alternative splicing signatures that are predictive of tumors. Moreover, the same methodology has led to uncover recurrent isoform switches in tumors, which may provide novel prognostic and therapeutic targets.\n\nSoftware and data are available at: https://bitbucket.org/regulatorygenomicsupf/iso-ktsp and http://dx.doi.org/10.6084/m9.figshare.1061917

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bioRxiv2014

Benchmark Analysis of Algorithms for Determining and Quantifying Full-length mRNA Splice Forms from RNA-Seq Data

The advantages of RNA sequencing (RNA-Seq) suggest it will replace microarrays for highly parallel gene expression analysis. For example, in contrast to arrays, RNA-Seq is expected to be able to provide accurate identification and quantification of full-length transcripts. A number of methods have been developed for this purpose, but short error prone reads makes it a difficult problem in practice. It is essential to determine which algorithms perform best, and where and why they fail. However, there is a dearth of independent and unbiased benchmarking studies of these algorithms. Here we take an approach using both simulated and experimental benchmark data to evaluate their accuracy. We conclude that most methods are inaccurate even using idealized data, and that no is method sufficiently accurate once complicating factors such as polymorphisms, intron signal, sequencing error, and multiple splice forms are present. These results point to the pressing need for further algorithm development.

Bioinformatics↗

bioRxiv2014

MINI REVIEW: Statistical methods for detecting differentially methylated loci and regions

DNA methylation, the reversible addition of methyl groups at CpG dinucleotides, represents an important regulatory layer associated with gene expression. Changed methylation status has been noted across diverse pathological states, including cancer. The rapid development and uptake of microarrays and large scale DNA sequencing has prompted an explosion of data analytic methods for processing and discovering changes in DNA methylation across varied data types. In this mini-review, we present a compact and accessible discussion of many of the salient challenges, such as experimental design, statistical methods for differential methylation detection, critical considerations such as cell type composition and the potential confounding that can arise from batch effects. From a statistical perspective, our main interests include the use of empirical Bayes or hierarchical models, which have proved immensely powerful in genomics, and the procedures by which false discovery control is achieved.

Bioinformatics↗

bioRxiv2014

Assessing allele specific expression across multiple tissues from RNA-seq read data

Motivation: RNA sequencing enables allele specific expression (ASE) studies that complement standard genotype expression studies for common variants and, importantly, also allow measuring the regulatory impact of rare variants. The Genotype-Tissue Expression project (GTEx) is collecting RNA-seq data on multiple tissues of a same set of individuals and novel methods are required for the analysis of these data. Results: We present a statistical method to compare different patterns of ASE across tissues and to classify genetic variants according to their impact on the tissue-wide expression profile. We focus on strong ASE effects that we are expecting to see for protein-truncating variants, but our method can also be adjusted for other types of ASE effects. We illustrate the method with a real data example on a tissue-wide expression profile of a variant causal for lipoid proteinosis, and with a simulation study to assess our method more generally. Availability: MAMBA software: http://birch.well.ox.ac.uk/~rivas/mamba/ R source code and data examples: http://www.iki.fi/mpirinen/ Contact: matti.pirinen@helsinki.fi rivas@well.ox.ac.uk

Bioinformatics↗

bioRxiv2014

Conservation of expression regulation throughout the animal kingdom

Following the increase in available sequenced genomes, tissue-specific transcriptomes are being determined for a rapidly growing number of highly diverse species. Traditionally, only the transcriptomes of related species with equivalent tissues have been compared. Such an analysis is much more challenging over larger evolutionary distances when complementary tissues cannot readily be defined. Here, we present a method for the cross-species mapping of tissue-specific and developmental gene expression patterns across a wide range of animals, including many non-model species. Our approach maps gene expression patterns between species without requiring the definition of homologous tissues. With the help of this mapping, gene expression patterns can be compared even across distantly related species. In our survey of 36 datasets across 27 species, we detected conserved expression programs on all taxonomic levels, both within animals and between the animals and their closest unicellular relatives, the choanoflagellates. We found that the rate of change in tissue expression patterns is a property of gene families. Our findings open new avenues of study for the comparison and transfer of knowledge between different species.

Bioinformatics↗

bioRxiv2014

Aligning sequence from molecular inversion probes

Summary: Molecular inversion probes (MIPs) allow efficient enrichment of genomic regions of interest for the purpose of targeted sequencing. To date, there is a paucity of simple-to-use software to align sequences derived from this method. Here, we describe a single program that performs mapping, arm removal, and deduplication before outputting alignments in SAM format. Availability: bwa-mips is available at https://github.com/brentp/bwa-mips under the MIT license. Contact: bpederse@gmail.com

Bioinformatics↗

bioRxiv2014

Rapid Core-Genome Alignment and Visualization for Thousands of Intraspecific Microbial Genomes

Though many microbial species or clades now have hundreds of sequenced genomes, existing whole-genome alignment methods do not efficiently handle comparisons on this scale. Here we present the Harvest suite of core-genome alignment and visualization tools for quickly analyzing thousands of intraspecific microbial strains. Harvest includes Parsnp, a fast core-genome multi-aligner, and Gingr, a dynamic visual platform. Combined they provide interactive core-genome alignments, variant calls, recombination detection, and phylogenetic trees. Using simulated and real data we demonstrate that our approach exhibits unrivaled speed while maintaining the accuracy of existing methods. The Harvest suite is open-source and freely available from: http://github.com/marbl/harvest.

Bioinformatics↗

bioRxiv2014

Poretools: a toolkit for analyzing nanopore sequence data

Motivation: Nanopore sequencing may be the next disruptive technology in genomics. Nanopore sequencing has many attractive properties including the ability to detect single DNA molecules without prior amplification, the lack of reliance on expensive optical components, and the ability to sequence very long fragments. The MinION from Oxford Nanopore Technologies (ONT) is the first nanopore sequencer to be commercialised and made available to early-access users. The MinION(TM) is a USB-connected, portable nanopore sequencer which permits real-time analysis of streaming event data. A cloud-based service is available to translate events into nucleotide base calls. However, software support to deal with such data is limited, and the community lacks a standardized toolkit for the analysis of nanopore datasets. Results: We introduce poretools, a flexible toolkit for manipulating and exploring datasets generated by nanopore sequencing devices from MinION for the purposes of quality control and downstream analysis. Poretools operates directly on the native FAST5 (a variant of the HDF5 standard) file format produced by ONT and provides a wealth of format conversion utilities and data exploration and visualization tools. Availability and implementation: Poretools is open source software and is written in Python as both a suite of command line utilities and a Python application programming interface. Source code and user documentation are freely available in Github at https://github.com/arq5x/poretools Contact: n.j.loman@bham.ac.uk, aaronquinlan@gmail.com Supplementary information: An IPython notebook demonstrating the use and functionality of poretools in greater detail is available from the Github repository.

Bioinformatics↗

bioRxiv2014

Butter: High-precision genomic alignment of small RNA-seq data

Eukaryotes produce large numbers of small non-coding RNAs that act as specificity determinants for various gene-regulatory complexes. These include microRNAs (miRNAs), endogenous short interfering RNAs (siRNAs), and Piwi-associated RNAs (piRNAs). These RNAs can be discovered, annotated, and quantified using small RNA-seq, a variant RNA-seq method based on highly parallel sequencing. Alignment to a reference genome is a critical step in analysis of small RNA-seq data. Because of their small size (20-30 nts depending on the organism and sub-type) and tendency to originate from multi-gene families or repetitive regions, reads that align equally well to more than one genomic location are very common. Typical methods to deal with multi-mapped small RNA-seq reads sacrifice either precision or sensitivity. The tool butter balances precision and sensitivity by placing multi-mapped reads using an iterative approach, where the decision between possible locations is dictated by the local densities of more confidently aligned reads. Butter displays superior performance relative to other small RNA-seq aligners. Treatment of multi-mapped small RNA-seq reads has substantial impacts on downstream analyses, including quantification of MIRNA paralogs, and discovery of endogenous siRNA loci. Butter is freely available under a GNU general public license.

Bioinformatics↗

bioRxiv2014

Extraction of relations between genes and diseases from text and large-scale data analysis: implications for translational research

Background Current biomedical research needs to leverage and exploit the large amount of information reported in publications. Automated text mining approaches, in particular those aimed at finding relationships between entities, are key for identification of actionable knowledge from free text repositories. We present the BeFree system aimed at identifying relationships between biomedical entities with a special focus on genes and their associated diseases. Results By exploiting morpho-syntactic information of the text BeFree is able to identify gene-disease, drug-disease and drug-target associations with state-of-the-art performance. The application of BeFree to real-case scenarios shows its effectiveness in extracting information relevant for translational research. We show the value of the gene-disease associations extracted by BeFree through a number of analyses and integration with other data sources. BeFree succeeds in identifying genes associated to a major cause of morbidity worldwide, depression, which are not present in other public resources. Moreover, large-scale extraction and analysis of gene-disease associations, and integration with current biomedical knowledge, provided interesting insights on the kind of information that can be found in the literature, and raised challenges regarding data prioritization and curation. We found that only a small proportion of the gene-disease associations discovered by using BeFree is collected in expert-curated databases. Thus, there is a pressing need to find alternative strategies to manual curation to review, prioritize and curate text-mining data and incorporate it into domain-specific databases. We present our strategy for data prioritization and discuss its implications for supporting biomedical research and applications. Conclusions BeFree is a novel text mining system that performs competitively for the identification of gene-disease, drug-disease and drug-target associations. Our analyses show that mining only a small fraction of MEDLINE results in a large dataset of gene-disease associations, and only a small proportion of this dataset is actually recorded in curated resources, raising several issues on data prioritization and curation. We propose that joint analysis of text mined data with data curated by experts appears as a suitable approach to both assess data quality and highlight novel and interesting information.

Bioinformatics↗

bioRxiv2014

QuASAR: Quantitative Allele Specific Analysis of Reads

MotivationExpression quantitative trait loci (eQTL) studies have discovered thousands of genetic variants that regulate gene expression, enabling a better understanding of the functional role of non-coding sequences. However, eQTL studies are costly, requiring large sample sizes and genome-wide genotyping of each sample. In contrast, analysis of allele specific expression (ASE) is becoming a popular approach to detect the effect of genetic variation on gene expression, even within a single individual. This is typically achieved by counting the number of RNA-seq reads matching each allele at heterozygous sites and testing the null hypothesis of a 1:1 allelic ratio. In principle, when genotype information is not readily available it could be inferred from the RNA-seq reads directly. However, there are currently no existing methods that jointly infer genotypes and conduct ASE inference, while considering uncertainty in the genotype calls.\n\nResultsWe present QuASAR, Quantitative Allele Specific Analysis of Reads, a novel statistical learning method for jointly detecting heterozygous genotypes and inferring ASE. The proposed ASE inference step takes into consideration the uncertainty in the genotype calls while including parameters that model base-call errors in sequencing and allelic over-dispersion. We validated our method with experimental data for which high quality genotypes are available. Results for an additional dataset with multiple replicates at different sequencing depths demonstrate that QuASAR is a powerful tool for ASE analysis when genotypes are not available.\n\nAvailabilityhttp://github.com/piquelab/QuASAR\n\nContactfluca@wayne.edu; rpique@wayne.edu

Bioinformatics↗

bioRxiv2014

Classification of RNA-Seq Data via Bagging Support Vector Machines

BackgroundRNA sequencing (RNA-Seq) is a powerful technique for transcriptome profiling of the organisms that uses the capabilities of next-generation sequencing (NGS) technologies. Recent advances in NGS let to measure the expression levels of tens to thousands of transcripts simultaneously. Using such information, developing expression-based classification algorithms is an emerging powerful method for diagnosis, disease classification and monitoring at molecular level, as well as providing potential markers of disease. Here, we present the bagging support vector machines (bagSVM), a machine learning approach and bagged ensembles of support vector machines (SVM), for classification of RNA-Seq data. The bagSVM basically uses bootstrap technique and trains each single SVM separately; next it combines the results of each SVM model using majority-voting technique.\n\nResultsWe demonstrate the performance of the bagSVM on simulated and real datasets. Simulated datasets are generated from negative binomial distribution under different scenarios and real datasets are obtained from publicly available resources. A deseq normalization and variance stabilizing transformation (vst) were applied to all datasets. We compared the results with several classifiers including Poisson linear discriminant analysis (PLDA), single SVM, classification and regression trees (CART), and random forests (RF). In slightly overdispersed data, all methods, except CART algorithm, performed well. Performance of PLDA seemed to be best and RF as second best for very slightly and substantially overdispersed datasets. While data become more spread, bagSVM turned out to be the best classifier. In overall results, bagSVM and PLDA had the highest accuracies.\n\nConclusionsAccording to our results, bagSVM algorithm after vst transformation can be a good choice of classifier for RNA-Seq datasets mostly for overdispersed ones. Thus, we recommend researchers to use bagSVM algorithm for the purpose of classification of RNA-Seq data. PLDA algorithm should be a method of choice for slight and moderately overdispersed datasets. An R/BIOCONDUCTOR package MLSeq with a vignette is freely available at http://www.bioconductor.org/packages/2.14/bioc/html/MLSeq.html

Bioinformatics↗

bioRxiv2014

UniqTag: Content-derived unique and stable identifiers for gene annotation

When working on an ongoing genome sequencing and assembly project, it is rather inconvenient when gene identifiers change from one build of the assembly to the next. The gene labelling system described here, UniqTag, addresses this common challenge. UniqTag assigns a unique identifier to each gene that is a representative k -mer, a string of length k , selected from the sequence of that gene. Unlike serial numbers, these identifiers are stable between different assemblies and annotations of the same data without requiring that previous annotations be lifted over by sequence alignment. We assign UniqTag identifiers to nine builds of the Ensembl human genome spanning seven years to demonstrate this stability.

Bioinformatics↗

bioRxiv2014

A comparison of control samples for ChIP-seq of histone modifications

The advent of high-throughput sequencing has allowed genome wide profiling of histone modifications by Chromatin ImmunoPrecipitation (ChIP) followed by sequencing (ChIP-seq). In this assay the histone mark of interest is enriched through a chromatin pull-down assay using an antibody for the mark. Due to imperfect antibodies and other factors, many of the sequenced fragments do not originate from the histone mark of interest, and are referred to as background reads. Background reads are not uniformly distributed and therefore control samples are usually used to estimate the background distribution at any given genomic position. The Encyclopedia of DNA Elements (ENCODE) Consortium guidelines suggest sequencing a whole cell extract (WCE, or \"input\") sample, or a mock ChIP reaction such as an IgG control, as a background sample. However, for a histone modification ChIP-seq investigation it is also possible to use a Histone H3 (H3) pull-down to map the underlying distribution of histones.\n\nIn this paper we generated data from a hematopoietic stem and progenitor cell population isolated from mouse foetal liver to compare WCE and H3 ChIP-seq as control samples. The quality of the control samples is estimated by a comparison to pull-downs of histone modifications and to expression data. We find minor differences between WCE and H3 ChIP-seq, such as coverage in mitochondria and behaviour close to transcription start sites. Where the two controls differ, the H3 pull-down is generally more similar to the ChIP-seq of histone modifications. However, the differences between H3 and WCE have a negligible impact on the quality of a standard analysis.

Bioinformatics↗

bioRxiv2014

Faster sequence alignment through GPU-accelerated restriction of the seed-and-extend search space

MotivationIn computing pairwise alignments of biological sequences, software implementations employ a variety of heuristics that decrease the computational effort involved in computing potential alignments. A key element in achieving high processing throughput is to identify and prioritize potential alignments where high-scoring mappings can be expected. These tasks involve listprocessing operations that can be efficiently performed on GPU hardware.\n\nResultsWe implemented a read aligner called A21 that exploits GPU-based parallel sort and reduction techniques to restrict the number of locations where potential alignments may be found. When compared with other high-throughput aligners, this approach finds more high-scoring mappings without sacrificing speed or accuracy. A21 running on a single GPU is about 10 times faster than comparable CPU-based tools; it is also faster and more sensitive in comparison with other recent GPU-based aligners.\n\nAvailabilityThe A21 software is open source and available at https://github.com/RWilton/A21.\n\nContactrwilton@pha.jhu.edu\n\nSupplementary informationSupplementary results are available at << >>

Bioinformatics↗

bioRxiv2014

Gene regulatory network inference from perturbed time-series expression data via ordered dynamical expansion of non-steady state actors

The reconstruction of gene regulatory networks from gene expression data has been the subject of intense research activity over the past many decades. A variety of models and methods have been developed to address different aspects of this important problem. However, these techniques are often difficult to scale, are narrowly focused on particular biological and experimental platforms, and require experimental data that are typically unavailable and difficult to ascertain. The more recent availability of higher-throughput sequencing platforms, combined with more precise modes of genetic perturbation, presents an opportunity to formulate more robust and comprehensive approaches to gene network inference. Here, we propose a step-wise framework for identifying gene-gene regulatory interactions that expand from a known point of genetic or chemical perturbation using time series gene expression data. This novel approach sequentially identifies non-steady state genes post-perturbation and incorporates them into a growing series of low-complexity optimization problems. The governing ordinary differential equations of this model are rooted in the biophysics of stochastic molecular events that underlie gene regulation, delineating roles for both protein and RNA-mediated gene regulation. We show the successful application of our core algorithms for network inference using simulated and real datasets.

Bioinformatics↗