MOTIVATION: Genotyping by sequencing is a powerful tool for investigating genetic variation in plants, but many economically important plants are allopolyploids, where homoeologous similarity obscures the subgenomic origin of reads and confounds allelic and homoeologous SNPs. Recent polyploid genotyping methods use allelic frequencies, rate of heterozygosity, parental cross or other information to resolve read assignment, but good subgenomic references offer the most direct information. The typical strategy aligns reads to the joint reference, performs diploid genotyping within each subgenome, and filters the results, but persistent read misassignment results in an excess of false heterozygous calls. RESULTS: We introduce the Comprehensive Allopolyploid Genotyper (CAPG), which formulates an explicit likelihood to weight read alignments against both subgenomic references and genotype individual allopolyploids from whole-genome resequencing data. We demonstrate CAPG in allotetraploids, where it performs better than Genome Analysis Toolkit's HaplotypeCaller applied to reads aligned to the combined subgenomic references. AVAILABILITY AND IMPLEMENTATION: Code and tutorials are available at https://github.com/Kkulkarni1/CAPG.git. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

MOTIVATION: Allostery enables changes to the dynamic behavior of a protein at distant positions induced by binding. Here, we present APOP, a new allosteric pocket prediction method, which perturbs the pockets formed in the structure by stiffening pairwise interactions in the elastic network across the pocket, to emulate ligand binding. Ranking the pockets based on the shifts in the global mode frequencies, as well as their mean local hydrophobicities, leads to high prediction success when tested on a dataset of allosteric proteins, composed of both monomers and multimeric assemblages. RESULTS: Out of the 104 test cases, APOP predicts known allosteric pockets for 92 within the top 3 rank out of multiple pockets available in the protein. In addition, we demonstrate that APOP can also find new alternative allosteric pockets in proteins. Particularly interesting findings are the discovery of previously overlooked large pockets located in the centers of many protein biological assemblages; binding of ligands at these sites would likely be particularly effective in changing the protein's global dynamics. AVAILABILITY AND IMPLEMENTATION: APOP is freely available as an open-source code (https://github.com/Ambuj-UF/APOP) and as a web server at https://apop.bb.iastate.edu/.

MOTIVATION: Amplicon sequencing is widely applied to explore heterogeneity and rare variants in genetic populations. Resolving true biological variants and quantifying their abundance is crucial for downstream analyses, but measured abundances are distorted by stochasticity and bias in amplification, plus errors during polymerase chain reaction (PCR) and sequencing. One solution attaches unique molecular identifiers (UMIs) to sample sequences before amplification. Counting UMIs instead of sequences provides unbiased estimates of abundance. While modern methods improve over naïve counting by UMI identity, most do not account for UMI reuse or collision, and they do not adequately model PCR and sequencing errors in the UMIs and sample sequences. RESULTS: We introduce Deduplication and Abundance estimation with UMIs (DAUMI), a probabilistic framework to detect true biological amplicon sequences and accurately estimate their deduplicated abundance. DAUMI recognizes UMI collision, even on highly similar sequences, and detects and corrects most PCR and sequencing errors in the UMI and sampled sequences. DAUMI performs better on simulated and real data compared to other UMI-aware clustering methods. AVAILABILITY AND IMPLEMENTATION: Source code is available at https://github.com/DormanLab/AmpliCI. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

SUMMARY: A new dynamic community identifier (DCI) is presented that relies upon protein residue dynamic cross-correlations generated by Gaussian elastic network models to identify those residue clusters exhibiting motions within a protein. A number of examples of communities are shown for diverse proteins, including GPCRs. It is a tool that can immediately simplify and clarify the most essential functional moving parts of any given protein. Proteins usually can be subdivided into groups of residues that move as communities. These are usually densely packed local sub-structures, but in some cases can be physically distant residues identified to be within the same community. The set of these communities for each protein are the moving parts. The ways in which these are organized overall can aid in understanding many aspects of functional dynamics and allostery. DCI enables a more direct understanding of functions including enzyme activity, action across membranes and changes in the community structure from mutations or ligand binding. The DCI server is freely available on a web site (https://dci.bb.iastate.edu/). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

MOTIVATION: Next-generation amplicon sequencing is a powerful tool for investigating microbial communities. A main challenge is to distinguish true biological variants from errors caused by amplification and sequencing. In traditional analyses, such errors are eliminated by clustering reads within a sequence similarity threshold, usually 97%, and constructing operational taxonomic units, but the arbitrary threshold leads to low resolution and high false-positive rates. Recently developed 'denoising' methods have proven able to resolve single-nucleotide amplicon variants, but they still miss low-frequency sequences, especially those near more frequent sequences, because they ignore the sequencing quality information. RESULTS: We introduce AmpliCI, a reference-free, model-based method for rapidly resolving the number, abundance and identity of error-free sequences in massive Illumina amplicon datasets. AmpliCI considers the quality information and allows the data, not an arbitrary threshold or an external database, to drive conclusions. AmpliCI estimates a finite mixture model, using a greedy strategy to gradually select error-free sequences and approximately maximize the likelihood. AmpliCI has better performance than three popular denoising methods, with acceptable computation time and memory usage. AVAILABILITY AND IMPLEMENTATION: Source code is available at https://github.com/DormanLab/AmpliCI. SUPPLEMENTARY INFORMATION: Supplementary material are available at Bioinformatics online.

Protein-protein interaction networks are one of the most effective representations of cellular behavior. In order to build these models, high-throughput techniques are required. Next-generation interaction screening (NGIS) protocols that combine yeast two-hybrid (Y2H) with deep sequencing are promising approaches to generate interactome networks in any organism. However, challenges remain to mining reliable information from these screens and thus, limit its broader implementation. Here, we present a computational framework, designated Y2H-SCORES, for analyzing high-throughput Y2H screens. Y2H-SCORES considers key aspects of NGIS experimental design and important characteristics of the resulting data that distinguish it from RNA-seq expression datasets. Three quantitative ranking scores were implemented to identify interacting partners, comprising: 1) significant enrichment under selection for positive interactions, 2) degree of interaction specificity among multi-bait comparisons, and 3) selection of in-frame interactors. Using simulation and an empirical dataset, we provide a quantitative assessment to predict interacting partners under a wide range of experimental scenarios, facilitating independent confirmation by one-to-one bait-prey tests. Simulation of Y2H-NGIS enabled us to identify conditions that maximize detection of true interactors, which can be achieved with protocols such as prey library normalization, maintenance of larger culture volumes and replication of experimental treatments. Y2H-SCORES can be implemented in different yeast-based interaction screenings, with an equivalent or superior performance than existing methods. Proof-of-concept was demonstrated by discovery and validation of novel interactions between the barley nucleotide-binding leucine-rich repeat (NLR) immune receptor MLA6, and fourteen proteins, including those that function in signaling, transcriptional regulation, and intracellular trafficking.

MOTIVATION: The goal of phylostratigraphy is to infer the evolutionary origin of each gene in an organism. This is done by searching for homologs within increasingly broad clades. The deepest clade that contains a homolog of the protein(s) encoded by a gene is that gene's phylostratum. RESULTS: We have created a general R-based framework, phylostratr, to estimate the phylostratum of every gene in a species. The program fully automates analysis: selecting species for balanced representation, retrieving sequences, building databases, inferring phylostrata and returning diagnostics. Key diagnostics include: detection of genes with inferred homologs in old clades, but not intermediate ones; proteome quality assessments; false-positive diagnostics, and checks for missing organellar genomes. phylostratr allows extensive customization and systematic comparisons of the influence of analysis parameters or genomes on phylostrata inference. A user may: modify the automatically generated clade tree or use their own tree; provide custom sequences in place of those automatically retrieved from UniProt; replace BLAST with an alternative algorithm; or tailor the method and sensitivity of the homology inference classifier. We show the utility of phylostratr through case studies in Arabidopsis thaliana and Saccharomyces cerevisiae. AVAILABILITY AND IMPLEMENTATION: Source code available at https://github.com/arendsee/phylostratr. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

In this work we present a flexible, probabilistic and reference-free method of error correction for high throughput DNA sequencing data. The key is to exploit the high coverage of sequencing data and model short sequence outputs as independent realizations of a Hidden Markov Model (HMM). We pose the problem of error correction of reads as one of maximum likelihood sequence detection over this HMM. While time and memory considerations rule out an implementation of the optimal Baum-Welch algorithm (for parameter estimation) and the optimal Viterbi algorithm (for error correction), we propose low-complexity approximate versions of both. Specifically, we propose an approximate Viterbi and a sequential decoding based algorithm for the error correction. Our results show that when compared with Reptile, a state-of-the-art error correction method, our methods consistently achieve superior performances on both simulated and real data sets.

BACKGROUND: High-throughput short read sequencing is revolutionizing genomics and systems biology research by enabling cost-effective deep coverage sequencing of genomes and transcriptomes. Error detection and correction are crucial to many short read sequencing applications including de novo genome sequencing, genome resequencing, and digital gene expression analysis. Short read error detection is typically carried out by counting the observed frequencies of kmers in reads and validating those with frequencies exceeding a threshold. In case of genomes with high repeat content, an erroneous kmer may be frequently observed if it has few nucleotide differences with valid kmers with multiple occurrences in the genome. Error detection and correction were mostly applied to genomes with low repeat content and this remains a challenging problem for genomes with high repeat content. RESULTS: We develop a statistical model and a computational method for error detection and correction in the presence of genomic repeats. We propose a method to infer genomic frequencies of kmers from their observed frequencies by analyzing the misread relationships among observed kmers. We also propose a method to estimate the threshold useful for validating kmers whose estimated genomic frequency exceeds the threshold. We demonstrate that superior error detection is achieved using these methods. Furthermore, we break away from the common assumption of uniformly distributed errors within a read, and provide a framework to model position-dependent error occurrence frequencies common to many short read platforms. Lastly, we achieve better error correction in genomes with high repeat content. AVAILABILITY: The software is implemented in C++ and is freely available under GNU GPL3 license and Boost Software V1.0 license at "http://aluru-sun.ece.iastate.edu/doku.php?id = redeem". CONCLUSIONS: We introduce a statistical framework to model sequencing errors in next-generation reads, which led to promising results in detecting and correcting errors for genomes with high repeat content.

MOTIVATION: Error correction is critical to the success of next-generation sequencing applications, such as resequencing and de novo genome sequencing. It is especially important for high-throughput short-read sequencing, where reads are much shorter and more abundant, and errors more frequent than in traditional Sanger sequencing. Processing massive numbers of short reads with existing error correction methods is both compute and memory intensive, yet the results are far from satisfactory when applied to real datasets. RESULTS: We present a novel approach, termed Reptile, for error correction in short-read data from next-generation sequencing. Reptile works with the spectrum of k-mers from the input reads, and corrects errors by simultaneously examining: (i) Hamming distance-based correction possibilities for potentially erroneous k-mers; and (ii) neighboring k-mers from the same read for correct contextual information. By not needing to store input data, Reptile has the favorable property that it can handle data that does not fit in main memory. In addition to sequence data, Reptile can make use of available quality score information. Our experiments show that Reptile outperforms previous methods in the percentage of errors removed from the data and the accuracy in true base assignment. In addition, a significant reduction in run time and memory usage have been achieved compared with previous methods, making it more practical for short-read error correction when sampling larger genomes. AVAILABILITY: Reptile is implemented in C++ and is available through the link: http://aluru-sun.ece.iastate.edu/doku.php?id=software CONTACT: [email protected].

Abstract Summary: Gaussian processes (GPs) are flexible statistical models commonly used for predicting output from complex computer codes. As such, GPs are well suited for the analysis of computer models of biological systems, which have been traditionally difficult to analyze due to their high-dimensional, non-linear and resource-intensive nature. We describe an R package, mlegp, that fits GPs to computer model outputs and performs sensitivity analysis to identify and characterize the effects of important model inputs. Availability: http://www.biomath.org/mlegp Contact: [email protected] Supplementary information: See http://www.biomath.org/mlegp for a user manual and examples.

Coinfection of a single cell with two or more HIV strains may produce recombinant viruses upon template switching by the replication machinery. We applied a hierarchical multiple change point model to simultaneously infer inter-subtype recombination breakpoints and spatial variation in the recombination rate along the HIV-1 genome. We examined thousands of publicly available HIV-1 sequences representing the worldwide epidemic and focused on 544 unique recombinants with 1,701 recombination breakpoints. Estimates of per site recombination rate revealed the presence of a novel hotspot in the pol gene, surrounded by a cluster of mutations associated with resistance to reverse transcriptase inhibitors. We also confirm the presence of a known hotspot in the env gene and a previously hypothesized hotspot in the gag gene.

UNLABELLED: Bayesian multiple change-point models accurately detect recombination in molecular sequence data. Previous Java-based implementations assume a fixed topology for the representative parental data. cBrother is a novel C language implementation that capitalizes on reduced computational time to relax the fixed tree assumption. We show that cBrother is 19 times faster than its predecessor and the fixed tree assumption can influence estimates of recombination in a medically-relevant dataset. AVAILABILITY: cBrother can be freely downloaded from http://www.biomath.org/dormanks/ and can be compiled on Linux, Macintosh and Windows operating systems. Online documentation and a tutorial are also available at the site.

Motivation: We introduce a dual multiple change-point (MCP) model for recombination detection among aligned nucleotide sequences. The dual MCP model is an extension of the model introduced previously by Suchard and co-workers. In the original single MCP model, one change-point process is used to model spatial phylogenetic variation. Here, we show that using two change-point processes, one for spatial variation of tree topologies and the other for spatial variation of substitution process parameters, increases recombination detection accuracy. Statistical analysis is done in a Bayesian framework using reversible jump Markov chain Monte Carlo sampling to approximate the joint posterior distribution of all model parameters. Results: We use primate mitochondrial DNA data with simulated recombination break-points at specific locations to compare the two models. We also analyze two real HIV sequences to identify recombination break-points using the dual MCP model. Availability: A software program ‘DualBrothers’ implementing the dual MCP model is available in the form of a Java package at http://www.biomath.ucla.edu/msuchard/DualBrothers Contact: [email protected] Supplementary information: http://www.biomath.ucla.edu/msuchard/DualBrothers