Motivation: In metagenomics, Kraken is one of the most widely used tools due to its robustness and speed. Yet, the overall turnaround time of metagenomic analysis is hampered by the sequential paradigm of wet and dry lab. In urgent experiments, it can be crucial to gain a timely insight into a dataset. Results: Here, we present LiveKraken, a real-time read classification tool based on the core algorithm of Kraken. LiveKraken uses streams of raw data from Illumina sequencers to classify reads taxonomically. This way, we are able to produce results identical to those of Kraken the moment the sequencer finishes. We are furthermore able to provide comparable results in early stages of a sequencing run, allowing saving up to a week of sequencing time on an Illumina HiSeq in High Throughput Mode. While the number of classified reads grows over time, false classifications appear in negligible numbers and proportions of identified taxa are only affected to a minor extent. Availability and implementation: LiveKraken is available at https://gitlab.com/rki_bioinformatics/LiveKraken. Supplementary information: Supplementary data are available at Bioinformatics online.

Motivation: Next Generation Sequencing is increasingly used in time critical, clinical applications. While read mapping algorithms have always been optimized for speed, they follow a sequential paradigm and only start after finishing of the sequencing run and conversion of files. Since Illumina machines write intermediate output results, HiLive performs read mapping while still sequencing and thereby drastically reduces crucial overall sample analysis time, e.g. in precision medicine. Methods: We present HiLive as a novel real time read mapper that implements a k-mer based alignment strategy. HiLive continuously reads intermediate BCL files produced by Illumina sequencers and then extends initial k-mer matches by increasingly produced data from the sequencer. Results: We applied HiLive on real human transcriptome data to show that final read alignments are reported within few minutes after the end of a full Illumina HiSeq 1500 run, while already the necessary conversion to FASTQ files as the standard input to current read mapping methods takes roughly five times as long. Further, we show on simulated and real data that HiLive has comparable accuracy to recent read mappers. Availability and Implementation: HiLive and its source code are freely available from https://gitlab.com/SimonHTausch/HiLive . Contact: [email protected]. Supplementary information: Supplementary data are available at Bioinformatics online.

MOTIVATION: Species identification and quantification are common tasks in metagenomics and pathogen detection studies. The most recent techniques are built on mapping the sequenced reads against a reference database (e.g. whole genomes, marker genes, proteins) followed by application-dependent analysis steps. Although these methods have been proven to be useful in many scenarios, there is still room for improvement in species and strain level detection, mainly for low abundant organisms. RESULTS: We propose a new method: DUDes, a reference-based taxonomic profiler that introduces a novel top-down approach to analyze metagenomic Next-generation sequencing (NGS) samples. Rather than predicting an organism presence in the sample based only on relative abundances, DUDes first identifies possible candidates by comparing the strength of the read mapping in each node of the taxonomic tree in an iterative manner. Instead of using the lowest common ancestor we propose a new approach: the deepest uncommon descendent. We showed in experiments that DUDes works for single and multiple organisms and can identify low abundant taxonomic groups with high precision. AVAILABILITY AND IMPLEMENTATION: DUDes is open source and it is available at http://sf.net/p/dudes SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. CONTACT: [email protected].

MOTIVATION: Metaproteomic analysis allows studying the interplay of organisms or functional groups and has become increasingly popular also for diagnostic purposes. However, difficulties arise owing to the high sequence similarity between related organisms. Further, the state of conservation of proteins between species can be correlated with their expression level, which can lead to significant bias in results and interpretation. These challenges are similar but not identical to the challenges arising in the analysis of metagenomic samples and require specific solutions. RESULTS: We introduce Pipasic (peptide intensity-weighted proteome abundance similarity correction) as a tool that corrects identification and spectral counting-based quantification results using peptide similarity estimation and expression level weighting within a non-negative lasso framework. Pipasic has distinct advantages over approaches only regarding unique peptides or aggregating results to the lowest common ancestor, as demonstrated on examples of viral diagnostics and an acid mine drainage dataset. AVAILABILITY AND IMPLEMENTATION: Pipasic source code is freely available from https://sourceforge.net/projects/pipasic/. CONTACT: [email protected] SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

MOTIVATION: The reliable identification of genes is a major challenge in genome research, as further analysis depends on the correctness of this initial step. With high-throughput RNA-Seq data reflecting currently expressed genes, a particularly meaningful source of information has become commonly available for gene finding. However, practical application in automated gene identification is still not the standard case. A particular challenge in including RNA-Seq data is the difficult handling of ambiguously mapped reads. RESULTS: We present GIIRA (Gene Identification Incorporating RNA-Seq data and Ambiguous reads), a novel prokaryotic and eukaryotic gene finder that is exclusively based on a RNA-Seq mapping and inherently includes ambiguously mapped reads. GIIRA extracts candidate regions supported by a sufficient number of mappings and reassigns ambiguous reads to their most likely origin using a maximum-flow approach. This avoids the exclusion of genes that are predominantly supported by ambiguous mappings. Evaluation on simulated and real data and comparison with existing methods incorporating RNA-Seq information highlight the accuracy of GIIRA in identifying the expressed genes. AVAILABILITY AND IMPLEMENTATION: GIIRA is implemented in Java and is available from https://sourceforge.net/projects/giira/.

MOTIVATION: Genome coverage, the number of sequencing reads mapped to a position in a genome, is an insightful indicator of irregularities within sequencing experiments. While the average genome coverage is frequently used within algorithms in computational genomics, the complete information available in coverage profiles (i.e. histograms over all coverages) is currently not exploited to its full extent. Thus, biases such as fragmented or erroneous reference genomes often remain unaccounted for. Making this information accessible can improve the quality of sequencing experiments and quantitative analyses. RESULTS: We introduce a framework for fitting mixtures of probability distributions to genome coverage profiles. Besides commonly used distributions, we introduce distributions tailored to account for common artifacts. The mixture models are iteratively fitted based on the Expectation-Maximization algorithm. We introduce use cases with focus on metagenomics and develop new analysis strategies to assess the validity of a reference genome with respect to (meta-) genomic read data. The framework is evaluated on simulated data as well as applied to a large-scale metagenomic study, for which we compute the validity of 75 microbial genomes. The results indicate that the choice and quality of reference genomes is vital for metagenomic analyses and that validation of coverage profiles is crucial to avoid incorrect conclusions. AVAILABILITY: The code is freely available and can be downloaded from http://sourceforge.net/projects/fitgcp/. CONTACT: [email protected] SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

MOTIVATION: Alignment of multiple liquid chromatography/mass spectrometry (LC/MS) experiments is a necessity today, which arises from the need for biological and technical repeats. Due to limits in sampling frequency and poor reproducibility of retention times, current LC systems suffer from missing observations and non-linear distortions of the retention times across runs. Existing approaches for peak correspondence estimation focus almost exclusively on solving the pairwise alignment problem, yielding straightforward but suboptimal results for multiple alignment problems. RESULTS: We propose SIMA, a novel automated procedure for alignment of peak lists from multiple LC/MS runs. SIMA combines hierarchical pairwise correspondence estimation with simultaneous alignment and global retention time correction. It employs a tailored multidimensional kernel function and a procedure based on maximum likelihood estimation to find the retention time distortion function that best fits the observed data. SIMA does not require a dedicated reference spectrum, is robust with regard to outliers, needs only two intuitive parameters and naturally incorporates incomplete correspondence information. In a comparison with seven alternative methods on four different datasets, we show that SIMA yields competitive and superior performance on real-world data. AVAILABILITY: A C++ implementation of the SIMA algorithm is available from http://hci.iwr.uni-heidelberg.de/MIP/Software.