Characterizing the taxonomic diversity of microbial communities is one of the primary objectives of metagenomic studies. Taxonomic analysis of microbial communities, a process referred to as binning, is challenging for the following reasons. Primarily, query sequences originating from the genomes of most microbes in an environmental sample lack taxonomically related sequences in existing reference databases. This absence of a taxonomic context makes binning a very challenging task. Limitations of current sequencing platforms, with respect to short read lengths and sequencing errors/artifacts, are also key factors that determine the overall binning efficiency. Furthermore, the sheer volume of metagenomic datasets also demands highly efficient algorithms that can operate within reasonable requirements of compute power. This review discusses the premise, methodologies, advantages, limitations and challenges of various methods available for binning of metagenomic datasets obtained using the shotgun sequencing approach. Various parameters as well as strategies used for evaluating binning efficiency are then reviewed.

SUMMARY: An unprecedented quantity of genome sequence data is currently being generated using next-generation sequencing platforms. This has necessitated the development of novel bioinformatics approaches and algorithms that not only facilitate a meaningful analysis of these data but also aid in efficient compression, storage, retrieval and transmission of huge volumes of the generated data. We present a novel compression algorithm (DELIMINATE) that can rapidly compress genomic sequence data in a loss-less fashion. Validation results indicate relatively higher compression efficiency of DELIMINATE when compared with popular general purpose compression algorithms, namely, gzip, bzip2 and lzma. AVAILABILITY AND IMPLEMENTATION: Linux, Windows and Mac implementations (both 32 and 64-bit) of DELIMINATE are freely available for download at: http://metagenomics.atc.tcs.com/compression/DELIMINATE. CONTACT: [email protected] SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

MOTIVATION: Compared with composition-based binning algorithms, the binning accuracy and specificity of alignment-based binning algorithms is significantly higher. However, being alignment-based, the latter class of algorithms require enormous amount of time and computing resources for binning huge metagenomic datasets. The motivation was to develop a binning approach that can analyze metagenomic datasets as rapidly as composition-based approaches, but nevertheless has the accuracy and specificity of alignment-based algorithms. This article describes a hybrid binning approach (SPHINX) that achieves high binning efficiency by utilizing the principles of both 'composition'- and 'alignment'-based binning algorithms. RESULTS: Validation results with simulated sequence datasets indicate that SPHINX is able to analyze metagenomic sequences as rapidly as composition-based algorithms. Furthermore, the binning efficiency (in terms of accuracy and specificity of assignments) of SPHINX is observed to be comparable with results obtained using alignment-based algorithms. AVAILABILITY: A web server for the SPHINX algorithm is available at http://metagenomics.atc.tcs.com/SPHINX/.

BACKGROUND: One of the primary goals of comparative metagenomic projects is to study the differences in the microbial communities residing in diverse environments. Besides providing valuable insights into the inherent structure of the microbial populations, these studies have potential applications in several important areas of medical research like disease diagnostics, detection of pathogenic contamination and identification of hitherto unknown pathogens. Here we present a novel and rapid, alignment-free method called HabiSign, which utilizes patterns of tetra-nucleotide usage in microbial genomes to bring out the differences in the composition of both diverse and related microbial communities. RESULTS: Validation results show that the metagenomic signatures obtained using the HabiSign method are able to accurately cluster metagenomes at biome, phenotypic and species levels, as compared to an average tetranucleotide frequency based approach and the recently published dinucleotide relative abundance based approach. More importantly, the method is able to identify subsets of sequences that are specific to a particular habitat. Apart from this, being alignment-free, the method can rapidly compare and group multiple metagenomic data sets in a short span of time. CONCLUSIONS: The proposed method is expected to have immense applicability in diverse areas of metagenomic research ranging from disease diagnostics and pathogen detection to bio-prospecting. A web-server for the HabiSign algorithm is available at http://metagenomics.atc.tcs.com/HabiSign/.

BACKGROUND: In metagenomic sequence data, majority of sequences/reads originate from new or partially characterized genomes, the corresponding sequences of which are absent in existing reference databases. Since taxonomic assignment of reads is based on their similarity to sequences from known organisms, the presence of reads originating from new organisms poses a major challenge to taxonomic binning methods. The recently published SOrt-ITEMS algorithm uses an elaborate work-flow to assign reads originating from hitherto unknown genomes with significant accuracy and specificity. Nevertheless, a significant proportion of reads still get misclassified. Besides, the use of an alignment-based orthology step (for improving the specificity of assignments) increases the total binning time of SOrt-ITEMS. RESULTS: In this paper, we introduce a rapid binning approach called DiScRIBinATE (Distance Score Ratio for Improved Binning And Taxonomic Estimation). DiScRIBinATE replaces the orthology approach of SOrt-ITEMS with a quicker 'alignment-free' approach. We demonstrate that incorporating this approach reduces binning time by half without any loss in the specificity and accuracy of assignments. Besides, a novel reclassification strategy incorporated in DiScRIBinATE results in reducing the overall misclassification rate to around 3 - 7%. This misclassification rate is 1.5 - 3 times lower as compared to that by SOrt-ITEMS, and 3 - 30 times lower as compared to that by MEGAN. CONCLUSIONS: A significant reduction in binning time, coupled with a superior assignment accuracy (as compared to existing binning methods), indicates the immense applicability of the proposed algorithm in rapidly mapping the taxonomic diversity of large metagenomic samples with high accuracy and specificity. AVAILABILITY: The program is available on request from the authors.

MOTIVATION: One of the first steps in metagenomic analysis is the assignment of reads/contigs obtained from various sequencing technologies to their correct taxonomic bins. Similarity-based binning methods assign a read to a taxon/clade, based on the pattern of significant BLAST hits generated against sequence databases. Existing methods, which use bit-score as the sole parameter to ascertain the significance of BLAST hits, have limited specificity and accuracy of binning. A new binning algorithm, called SOrt-ITEMS is introduced, which addresses these limitations. The method uses alignment parameters besides the bit score to first identify an appropriate taxonomic level where the read can be assigned. An orthology-based approach is subsequently used by the method for the final assignment. RESULTS: The performance of SOrt-ITEMS has been validated with reads simulating sequences from 454 and Sanger sequencing technologies. In addition, the taxonomic composition of the Sargasso Sea data set has been analyzed using SOrt-ITEMS. SOrt-ITEMS shows improved specificity and accuracy of assignments especially in simulated scenarios, wherein sequences corresponding to the source organism of the reads are absent in the reference database. AVAILABILITY: SOrt-ITEMS software is available for download from: http://metagenomics.atc.tcs.com/binning/SOrt-ITEMS. No license is needed for academic and nonprofit use.