Metagenomics enables the study of unculturable microorganisms in different environments directly. Discriminating between the compositional differences of metagenomes is an important and challenging problem. Several distance functions have been proposed to estimate the differences based on functional profiles or taxonomic distributions; however, the strengths and limitations of such functions are still unclear. Initially, we analyzed three well-known distance functions and found very little difference between them in the clustering of samples. This motivated us to incorporate suitable normalizations and phylogenetic information into the functions so that we could cluster samples from both real and synthetic data sets. The results indicate significant improvement in sample clustering over that derived by rank-based normalization with phylogenetic information, regardless of whether the samples are from real or synthetic microbiomes. Furthermore, our findings suggest that considering suitable normalizations and phylogenetic information is essential when designing distance functions for estimating the differences between metagenomes. We conclude that incorporating rank-based normalization with phylogenetic information into the distance functions helps achieve reliable clustering results.

SUMMARY: MetaABC is a metagenomic platform that integrates several binning tools coupled with methods for removing artifacts, analyzing unassigned reads and controlling sampling biases. It allows users to arrive at a better interpretation via series of distinct combinations of analysis tools. After execution, MetaABC provides outputs in various visual formats such as tables, pie and bar charts as well as clustering result diagrams. AVAILABILITY: MetaABC source code and documentation are available at http://bits2.iis.sinica.edu.tw/MetaABC/ CONTACT: [email protected]; [email protected] SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

MOTIVATION: Metagenomics involves sampling and studying the genetic materials in microbial communities. Several statistical methods have been proposed for comparative analysis of microbial community compositions. Most of the methods are based on the estimated abundances of taxonomic units or functional groups from metagenomic samples. However, such estimated abundances might deviate from the true abundances in habitats due to sampling biases and other systematic artifacts in metagenomic data processing. RESULTS: We developed the MetaRank scheme to convert abundances into ranks. MetaRank employs a series of statistical hypothesis tests to compare abundances within a microbial community and determine their ranks. We applied MetaRank to synthetic samples and real metagenomes. The results confirm that MetaRank can reduce the effects of sampling biases and clarify the characteristics of metagenomes in comparative studies of microbial communities. Therefore, MetaRank provides a useful rank-based approach to analyzing microbiomes. CONTACT: [email protected] SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

BACKGROUND: Investigation of metagenomes provides greater insight into uncultured microbial communities. The improvement in sequencing technology, which yields a large amount of sequence data, has led to major breakthroughs in the field. However, at present, taxonomic binning tools for metagenomes discard 30-40% of Sanger sequencing data due to the stringency of BLAST cut-offs. In an attempt to provide a comprehensive overview of metagenomic data, we re-analyzed the discarded metagenomes by using less stringent cut-offs. Additionally, we introduced a new criterion, namely, the evolutionary conservation of adjacency between neighboring genes. To evaluate the feasibility of our approach, we re-analyzed discarded contigs and singletons from several environments with different levels of complexity. We also compared the consistency between our taxonomic binning and those reported in the original studies. RESULTS: Among the discarded data, we found that 23.7 ± 3.9% of singletons and 14.1 ± 1.0% of contigs were assigned to taxa. The recovery rates for singletons were higher than those for contigs. The Pearson correlation coefficient revealed a high degree of similarity (0.94 ± 0.03 at the phylum rank and 0.80 ± 0.11 at the family rank) between the proposed taxonomic binning approach and those reported in original studies. In addition, an evaluation using simulated data demonstrated the reliability of the proposed approach. CONCLUSIONS: Our findings suggest that taking account of conserved neighboring gene adjacency improves taxonomic assignment when analyzing metagenomes using Sanger sequencing. In other words, utilizing the conserved gene order as a criterion will reduce the amount of data discarded when analyzing metagenomes.