SUMMARY: Accurate gene prediction is essential for successful metagenome analysis. We present KOunt, a Snakemake pipeline, that precisely quantifies KEGG orthologue abundance. AVAILABILITY AND IMPLEMENTATION: KOunt is available on GitHub: https://github.com/WatsonLab/KOunt. The KOunt reference database is available on figshare: https://doi.org/10.6084/m9.figshare.21269715. Test data are available at https://doi.org/10.6084/m9.figshare.22250152 and version 1.2.0 of KOunt at https://doi.org/10.6084/m9.figshare.23607834.

Reducing rumen methane emissions based on the basal diet is one primary approach for livestock production. However, microbiological mechanisms of rumen methane emissions under different diets are very complex and lack comprehensive understanding. This research developed an innovative multilayer network framework to investigate relationships of rumen microbes, transformations of metabolites and functions of rumen microbial genes. The multilayer network model explored the diets-based variation in rumen microbial system differences. The topological structure of the multilayer network reflects volatile fatty acid (VFA) fermentation characteristics between diets. Propionate is the highest degree node of the concentrate-diet (CONC) multilayer network while acetate has the highest degree in the forage-diet (FOR) multilayer network. The microbial communities under FOR diet are more diverse and have more complicated interactions. Methanobrevibacter copresented with Methanosphaera only in the CONC network. There are 33 microbial functional genes in the FOR network that are enriched in the methane synthesize functional module, including the CO2 reduction and the enzymatic reaction of acetyl phosphate to synthesize acetyl-CoA. The microbial genes of the CONC network are found to be associated with the serine biosynthetic and the biochemical process of acetate convert to acetyl-CoA and acetyl phosphate. This study verified the variations of diet-based methane phenotypic are results of systemic changing in the metabolism of the entire rumen microbiome.

The importance of the composition and signature of rumen microbial communities has gained increasing attention. One of the key techniques was to infer co-abundance networks through correlation analysis based on relative abundances. While substantial insights and progress have been made, it has been found that due to the compositional nature of data, correlation analysis derived from relative abundance could produce misleading results and spurious associations. In this study, we proposed the use of a framework including a compendium of two correlation measures and three dissimilarity metrics in an attempt to mitigate the compositional effect in the inference of significant associations in the bovine rumen microbiome. We tested the framework on rumen microbiome data including both 16S rRNA and KEGG genes associated with methane production in cattle. Based on the identification of significant positive and negative associations supported by multiple metrics, two co-occurrence networks, e.g., co-presence and mutual-exclusion networks, were constructed. Significant modules associated with methane emissions were identified. In comparison to previous studies, our analysis demonstrates that deriving microbial associations based on the correlations between relative abundances may not only lead to missing information but also produce spurious associations. To bridge together different co-presence and mutual-exclusion relations, a multiplex network model has been proposed for integrative analysis of co-occurrence networks which has great potential to support the prediction of animal phytotypes and to provide additional insights into biological mechanisms of the microbiome associated with the traits.

It can be an arduous and time consuming task to manually curate video data for tracking the movement of an object. This is particularly challenging when tracking animals whose behaviour is sporadic and unpredictable, but it can provide pertinent information when monitoring health, conditions and treatments. Here, we evaluate the use of machine learning retroactively applied to automatically track specific steers in hours of video with minimal manual interaction. This is approached using the Faster R-CNN Object Detection algorithm with VGG-16 acting as a feature extractor. Performance on a number of video segments is presented and discussed, and the issues encountered are outlined. This highlights a number of guidelines that should be taken under consideration when generating video data to improve object detection performance, and helps define the applicability of the approach to pre-existing data.

Metabolites are the final product of biochemical reactions in the rumen micro-ecological system and very sensitive to changes of microbial genes. However, limited by the spectra library and the computational techniques of structure identification, the identification of metabolites from non-targeted metabolomics is time-consuming and inefficient. The absence of specific information about metabolites makes the biological interpretation of the quantitative analysis of metabolomics meaningless. Based on the nonlinear association between microbial genes and metabolites, combined with knowledge of metabolic pathways from the KEGG database, this study developed a knowledge driven mutual information-based analytical framework for identifying metabolites associated with integrals derived from NMR analysis results. In this study, one known metabolite and three sets of integrals with unknow metabolites were identified within the novel framework. The results showed that this mutual information-based framework could very efficiently target metabolites that may correspond to integrals from NMR spectra.

Metagenomics is proliferating for studying environmental microbial communities and their role in animal functions. This paper aims to study the role of functions of microbial communities present in cattle (Bos taurus) and their relation to dietary supplement usage. The functional study was conducted as part of the EU H2020 MetaPlat project11MetaPlat, http://www.metaplat.eu. In this research, we proposed a novel phylogeny-driven approach to classify 16S rRNA samples from cattle rumen microbiome and relate them to the functional phenotype of diet (referred to as functional analysis). Phylogeny covers biological relationships from different taxonomical levels combined with their respective evolutionary measures. We performed this analysis by proposing a novel method based on phylogeny-adjusted distance-based indices. These indices are used in ranking microbial feature space derived from the topology of the phylogenetic tree. The integrative approach incorporating phylogeny into feature engineering as part of machine learning (ML) modeling, achieved high predictive performance with Accuracy of 0.962 and Kappa of 0.950 for classifying cattle microbiome into the phenotype of a diet supplemented with oil, nitrate, combined (with oil and nitrate) and controls.

MOTIVATION: Metagenomics is a powerful tool for assaying the DNA from every genome present in an environment. Recent advances in bioinformatics have enabled the rapid assembly of near-complete metagenome-assembled genomes (MAGs), and there is a need for reproducible pipelines that can annotate and characterize thousands of genomes simultaneously, to enable identification and functional characterization. RESULTS: Here we present MAGpy, a scalable and reproducible pipeline that takes multiple genome assemblies as FASTA and compares them to several public databases, checks quality, suggests a taxonomy and draws a phylogenetic tree. AVAILABILITY AND IMPLEMENTATION: MAGpy is available on github: https://github.com/WatsonLab/MAGpy. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The recognized significance of rumen microbiome has inspired efforts to examine the composition of rumen microbial communities in a large scale. One of the key research areas is to infer association and dependencies between members of rumen microbial communities through correlation analysis. However, it has been found that due to the compositional nature of data, simply applying correlation-based techniques to the analysis of relative abundance of microbial genes may produce artefactual correlation and loss of information. In an attempt to mitigate the compositional effect on the analysis of rumen microbiome data, this study applied a framework including a compendium of two correlation measures and three dissimilarity metrics that are intrinsically robust to compositionality. Based on the inference of significant positive and negative associations, co-presence and mutual-exclusion networks were constructed. The corresponding modules associated with methane production were identified. The modules are highly enriched with microbial genes associated with methane emissions and encoding enzymes involved in the methane methanogensis pathway. In comparisons to previous studies, our analysis demonstrates that deriving microbial associations based on the correlations between relative abundances may not only lead to missing information but also produce spurious associations.

Metagenomics involves the genetic analysis of microbial DNA extracted from communities in an environment sample. Advent and falling costs of next-generation sequencing technologies has accelerated metagenomics research providing an improved understanding of microbial communities. In this study we investigate if the traits methane production and feed conversion rates in the rumen microbial community overlap with top genes ranked by topological metrics in a co-abundance network. A co-abundance network was constructed from abundance values of 1570 microbial genes in rumen samples of 8 cattle identified in a metagenomics study at the Beef and Sheep Research Centre of Scotland's Rural College. We used 4 different topological measures: Degree Centrality, Betweenness Centrality, Bonacich Power Centrality and PageRank to the network. Using permutation testing, we discovered, methane production trait genes significantly overlapped with top ranked genes obtained using the metrics PageRank and Bonacich Power Centrality. Feed conversion trait genes overlapped with top ranked genes using Bonacich Power Centrality and Betweenness. Furthermore, we observed the top ranked genes from PageRank and Bonacich Power Centrality significantly overlapped with genes involved in the KEGG methane metabolism pathway and ranked highly key methanogenesis genes such as mcrA and fmdB. Identified functional clusters containing most methane and feed conversion genes were also analyzed in terms of overlap with top ranked genes from topological metrics.

A better understanding of the composition of rumen microbial communities and the association between host genetic and microbial activities has important applications and implication in bioscience. Being capable of revealing the full extent of microbial gene diversity, metagenomics-based approaches hold great promises in this endeavor. This study investigates the rumen microbial community in cattle through the integration of metagenomic and network-based approaches. Based on the relative abundance of 1570 microbial genes identified in a metagenomics analysis, the co-abundance network was constructed and functional modules of microbial genes were identified. One of the main contributions of this study is to develop a random matrix theory-based approach to automatically determine the correlation threshold used to construct the co-abundance network. It has been shown that the network exhibits a highly modular structure with each of the three main modules well separated. The involvement of KEGG pathways in each module was analysed. A close look at the abundance profiles highlights that Module B is strongly associated with methane emissions while Module C is highly enriched with microbial genes associated with feed conversion efficiency.