Culture independent techniques, such as shotgun metagenomics and 16S rRNA amplicon sequencing have dramatically changed the way we can examine microbial communities. Recently, changes in microbial community structure and dynamics have been associated with a growing list of human diseases. The identification and comparison of bacteria driving those changes requires the development of sound statistical tools, especially if microbial biomarkers are to be used in a clinical setting. We present , a novel multivariate data analysis framework for metagenomic biomarker discovery. accounts for the compositional nature of 16S data and enables detection of subtle differences when high inter-subject variability is present due to microbial sampling performed repeatedly on the same subjects, but in multiple habitats. Through data dimension reduction the multivariate methods provide insightful graphical visualisations to characterise each type of environment in a detailed manner. We applied to 16S microbiome studies focusing on multiple body sites in healthy individuals, compared our results with existing statistical tools and illustrated added value of using multivariate methodologies to fully characterise and compare microbial communities.

We are amidst an ongoing flood of sequence data arising from the application of high-throughput technologies, and a concomitant fundamental revision in our understanding of how genomes evolve individually and within the biosphere. Workflows for phylogenomic inference must accommodate data that are not only much larger than before, but often more error prone and perhaps misassembled, or not assembled in the first place. Moreover, genomes of microbes, viruses and plasmids evolve not only by tree-like descent with modification but also by incorporating stretches of exogenous DNA. Thus, next-generation phylogenomics must address computational scalability while rethinking the nature of orthogroups, the alignment of multiple sequences and the inference and comparison of trees. New phylogenomic workflows have begun to take shape based on so-called alignment-free (AF) approaches. Here, we review the conceptual foundations of AF phylogenetics for the hierarchical (vertical) and reticulate (lateral) components of genome evolution, focusing on methods based on k-mers. We reflect on what seems to be successful, and on where further development is needed.

Natural history collections are repositories of biodiversity and are potentially used by molecular ecologists for comparative taxonomic, phylogenetic, biogeographic and forensic purposes. Specimens in fish collections are preserved using a combination of methods with many fixed in formalin and then preserved in ethanol for long-term storage. Formalin fixation damages DNA, thereby limiting genetic analyses. In this study, the authors compared the DNA barcoding and identification success for frozen and formalin-fixed tissues obtained from specimens in the CSIRO Australian National Fish Collection. They studied 230 samples from fishes (consisting of >160 fish species). An optimized formalin-fixed, paraffin-embedded DNA extraction method resulted in usable DNA from degraded tissues. Four mini barcoding assays of the mitochondrial DNA (mtDNA) were characterized with Sanger and Illumina amplicon sequencing. In the good quality DNA (without exposure to formalin), up to 88% of the specimens were correctly matched at the species level using the cytochrome oxidase subunit 1 (COI) mini barcodes, whereas up to 58% of the specimens exposed to formalin for less than 8 weeks were correctly identified to species. In contrast, 16S primers provided higher amplification success with formalin-exposed tissues, although the COI gene was more successful for identification. Importantly, the authors found that DNA of a certain size and quality can be amplified and sequenced despite exposure to formalin, and Illumina sequencing provided them with greater power of resolution for taxa identification even when there was little DNA present.Overall, within parameter constraints, this study highlights the possibilities of recovering DNA barcodes for identification from formalin-fixed fish specimens, and the authors provide guidelines for when successful identification could be expected.