Phylogenetic placement of query samples on an existing phylogeny is increasingly used in molecular ecology, including sample identification and microbiome environmental sampling. As the size of available reference trees used in these analyses continues to grow, there is a growing need for methods that place sequences on ultra‐large trees with high accuracy. Distance‐based placement methods have recently emerged as a path to provide such scalability while allowing flexibility to analyse both assembled and unassembled environmental samples. In this study, we introduce a distance‐based phylogenetic placement method, APPLES‐2, that is more accurate and scalable than existing distance‐based methods and even some of the leading maximum‐likelihood methods. This scalability is owed to a divide‐and‐conquer technique that limits distance calculation and phylogenetic placement to parts of the tree most relevant to each query. The increased scalability and accuracy enables us to study the effectiveness of APPLES‐2 for placing microbial genomes on a data set of 10,575 microbial species using subsets of 381 marker genes. APPLES‐2 has very high accuracy in this setting, placing 97% of query genomes within three branches of the optimal position in the species tree using 50 marker genes. Our proof‐of‐concept results show that APPLES‐2 can quickly place metagenomic scaffolds on ultra‐large backbone trees with high accuracy as long as a scaffold includes tens of marker genes. These results pave the path for a more scalable and widespread use of distance‐based placement in various areas of molecular ecology.

16S rRNA and shotgun metagenomics studies typically yield different results, traditionally thought to be due to biases in amplification. We show that differences in reference phylogeny are more important. By inserting sequences into a whole-genome phylogeny, we show that 16S rRNA and shotgun metagenomic data generated from the same samples agree in principal coordinates space, taxonomy, and in phenotype effect size when analyzed with the same tree.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by heterogeneous cognitive, behavioral and communication impairments. Disruption of the gut–brain axis (GBA) has been implicated in ASD although with limited reproducibility across studies. In this study, we developed a Bayesian differential ranking algorithm to identify ASD-associated molecular and taxa profiles across 10 cross-sectional microbiome datasets and 15 other datasets, including dietary patterns, metabolomics, cytokine profiles and human brain gene expression profiles. We found a functional architecture along the GBA that correlates with heterogeneity of ASD phenotypes, and it is characterized by ASD-associated amino acid, carbohydrate and lipid profiles predominantly encoded by microbial species in the genera Prevotella, Bifidobacterium, Desulfovibrio and Bacteroides and correlates with brain gene expression changes, restrictive dietary patterns and pro-inflammatory cytokine profiles. The functional architecture revealed in age-matched and sex-matched cohorts is not present in sibling-matched cohorts. We also show a strong association between temporal changes in microbiome composition and ASD phenotypes. In summary, we propose a framework to leverage multi-omic datasets from well-defined cohorts and investigate how the GBA influences ASD.

Studies using 16S rRNA and shotgun metagenomics typically yield different results, usually attributed to PCR amplification biases. We introduce Greengenes2, a reference tree that unifies genomic and 16S rRNA databases in a consistent, integrated resource. By inserting sequences into a whole-genome phylogeny, we show that 16S rRNA and shotgun metagenomic data generated from the same samples agree in principal coordinates space, taxonomy and phenotype effect size when analyzed with the same tree.

Placing new sequences onto reference phylogenies is increasingly used for analyzing environmental samples, especially microbiomes. Existing placement methods assume that query sequences have evolved under specific models directly on the reference phylogeny. For example, they assume single-gene data (e.g., 16S rRNA amplicons) have evolved under the GTR model on a gene tree. Placement, however, often has a more ambitious goal: extending a (genome-wide) species tree given data from individual genes without knowing the evolutionary model. Addressing this challenging problem requires new directions. Here, we introduce Deep-learning Enabled Phylogenetic Placement (DEPP), an algorithm that learns to extend species trees using single genes without pre-specified models. In simulations and on real data, we show that DEPP can match the accuracy of model-based methods without any prior knowledge of the model. We also show that DEPP can update the multi-locus microbial tree-of-life with single genes with high accuracy. We further demonstrate that DEPP can combine 16S and metagenomic data onto a single tree, enabling community structure analyses that take advantage of both sources of data.