Automated Reconstruction of Whole-Genome Phylogenies from Short-Sequence Reads

Abstract

Studies of microbial evolutionary dynamics are being transformed by the availability of affordable high-throughput sequencing technologies, which allow whole-genome sequencing of hundreds of related taxa in a single study. Reconstructing a phylogenetic tree of these taxa is generally a crucial step in any evolutionary analysis. Instead of constructing genome assemblies for all taxa, annotating these assemblies, and aligning orthologous genes, many recent studies 1) directly map raw sequencing reads to a single reference sequence, 2) extract single nucleotide polymorphisms (SNPs), and 3) infer the phylogenetic tree using maximum likelihood methods from the aligned SNP positions. However, here we show that, when using such methods to reconstruct phylogenies from sets of simulated sequences, both the exclusion of nonpolymorphic positions and the alignment to a single reference genome, introduce systematic biases and errors in phylogeny reconstruction. To address these problems, we developed a new method that combines alignments from mappings to multiple reference sequences and show that this successfully removes biases from the reconstructed phylogenies. We implemented this method as a web server named REALPHY (Reference sequence Alignment-based Phylogeny builder), which fully automates phylogenetic reconstruction from raw sequencing reads.

Comparison of Lactobacillus crispatus isolates from Lactobacillus-dominated vaginal microbiomes with isolates from microbiomes containing bacterial vaginosis-associated bacteria

“…The web-based phylogenetic analysis tool REALPHY (preference alignment-based phylogeny builder) was used to compare the L. crispatus genomes (Bertels et al, 2014). REALPHY generates phylogenetic trees by merging alignments obtained by mapping to multiple reference genomes.…”

“…Of these, 1307 genes that encoded proteins consisting of w75 amino acids were common to all strains and the remaining genes were present on only a subset of the genomes analysed. The phylogenetic relationship amongst these isolates and the 10 L. crispatus strains listed in NCBI was determined by comparing the whole genomes with REALPHY (Bertels et al, 2014). First, the genomes of all 18 strains were merged into a core genome that was used as a reference for the analysis.…”

Molecular Types of Methicillin-Resistant Staphylococcus aureus and Methicillin-Sensitive S. aureus Strains Causing Skin and Soft Tissue Infections and Nasal Colonization, Identified in Community Health Centers in New York City

“…The phylogenetic trees were manually constructed based on the discovered numbers of polymorphisms between the isolates. The more complex tree was constructed with the software REALPHY using its default settings (http://realphy.unibas.ch/fcgi /realphy) (29). Using the vernacular in the documentation for REALPHY, the reference genome was USA300-FPR3757, the query genomes were the reads for the CAMP isolates (this study) and the northern Manhattan isolates (30), the two reference genome alignments were merged in order to generate a single tree, and the tree was inferred by PhyML.…”

Evaluation of phylogenetic reconstruction methods using bacterial whole genomes: a simulation based study

“…A more recent, larger study in eukaryotes compared these an IQ-TREE in terms of best likelihood on both species and gene trees 5 . Other small-scale comparisons include a comparison of read-to-tree pipelines with other pieces of software 6 , and the production of “well characterised” reference datasets for testing methods 7 . A recent study instead used an Escherichia coli hypermutator to conduct experimental evolution along a defined balanced phylogeny, and then by sequencing the strains at the tips, the authors compared the ability of 12 combinations of methods to reconstruct the correct phylogenetic relationship 8 .…”