PHYRN: A Robust Method for Phylogenetic Analysis of Highly Divergent Sequences

Abstract

Both multiple sequence alignment and phylogenetic analysis are problematic in the “twilight zone” of sequence similarity (≤25% amino acid identity). Herein we explore the accuracy of phylogenetic inference at extreme sequence divergence using a variety of simulated data sets. We evaluate four leading multiple sequence alignment (MSA) methods (MAFFT, T-COFFEE, CLUSTAL, and MUSCLE) and six commonly used programs of tree estimation (Distance-based: Neighbor-Joining; Character-based: PhyML, RAxML, GARLI, Maximum Parsimony, and Bayesian) against a novel MSA-independent method (PHYRN) described here. Strikingly, at “midnight zone” genetic distances (∼7% pairwise identity and 4.0 gaps per position), PHYRN returns high-resolution phylogenies that outperform traditional approaches. We reason this is due to PHRYN's capability to amplify informative positions, even at the most extreme levels of sequence divergence. We also assess the applicability of the PHYRN algorithm for inferring deep evolutionary relationships in the divergent DANGER protein superfamily, for which PHYRN infers a more robust tree compared to MSA-based approaches. Taken together, these results demonstrate that PHYRN represents a powerful mechanism for mapping uncharted frontiers in highly divergent protein sequence data sets.

Reevaluation of the evolutionary events within recA/RAD51 phylogeny

“…Since all sequences in Set-1 and Set-2 share a common recA domain, these homologous domains were used to construct a recA/RAD51 specific PSSM library (see [18] and Methods for complete description of PHYRN implementation). Subsequently, full-length sequences from each data set were aligned with their respective recA/RAD51 PSSM library.…”

“…The pipeline for the PHYRN algorithm is described in detail in Bhardwaj et al [18]. The recA/RAD51 domain boundaries were defined in the full-length sequences using NCBI CDD with default settings [21].…”

“…To increase the specificity of the PSSM library, we first collected all putative recA/RAD51 containing proteins, and subsequently used these sequences as a target database for pssmgen script in the PHYRNv1.6 package. Previous results with PHYRN have shown that an e-value of 1e -6 provides the best results with the non-redundant (NR) NCBI database [18]. Since our target recA/RAD51 database is significantly smaller in size, and the e-value threshold scales are proportional to the size of target database, we used an e-value of 7e -13 for PSSM generation.…”

“…To discriminate between these two disparate phylogenetic results, we applied our recently developed Position Specific Scoring Matrix (PSSM)-driven algorithm, termed PHYlogenetic ReconstructioN (PHYRN), that is highly accurate and robust for tree inference in highly divergent protein families [18]. PHYRN was benchmarked in simulated data sets with average pairwise identity <8.5% and was shown to be more accurate than multiple sequence alignment using either Maximum Likelihood [19] or Bayesian [20] methods.…”

A new lineage of segmented RNA viruses infecting animals

“…We excluded birnaviruses and permutotetraviruses, which have a permuted RdRp that cannot be straightforwardly aligned (Wolf et al 2018). Phylogenetic inference is necessarily challenging with such high levels of divergence (mean pairwise protein identity of only 7.6%) and the inferred relationships among such distantly-related lineages are unlikely to be reliable (Bhardwaj et al 2012, Nute et al 2018. In particular, although current phylogenetic methods perform surprisingly well on simulated data with identities as low 8-10%, this is only true when homology is known (i.e.…”