Fully automated sequence alignment methods are comparable to, and much faster than, traditional methods in large data sets: an example with hepatitis B virus

Aligning sequences for phylogenetic analysis (multiple sequence alignment; MSA) is an important, but increasingly computationally expensive step with the recent surge in DNA sequence data. Much of this sequence data is publicly available, but can be extremely fragmentary (i.e., a combination of full genomes and genomic fragments), which can compound the computational issues related to MSA. Traditionally, alignments are produced with automated algorithms and then checked and/or corrected “by eye” prior to phylogenetic inference. However, this manual curation is inefficient at the data scales required of modern phylogenetics and results in alignments that are not reproducible. Recently, methods have been developed for fully automating alignments of large data sets, but it is unclear if these methods produce alignments that result in compatible phylogenies when compared to more traditional alignment approaches that combined automated and manual methods. Here we use approximately 33,000 publicly available sequences from the hepatitis B virus (HBV), a globally distributed and rapidly evolving virus, to compare different alignment approaches. Using one data set comprised exclusively of whole genomes and a second that also included sequence fragments, we compared three MSA methods: (1) a purely automated approach using traditional software, (2) an automated approach including by eye manual editing, and (3) more recent fully automated approaches. To understand how these methods affect phylogenetic results, we compared resulting tree topologies based on these different alignment methods using multiple metrics. We further determined if the monophyly of existing HBV genotypes was supported in phylogenies estimated from each alignment type and under different statistical support thresholds. Traditional and fully automated alignments produced similar HBV phylogenies. Although there was variability between branch support thresholds, allowing lower support thresholds tended to result in more differences among trees. Therefore, differences between the trees could be best explained by phylogenetic uncertainty unrelated to the MSA method used. Nevertheless, automated alignment approaches did not require human intervention and were therefore considerably less time-intensive than traditional approaches. Because of this, we conclude that fully automated algorithms for MSA are fully compatible with older methods even in extremely difficult to align data sets. Additionally, we found that most HBV diagnostic genotypes did not correspond to evolutionarily-sound groups, regardless of alignment type and support threshold. This suggests there may be errors in genotype classification in the database or that HBV genotypes may need a revision

Commentaries on viewpoint : physiology and fast marathons

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Genome_trees.zip

Tree files estimated from sequence alignments of hepatitis B virus genomes. Trees are best maximum likelihood (ML) trees with bootstrap support values. Includes trees based on MUSCLE, manual, and PASTA genome alignments

GI_Clustering.zip

Initial files of hepatitis B virus sequences clustered according to GenBank GI number

Genome_consensus_sequence.fasta

Consensus sequence of hepatitis B virus genomes. This sequence was used as a reference for HBV manual alignments

Supplementary_Information.docx

List of commands used in software programs for the alignment and tree estimation of hepatitis B virus sequences

Cleaned_GenBank_Files.zip

Hepatitis B virus GenBank files after initial data filtering steps

Genotype_trees.zip

Tree files used for genotype occupancy tests in hepatitis B viruses. Trees estimated from manual or PASTA genome alignments. Files include .tre and .xml format