Systematic overestimation of gene gain through false diagnosis of gene absence
© BioMed Central Ltd 2007
Published: 26 February 2007
The usual BLAST-based methods for assessing gene presence and absence lead to systematic overestimation of within-species gene gain by lateral transfer.
Genomes from different strains of the same bacterial species often differ substantially (up to 30%) in gene content [1–6]. There are two general ways to account for such gene content variability ('patchy distribution') among closely related genomes: strain-specific loss of genes after divergence from a common species ancestor that contained the genes, and strain-specific gain of genes after divergence from an ancestor that lacked them. Gain might be effected through lateral gene transfer (LGT), duplication (paralog creation) or, much less likely, de novo creation. Several recent publications have attempted to assess rates of within-species gain and loss using parsimony-based approaches applied to gene presence/absence data, in the context of a reference strain phylogeny [7–11]. Similar parsimony-based approaches have also been taken for inferences of gene gain/loss at larger phylogenetic distances [12–14].
Although prokaryotic genomes have traditionally been viewed as efficiently packed with functioning genes, and mutationally biased towards rapid deletion of dysfunctional regions , there are new indications that significant numbers of pseudogenes persist in some genomes [16–18]. In addition, detailed analyses show that in reduced genomes such as those of Rickettsia, intergenic regions often represent decaying remnants of genes . Some categorization more nuanced than 'presence' versus 'absence' might thus better capture genome history. But for gain-and-loss surveys of the sort cited there may seem to be no alternative to the binary approach. A gene is considered 'present' if represented by an open reading frame (ORF) showing significant similarity in sequence (with arbitrarily chosen significance cutoff) and having similar length to a query gene; otherwise it is scored as 'absent'. We systematically screened groups of closely related genomes (see Additional data files 1-3) for gene-family presence/absence patterns using several common criteria. When potential gene remnants detectable by less stringent methods are included, the number of gene families for which events of gain or loss within a species might be inferred (because they are scored as present only in some strains) can drop by as much as 90% (or as little as 7%) - on average about 60%. The extent to which recognition of such remnants will decrease estimates of the rates of gain of genes by LGT and increase estimates of the gene content of species' ancestors will depend on how recognition affects inferred patterns of presence and absence as displayed on a phylogeny of the species' strains. Each gene family must be individually examined, and where there is frequent between-strain recombination, not only is strain phylogeny a problematic concept , but it will sometimes be the case that gene remnants are themselves acquired by LGT.
Therefore, without agreed-upon definitions of presence/absence and reliable methods of detection, quantitation of rates of within-species gene gain have questionable meaning. It is both a practical concern and of theoretical interest that we really do not have a definition for gene loss. It is not clear where - along the line from the appearance of the first subtly deleterious regulatory or missense mutation to the deletion of the last nucleotide - we would agree to declare a gene to be lost. Parsimony-based inferences depend on how we make that declaration, but most quantitative treatments of gene loss in evolution avoid this question altogether. Moreover, in recombinogenic species, the possibility of exchange of remnants of inactivated genes between lineages means that there will be additional difficulties in reconstructing the decay process for individual genes. Indeed, in highly recombinogenic groups such as Neisseria, where homologous recombination, not mutation, is the principal source of between-strain sequence variation , it should seldom be possible to reconstruct the loss of an individual gene as a linear process of decay. These problems are of practical concern, as inferences about gain and loss dominate discussion of the evolution of pathogenicity and environmental adaptation within species. They are also of theoretical interest, bearing on the use of parsimony in evolutionary reconstruction.
As a matter of good practice, no claim that strains of the same species differ in gene content should be based on BLAST results alone, as differences in annotation abound and even BLASTing a single genome against itself does not recover all its annotated ORFs. No BLASTP+BLASTN-based estimate of the number of genes that a genome must have received by LGT (because they are absent from sister lineages in the same species) should be accepted without recognition that it is probably too high, possibly by several-fold. Species seem to differ in the extent to which such estimates are sensitive to BLAST parameters, and it is unlikely that optimal parameters - could these somehow be established - would be the same for all species groups. Ideally, all gene families would be examined for even highly decayed remnants.
Additional data files
The following additional data are available online with this paper. Additional data file 1 contains Materials and methods for the analyses performed. Additional data file 2 describes in detail the comparison of different BLAST-based criteria for presence/absence detection. Additional data file 3 is a table listing the composition of the analyzed genome groups.
This work was supported through CIHR (MOP-4467) and Genome Atlantic (Genome Canada) grants to W.F.D. O.Z. is supported through a CIHR Postdoctoral Fellowship and is an honorary Killam Postdoctoral Fellow at Dalhousie University. O.Z., C.L.N. and W.F.D. designed the study. O.Z. carried out all analyses. O.Z. and W.F.D. wrote the manuscript.
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