MUSCLE manual
Big alignments, do they make sense?
Probably the most common question I get about MUSCLE is “Im trying to align [big number here] sequences and I'm running out of memory. What should I do?”. There are various techniques that can reduce the amount of memory and time needed to make very large alignments, and I'll describe them below, but a more important question may be: why are you making the alignment? Is this the best approach to analyzing your sequences? Occasionally I’ve heard some good answers, and this is inspiring me to work on better methods for building and analyzing large alignments, but I suspect that sometimes people are doing this because this is the way sequence analysis has traditionally been done, without giving enough attention to the limitations of multiple alignment methods.

The key question is, can a multiple alignment represent homology between letters accurately enough to enable robust inferences to be made by downstream tools? It is almost impossible to review an alignment that large by eye, so inevitably automated tools are used. For example, if you want to estimate a phylogenetic tree, then will the tree be good enough to be informative? (Similar questions apply to building large trees, but let’s not go there now).

The answer is, probably not: it is almost never possible to build a good multiple alignment of that size. The matrix format (row and column) of a multiple alignment implicitly assumes that the only important mutations are substitutions, insertions of short random sequences and deletions. This is a reasonable simplification for a small number of closely related sequences, but is increasingly dubious as the divergence or the number of sequences increases.

The most common mutation producing short  insertions is tandem duplication. Tandem duplications tend to expand into arrays with three or more copies of repeated motifs. Tandems cause 2:1 homology (or n:1 for arrays)  between a sequence that has the duplication and a sequence that does not, and a multiple alignment cannot represent this because homology is implicitly required to be either 1:1 or non-existent. If your definition of alignment correctness is that homologous residues must be in the same column if and only if they are homologous, then correctness is impossible if tandems are present. If tandems are relatively rare, then it is reasonable to ignore the problem and arbitrarily choose one segment to align. But in more variable regions (e.g., surface loops in proteins or DNA that is evolving neutrally or under weak constraint), they are probably common. In that case, repeated, overlapping cycles of tandem duplications, inversions and deletions presumably leave a tangled mess where inference of homologous residues is impossible due to information loss. I call this “churn”. Further, even if you could infer homology, you would not be able to represent all the relationships in a conventional matrix multiple alignment form. Tandems and inversions obviously can’t be represented. Overlapping inversions can induce a translocation on the same strand (I guess a figure would be helpful, let me know if this isn’t clear). Global multiple alignment assumes that the order of letters is preserved, so translocations can’t be represented either. A potentially interesting special case is insertions at the same locus in independent lineages, as pointed out by Ari Löytynoja and Nick Goldman in a series of recent papers. These papers have many problems, which I hope to discuss in elsewhere (maybe in a later blog post), but here I want to point out that they overlook that the most likely cause of such insertions is tandem duplications, in which case the inserted bases will be homologous despite having been inserted independently. Such events would complicate the definition of a correct alignment and the inference of evolutionary history, though it should be noted that Löytynoja and Goldman fail to present any evidence that independent insertions at the same locus is common enough to be significant in this context. My own view is that independent insertions at a single locus is a minor example among several types of evolutionary event that undermine the traditional multiple alignment model.

Let's suppose that you have 10,000 sequences that are miraculously immune from tandem duplications, inversions and translocations, i.e. the evolutionary processes that mess up multiple alignments. Now can you make a good alignment? Sorry, but still probably not. Now we run into limitations of practical algorithms and problems due to information loss. Most popular global multiple alignment algorithms, including MUSCLE, PROBCONS, CLUSTALW, T-Coffee and others, are based on iterating global pair-wise alignment. These methods implicitly assume the simplified model of evolution (substitutions, deletions and short random insertions) as described above. The only available information is sequence similarity, and this has unavoidable limitations. As sequences diverge, the placement of gaps gets less certain, and the probability of an error in the positioning of a given gap endpoint increases. This can happen even in closely related sequences. Suppose your sequence is …ACCG…, and one of the C’s is deleted, leaving ACG. You can’t tell which C it was, and the alignment must make an arbitrary choice. If two other sequences have …ACG…, the parsimonious assumption (assuming it was in fact a deletion) is that one of the Cs was deleted in the common ancestor. That’s the best guess, but it will sometimes be wrong. Maybe there was an independent deletion at the same locus in two different lineages. Maybe one lineage deleted C1 and the other deleted C2. It is impossible in principle to distinguish between these histories, so sometimes the alignment will be wrong. Every time you add a sequence, there is some probability of introducing new errors. The probability (call it Pe) of introducing an error increases with divergence, but is never zero — even if the sequences are 100% identical, you can’t rule out that there are intermediate mutations that canceled each other out. So with 10,000 sequences, you’ll going to have at least 10,000 x Pe errors, and this is going to be a big number even if Pe is small. The problem is much worse than this because each time an error is introduced, it propagates to all higher levels in the tree due to the ‘progressive’ approach taken by all popular methods that are capable of making large alignments.

So what can you do? Using a 64-bit build of MUSCLE may allow you to build a single multiple alignment, if that does make sense after all.

Other approaches might make sense, depending on what you want to use the alignment for. In the 16S rRNA world, several methods use reference multiple alignments provided by databases such as Greengenes, RDP or SILVA. One method for producing these alignments is the NAST aligner, which deliberately introduces errors (!) in order to mitigate the problem of the growing number of columns in a very large alignment. I believe this is the wrong approach: NAST-like alignments are basically a response to the slow performance of search and de novo alignment programs, and I believe that the improved performance of the USEARCH algorithm enables better solutions to problems such as chimera detection where reference alignments have traditionally been used.

One option to consider is to reduce the data size by clustering. If you have 10,000 sequences, probably many of them are in closely related subfamilies. Then it makes sense to cluster at, say, 95% or 90% identity using UCLUST. That might leave a few hundred sequences to align, which would reveal the most highly conserved regions but might fail to give a meaningful alignment in the more variable regions. If needed, you could independently align members of each cluster to each other. I’m always interested to discuss this kind of problem, and I'm actively working on improved solutions.