PhastCons HOWTO

Adam Siepel (
Last Modified: June 14, 2005

NOTE: This is a specialized tutorial with extended usage and options for phastCons. For a basic tutorial for getting started with phastCons please visit phastCons Tutorial.


This document is intended to provide a reasonably detailed, "nuts-and-bolts" level introduction to the phastCons program. It should be regarded as a supplement to the phastCons paper, which focuses on scientific results and more theoretical modeling issues, and to the phastCons Tutorial, which provides basic information about using the program but does not address several important side issues. The questions of how to set the program's tuning parameters and how to estimate any remaining free parameters are discussed here, as are practical methods for applying the program to very large data sets. Some lesser-known features of the program are also briefly discussed, e.g., having to do with modeling rate variation or indels, and with producing coding-potential rather than conservation scores.

PhastCons is part of the PHAST (PHylogenetic Analysis with Space/Time models) package, which can be downloaded from the PHAST Website. The latest version of the phastCons help page can be obtained by running the program with the --help option.


  1. Introduction
  2. Getting Started
  3. State-Transition Parameters
  4. Working with Large Data Sets
  5. Advanced Topics [NOT YET WRITTEN]
  6. References
  7. Feedback
  8. Credits


PhastCons is a program for identifying evolutionarily conserved elements in a multiple alignment, given a phylogenetic tree. It is based on a statistical model of sequence evolution called a phylogenetic hidden Markov model (phylo-HMM). If you are familiar with the popular VISTA program (Mayor et al, 2000), which plots the percent identity between two aligned sequences along their length, you might think of phastCons as a kind of next-generation VISTA. The differences between phastCons and VISTA include that phastCons considers n species rather than two, it considers the phylogeny by which these species are related, and instead of measuring similarity/divergence simply in terms of percent identity, it uses statistical models of nucleotide substitution that allow for multiple substitutions per site and for unequal rates of substitution between different pairs of bases (e.g., a higher frequency of transitions than transversions). PhastCons is the analytical engine behind the increasingly popular conservation tracks in the UCSC Genome Browser.

PhastCons and the rest of the PHAST package are written in C. The PHAST package has been compiled and installed under various UNIX and linux platforms and under Mac OS X. It should be relatively easy to port the software to other platforms.


In this section, I'll discuss a few basic ways of using the program and some of its most commonly used options.

2.1 Overview of Usage

PhastCons essentially does three things: it produces base-by-base conservation scores (as displayed in the conservation tracks in the UCSC browser), it produces predictions of discrete conserved elements (as displayed in the "most conserved" tracks in the browser), and it estimates free parameters. (We'll assume in this section that the base-by-base scores are conservation scores and the discrete elements are conserved elements, despite that they may have alternative interpretations; see Section 5.)

As input, PhastCons takes a multiple alignment, a phylogenetic model for conserved regions, and a phylogenetic model for nonconserved regions. The model for conserved regions is optional. If it is not given, then this model is defined indirectly by "scaling" the model for nonconserved regions by a factor called rho (see the --rho option to phastCons). The basic idea of the program is to scan along the alignment for regions that are better "explained" by the conserved model than by the nonconserved model; such regions will be output as conserved elements, and the probability that each base is in such a region will be output as the conservation score for that base.

The program also needs a specification of the state-transition probabilities for the HMM. These probabilities determine how freely the model can switch between states, and as a result, they define important features such as the expected length of conserved elements, the expected coverage by conserved elements, and the "smoothness" of the conservation plot (see the phastCons paper for details). The transition probabilities can be set directly with the --transitions option or with the --expected-length and --target-coverage options (see Section 3). Alternatively, they may be left undefined, and will automatically be estimated by maximum likelihood. Probably the trickiest part of getting good results with phastCons is figuring out how to set these parameters. Let's gloss over this potentially complicated issue for now and focus on the basics of using the program. For the moment, assume the transition probabilities are set by the tuning parameters, say, by using --target-coverage 0.25 and --expected-length 12. We'll return to this issue in Section 3.

The program can be run with a command of the form:

    phastCons [OPTIONS] alignment [cons.mod,]noncons.mod > scores.wig
where alignment is a multiple alignment in an appropriate format (see --msa-format), cons.mod and noncons.mod are phylogenetic models for conserved and nonconserved regions, respectively, in PHAST's .mod format, and scores.wig is a file of conservation scores in the fixed-step WIG format used by the UCSC Genome Browser (

For example,

    phastCons --target-coverage 0.25 --expected-length 12 \
        --msa-format MAF alignment.maf cons.mod,noncons.mod \
        > scores.wig
    phastCons --target-coverage 0.25 --expected-length 12 \
        --rho 0.4 --msa-format MAF alignment.maf noncons.mod \
        > scores.wig
where in the second case the conserved model is defined as a scaled version of the nonconserved model, with scaling factor rho = 0.4. Here an alignment in the Multiple Alignment Format (MAF) used in the UCSC Genome Browser is assumed.

A set of discrete conserved elements can be predicted by adding the --most-conserved option, e.g.,

    phastCons --target-coverage 0.25 --expected-length 12 --rho 0.4 \
        --most-conserved most-cons.bed --msa-format MAF alignment.maf \
        noncons.mod > scores.wig
which will cause the coordinates of predicted elements to be written to the file "most-cons.bed" in BED format (see Alternatively, these elements can be written in GFF (see, by giving the output filename a suffix of ".gff".

If you don't care about the conservation scores, you can avoid computing them with the --no-post-probs option. (This can save some time.) If phastCons is run with --no-post-probs and without --most-conserved, then it will neither predict a set of discrete elements nor produce the conservation scores. However, it will still estimate free parameters, if directed to do so (see below).

In summary, the default behavior of the program is to estimate any unspecified free parameters, then to compute conservation scores and output them to stdout. However, it can be made to perform any combination of its three main tasks -- predicting elements, computing scores, and estimating parameters -- by running it with or without --most-conserved, with or without --no-post-probs, and with or without a specification of the values of its free parameters.

2.2 Obtaining Phylogenetic Models

There are three ways main ways to obtain the phylogenetic models (.mod files) used by phastCons:
  1. Let phastCons itself estimate the models directly from the data, using an "unsupervised" learning algorithm (the EM algorithm described in the phastCons paper).
  2. Separately estimate the nonconserved model from labeled training data, e.g., fourfold degenerate (4d) sites or ancestral repeats (ARs), and let phastCons estimate the scaling parameter rho.
  3. Separately estimate both the conserved and nonconserved models from labeled training data and don't do any estimation with phastCons.
In general, option 1 is recommended, but there are times when it may not be ideal. When option 1 is used, the nonconserved model is essentially estimated from sites outside of predicted conserved elements. If a data set contains very distant species, which align mostly in conserved regions (e.g., coding exons), then the estimates of the nonconserved branch lengths to these species will tend to be underestimated, because any "nonconserved" bases that do align are probably actually at least partially conserved. It may make sense in such a case to estimate a nonconserved model from 4d sites in coding regions (where alignments can reliably be obtained), and to estimate the conserved model either from labeled training data (option 3) or by letting phastCons estimate the parameter rho (option 2).

It is worth noting that in experiments we've done with vertebrate data (including human, mouse, rat, chicken, and Fugu sequences), estimating the nonconserved model from 4d sites rather than letting phastCons estimate them did not make too much of a difference in the predicted conserved elements, even though it did result in quite different branch-length estimates (see Supplementary Material to phastCons paper). The EM algorithm (option 1) seems to be able to learn a separation between conserved and nonconserved regions even if the model it learns for nonconserved regions is not an entirely accurate representation of the neutral substitution process. On the other hand, when the number of species is large (say, greater than a dozen), option 1 can be quite computationally intensive, and options 2 or 3 may be preferable purely for practical reasons (see the ENCODE example, below). As a rule of thumb, I recommend option 1 unless you have (a) more than 12 species or (b) many distant species with low alignment coverage and you are concerned about distortions in branch lengths. I recommend option 2 in these cases.

For options 2 or 3, you can estimate your models using the program phyloFit, also in the PHAST package. Start with an alignment and an annotation file indicating the sites of interest in your alignment. For example, suppose you have an alignment of human, mouse, and rat sequences in a file called data.fa (FASTA format), and suppose you have a file called AR.gff (GFF format) defining the locations of ancestral repeats (ARs) in the coordinate system of the first sequence (human). For example,

human   RepeatMasker    AR      14451829        14451925        .       .       .       type "L1MC4a.LINE.L1"
human   RepeatMasker    AR      14452228        14452443        .       .       .       type "L1MC4a.LINE.L1.2"
human   RepeatMasker    AR      14458153        14458278        .       .       .       type "L2.LINE.L2"
human   RepeatMasker    AR      14458258        14458386        .       .       .       type "L2.LINE.L2.2"
human   RepeatMasker    AR      14458781        14459349        .       .       .       type "L2.LINE.L2.3"
human   RepeatMasker    AR      14460129        14460311        .       .       .       type "L1ME.LINE.L1"
human   RepeatMasker    AR      14460948        14461016        .       .       .       type "L2.LINE.L2.4"
human   RepeatMasker    AR      14463023        14463356        .       .       .       type "MER89-int.LTR.ERV1"
You can estimate a phylogenetic model for these sites with a command such as:
        phyloFit --tree "(human,(mouse,rat))" --features AR.gff \
            --do-cats AR --out-root nonconserved data.fa
Here the model will be written to a file called nonconserved.AR.mod. See the phyloFit help page (run 'phyloFit --help') for additional details.

With 4d sites, the program msa_view may be helpful. Suppose instead of annotations of ARs, you have a file called genes.gff describing coding regions in the human sequence, e.g.,

human    UCSC        CDS     4562    4692    .       -       0       transcript_id "NM_198943" ; gene_id "NM_198943"
human    UCSC        CDS     7469    7605    .       -       0       transcript_id "NM_198943" ; gene_id "NM_198943"
human    UCSC        CDS     7778    7924    .       -       0       transcript_id "NM_198943" ; gene_id "NM_198943"
human    UCSC        CDS     8131    8242    .       -       1       transcript_id "NM_198943" ; gene_id "NM_198943"
human    UCSC        CDS     14601   14749   .       -       0       transcript_id "NM_198943" ; gene_id "NM_198943"
You can extract 4d sites from this alignment with a command like
    msa_view data.fa --4d --features genes.gff > 
This will create a representation in the "sufficient statistics" (SS) format used by several PHAST tools of whole codons containing 4d sites. You can extract just the 4d sites (in the 3rd codon positions) with a command like
    msa_view --in-format SS --out-format SS \
         --tuple-size 1 >
You can now estimate a phylogenetic model with a command like
    phyloFit --tree "(human,(mouse,rat))" --msa-format SS \
        --out-root nonconserved-4d
If you have several alignments and several (corresponding) GFF files, e.g., for separate human chromosomes, you can extract 4d sites from each one using msa_view, then combine all the 4d sites using msa_view --aggregate, then estimate a phylogenetic model from the pooled data:
    for set in set1 set2 set3 ; do 
        msa_view $set.maf --in-format MAF --4d --features $set.gff \
            > $ 
        msa_view $ --in-format SS --out-format SS \
            --tuple-size 1 > $
    msa_view --aggregate human,mouse,rat * >
    phyloFit --tree "(human,(mouse,rat))" --msa-format SS \
        --out-root nonconserved-all-4d
(Here we have assumed the input alignments are in MAF format.)

Similar methods could be used to estimate a model for conserved regions, say, from 1st codon positions. This gets slightly more complicated, e.g.:

    msa_view data.maf --in-format MAF --features genes.gff \
        --catmap "NCATS = 3; CDS 1-3" --out-format SS --unordered-ss \
        --reverse-groups transcript_id >
    phyloFit --tree "(human,(mouse,rat))" --msa-format SS --do-cats 1 \
        --out-root conserved-codon1
See the msa_view and phyloFit help pages for details.

It should now be clear how option 3 would be accomplished: estimate conserved and nonconserved models using appropriate annotations files and phyloFit, then run phastCons as shown in Section 2.1. Option 2 is similar, but requires the use of the --estimate-rho option, e.g.,

    phastCons --target-coverage 0.25 --expected-length 12 \
        --rho 0.4 --estimate-rho mytrees --msa-format MAF \
        alignment.maf nonconserved-4d.mod > scores.wig
where nonconserved-4d.mod is a nonconserved model estimated from 4d sites. The parameter rho will be initialized to 0.4, then iteratively improved using an EM algorithm. (Note that a careful choice of initialization can improve running time substantially.) After rho has been estimated, the new conserved and nonconserved models will be written to mytrees.cons.mod and mytrees.noncons.mod, respectively, and program will carry on as before. (Here the new nonconserved model will be identical to nonconserved-4d.mod, but in some cases, e.g., if --gc is used, it will not be.) In this case, phastCons will output conservation scores but not discrete conserved elements, but it could be made to produce both types of output, or neither type of output (if only the estimate of rho were of interest).

Option 1 requires the use of --estimate-trees, which is similar to --estimate-rho:

    phastCons --target-coverage 0.25 --expected-length 12 \
        --estimate-trees mytrees --msa-format MAF alignment.maf \
        init.mod > scores.wig
In this case, phastCons will estimate all free parameters of the phylogenetic models by maximum likelihood, and will output the new models to mytrees.cons.mod and mytrees.noncons.mod. The given tree model, init.mod, will simply be used for initialization of the nonconserved model. It can be obtained with phyloFit, either using 4d sites, ARs, or similar (as above), or simply using the entire data set, e.g.,
    phyloFit --tree "(human,(mouse,rat))" --msa-format MAF \
        --out-root init alignment.maf
With option 2 or option 1, it may make sense to run phastCons twice, once for "training" (parameter estimation) and once for prediction. For example,
    phastCons --target-coverage 0.25 --expected-length 12 \
        --estimate-trees mytrees --msa-format MAF alignment.maf \
        init.mod --no-post-probs
    phastCons --target-coverage 0.25 --expected-length 12 \
        --msa-format MAF alignment.maf --most-conserved most-cons.bed \
        mytrees.cons.mod,mytrees.noncons.mod > scores.wig
The training step is typically much slower, and in some cases, it may be done with a subset of the data, for example, a random sample of 100-200 fragments from genome-wide alignments. Also, with large data sets, it may make sense to train separately on small fragments (possibly in parallel, on a compute cluster), then to combine the parameter estimates, then to do prediction with the combined estimates (see Section 4).

Note that options 1, 2, and 3 can all be used with or without --target-coverage and --expected-length (see Section 3).


3.1 Basics

The probabilities of transitioning between the states in the HMM are defined by two state-transition parameters, called mu and nu. The parameter mu is the probability of entering the nonconserved state from the conserved state, and nu is the probability of entering the conserved state from the nonconserved state; thus 1-mu and 1-nu are the probabilities of remaining in the conserved and nonconserved states, respectively. It turns out to be useful in some cases to reparameterize the state-transition probabilities in terms of a parameter gamma, which is the expected coverage by conserved elements (i.e., the expected fraction of bases that would be conserved if the model were used to generate the data), and a parameter omega, which is the expected length of a conserved element (again, if the model were used to generate the data). There is a one-to-one mapping between mu and nu on the one hand and gamma and omega on the other; that is, if you have mu and nu, you can easily compute gamma and omega, and vice versa. The details of how this works are given in the phastCons paper and are not important here.

Here is what is important to understand in order to use the program:

3.2 Tuning Parameters

While full maximum-likelihood estimation may seem at first glance to be the ideal way to approach the state-transition parameters, it does not work very well in practice -- especially with genomes having relatively low fractions of conserved bases (such as vertebrate genomes). The maximum likelihood estimate (mle) of the "expected length" parameter omega tends to be very large, leading to a small number of very long predicted conserved elements and "over-smoothed" conservation scores. Why this occurs is beyond the scope of this HOWTO; interested readers should consult the Supplementary Material to the phastCons paper, where mle's are discussed in some detail. Suffice it to say that real genomic sequences can only be described very approximately by a two-state hidden Markov model, and because of limitations of this approximation, the likelihood is often maximized in a biologically uninteresting region of the parameter space. For this reason, I recommend treating the state-transition parameters as tuning parameters.

The question then becomes what criteria should be used for adjusting the tuning parameters. There are two natural features to focus on. The first, called "coverage," is the fraction of bases in a data set that fall within predicted conserved elements. You might have a reason to aim for a particular level of coverage; for example, with mammalian data, a target of 5% might be selected, since this is considered a reasonable ballpark estimate of the fraction of sites under purifying selection in mammalian genomes. If you don't have a target in mind for the overall level of coverage, you can aim for a particular level of coverage of known functional elements such as protein-coding exons. (This was the method we used in the phastCons paper to calibrate the model in similar ways for vertebrate, insect, worm, and yeast data sets; we chose a target of 65% coverage of coding exons by conserved elements.) The target-coverage parameter (gamma) is a "knob" you can turn to reach a particular level of actual coverage. As you increase this parameter, more bases will be predicted to be in conserved elements, and as you decrease it fewer bases will be predicted to be in conserved elements. (Gamma can be thought of in a Bayesian sense as a prior, which has the effect of imposing a bias on the actual coverage.)

The second feature is called "smoothing." Smoothing describes the degree to which the conservation scores tend to be similar from one base to the next. A high degree of smoothing occurs when the probabilities mu and nu are small -- i.e., when changing states is unlikely -- and a lower degree of smoothing occurs when mu and nu are larger. Higher smoothing means the predicted conserved elements tend to be longer on average, and are more likely to include short sequences of nonconserved bases. Lower smoothing implies shorter, more fragmented conserved elements, containing fewer nonconserved sites. Increasing the expected length parameter (omega) tends to increase the level of smoothing and the average length of predicted conserved elements.

What is the right level of smoothing? This is perhaps a more difficult question than what is the right level of coverage. In many cases, it may be best just to set the smoothing so that the conservation plot "looks right." By plotting the scores next to a multiple alignment, as in the UCSC browser, it is usually not too hard to see if the plot is dramatically "over-smoothed" (in the extreme, it resembles a square wave function, with alternating regions of scores near 0 and scores near 1) or "under-smoothed" (in the extreme, the scores at adjacent sites appear essentially uncorrelated). It may also be useful to look at the shortest predicted conserved elements and see whether they are believable -- are they long enough and conserved enough to reasonably be called "conserved elements"? If not, perhaps the degree of smoothing should be increased. Similarly, one might look at sequences of nonconserved sites within conserved elements. If they are quite long and poorly conserved, yet the elements are not being broken, then perhaps the smoothing should be reduced. One might also look at the breaks between conserved elements (if they are too short, the smoothing should be increased) and sequences of conserved sites that have not been predicted as conserved elements (if they are too long, the smoothing should be decreased).

An alternative, perhaps more principled, way of setting the level of smoothing is to use the "phylogenetic information threshold" (PIT) method described in the phastCons paper. The paper should be consulted for details, but the PIT is essentially a threshold for the amount of "information" (in an information theoretic sense) that is required to predict a conserved element, on average. It is an alternative measure of the degree of smoothing (a higher PIT means more smoothing), which can be meaningfully compared across different data sets and phylogenies. For example, to achieve a given PIT -- say, 10 bits -- a larger expected length might be needed for a two-species data set, for which there is less information about conservation at each site, than for a ten-species data set. (The PIT actually considers the shape of the phylogeny and its branch lengths, not just the number of species.) A program in PHAST called consEntropy can be used to compute the PIT for a given set of parameters. The inputs to the program are the target-coverage and expected-length parameters and the conserved and nonconserved phylogenetic models (see the consEntropy help page and examples below). One can simply run consEntropy after adjusting the tuning parameters and re-estimating any free parameters, and compare the reported PIT to the target value; if necessary, the parameters can be adjusted and the procedure can be repeated (see below). There are cases in which the PIT method will not be appropriate. For example, when an alignment contains a lot of missing data, the PIT will be misleading, because there is actually less information per site than the phylogenetic models would imply. Also, consEntropy will not work with large trees, because, as currently implemented, its running time is exponential in the number of species.

Coverage and smoothing are to some degree separate, with coverage being primarily influenced by the --target-coverage option and smoothing being primarily influenced by the --expected-length option, but they are also interrelated. The final set of predictions depends on all parameters and on the data in a complicated way, and will be affected, sometimes unpredictably, by a change to any parameter. For example, changing the --expected-length option while holding --target-coverage fixed generally will alter coverage somewhat as well as smoothing. For this reason, the --target-coverage and --expected-length parameters should be tuned simultaneously, until constraints on both coverage and smoothing are satisfied.

3.3 An Example

Suppose we have an alignment of human, mouse, and rat sequences (with human as the reference sequence) and we wish to estimate the conserved and nonconserved models from the data using phastCons (option 3, above). We want to adjust the tuning parameters such that 65% of bases in coding exons are covered by conserved elements and the PIT is 10 bits. We already have a starting model called init.mod.

We begin with a guess at the values of the tuning parameters. In this case, a starting target-coverage of 0.05 seems appropriate, since our prior belief is that about 5% of bases are conserved, but we have to consider the fact that only about 40% of the human bases align to mouse or rat, and phastCons will not predict conserved elements in regions of no alignment. Therefore, we start instead with a target coverage of 0.05/0.4 = 0.125, the fraction of aligned bases we expect to be conserved. We'll guess at a value of 20bp for the expected length. The average length of conserved elements is likely to be considerably longer -- probably closer to 100bp -- but we want to influence the model toward the detection of shorter conserved elements, e.g., possible transcription factor binding sites. One way of thinking about it is that we want the conservation score at each base to reflect an average "window" of about 20bp centered at that site. Remember, these are just rough starting values; we're going to adjust them as we go.

A starting estimate of the tree models can be obtained with a command such as:

    phastCons --target-coverage 0.125 --expected-length 20 \
        --estimate-trees v1 --most-conserved most-cons.bed \
        --log optim.log --no-post-probs --msa-format MAF \
        alignment.maf init.mod 
(The progress of the program can be charted by monitoring the log file, optim.log.) Now we need to see how close we are to our coverage and smoothing targets. We'll need a program to compute the intersection of a set of coding regions and the predicted conserved elements. Jim Kent's featureBits is ideal for this, but other tools could be used as well. Suppose we have a bed file CDS.bed, containing the coordinates of coding exons in the region of interest. A featureBits command such as
    featureBits -enrichment hg17 CDS.bed most-cons.bed
will measure the coverage of the regions in CDS.bed by the regions in most-cons.bed. In this case, featureBits reports
    CDS.bed 0.159%, most-cons.bed 0.233%, both 0.102%, cover 64.25%, enrich 275.73x
indicating coverage of 64.25%, only slightly below our target. To test smoothness, we run consEntropy:
    consEntropy 0.125 20 v1.cons.mod v1.noncons.mod
and obtain the following output
    Transition parameters: gamma=0.125000, omega=20.000000, mu=0.050000, nu=0.007143
    Relative entropy: H=0.295299 bits/site
    Expected min. length: L_min=49.071119 sites
    Expected max. length: L_max=24.350034 sites
    Phylogenetic information threshold: PIT=L_min*H=14.490669 bits
Thus, the PIT is about 14.5 bits, somewhat higher than desired.

Now we rerun the program with new values for the tuning parameters. We might try reducing the expected length to 15 and leaving the target-coverage the same, because we don't know how the new expected length will affect the coverage.

    phastCons --target-coverage 0.125 --expected-length 12 \
        --estimate-trees v2 --most-conserved most-cons.bed \
        --log optim.log --no-post-probs --msa-format MAF \
        alignment.maf v1.noncons.mod 
An important point here is that the free parameters (in this case the tree models) are being re-estimated with the new value of the tuning parameters. One might be tempted just to redo the prediction step, with the previously estimated tree models, but I don't recommend this shortcut: you'd be mixing tree models trained under one set of assumptions with a prediction method based on another set of assumptions. Note also that the previous estimate of the nonconserved model has been used for initialization; this can sometimes lead to faster convergence.

This time we measure a coverage of 58.7% and a PIT of 14.3 bits. Apparently, the reduced smoothing (i.e., lower expected length) has resulted in a greater separation between the conserved and nonconserved models: there are fewer nonconserved sites mixed in with the conserved ones, making the conserved model more conserved, and there are fewer conserved sites mixed in with the nonconserved ones, making the nonconserved model less conserved. As a result, the phylogenetic information per base has increased, and despite the reduced smoothing, the PIT has stayed roughly the same. In addition, the coverage has decreased a bit, possibly because fewer nonconserved sites flanked by conserved sites are being pulled into conserved elements. It seems as though we've lost ground, but we just need to be persistent. Let's try reducing the expected length a bit more and cranking up the target coverage to compensate for it:

    phastCons --target-coverage 0.2 --expected-length 8 \
        --estimate-trees v3 --most-conserved most-cons.bed \
        --log optim.log --no-post-probs --msa-format MAF \
        alignment.maf v2.noncons.mod 
This time we obtain a coverage of 59.2% and a PIT of 12.6 bits -- closer, but still not quite there. At any rate, you see how it works: we simply iterate this procedure until we're close enough to our target values. Usually this doesn't take more than six or seven iterations.

Tuning the program is a bit of an art: the parameters are all interrelated, and it takes a little time to get a feeling for what effect each change will have. As you get better at it, you'll need fewer iterations to reach convergence.


The examples above have all assumed that phastCons is to be run on a small to moderate-sized data set, consisting of at most, say, a dozen sequences and a few million sites (alignment columns). Additional care needs to be taken with larger data sets. The main problem is parameter estimation. If all parameters are fixed, the running time of the program is proportional to the product of the number of species, n, and the number of sites in an alignment, L, with a reasonably small proportionality constant. This O(nL) algorithm is practical for use use with quite large data sets, given sufficient RAM. The EM algorithm for parameter estimation, however, must perform a similar O(nL)-time computation many times and between iterations must also execute a potentially expensive numerical maximization routine. Straightforward parameter estimation (as discussed above) will not be practical with extremely large data sets.

The techniques I recommend are, basically, (1) to "divide and conquer" and make as much use as possible of parallel processing, and (2) to make the estimation problem as easy as possible. In this section, I will discuss two different examples of large data sets, one consisting of a moderate number of species and a large number of sites, and the other consisting of a large number of species and a moderate number of sites. A "divide and conquer" strategy is the key to addressing the first problem and making the estimation problem easier is the key to addressing the second problem. Note that the two strategies I will describe are essentially orthogonal, and could be used in combination with a data set having many sequences *and* many sites.

4.1 Many Sites, Not Too Many Sequences

First let's consider the case of a set of alignments with a modest number of species and a large number of sites. Assume these are whole-genome alignments, referenced to the human genome. Physically, they are represented by a MAF file for each human chromosome. This is essentially the situation we face for the conservation tracks in the UCSC Genome Browser: given a set of genome-wide MAFs, obtain corresponding base-by-base conservation scores and predicted conserved elements.

Here's the recommended "divide and conquer" approach for this case:

  1. Split the alignments into small fragments (each spanning about 1Mb of the human genome)
  2. Estimate parameters for each fragment, separately and in parallel.
  3. Combine the separately estimated parameters by averaging.
  4. Predict conserved elements and conservation scores globally using the combined estimates
  5. Adjust tuning parameters and return to step 2, if necessary. Repeat until coverage and smoothing targets are met.
Note that the strategy of separately estimating the parameters locally and then averaging them genome wide is an approximation -- it's not guaranteed to result in true maximum likelihood estimates of free parameters. In practice, however, the estimates seem to be quite close to true mle's, and the method is much more logistically straightforward than trying to do true maximum likelihood estimation at the whole-genome level. For this approach to be reasonable, it's important that the fragments contain similar amounts of data (so that an unweighted average is reasonable) and that they are as large as possible (so that sampling error is minimized).

Let's discuss the steps one by one.

  1. Split the alignments into small fragments

    This can be accomplished by running a program called msa_split on each MAF. Use a command like the following:

            mkdir -p CHUNKS            # put fragments here
            for file in chr*.maf ; do
                root=`basename file .maf`
                msa_split $file --in-format MAF --refseq $root.fa \
                    --windows 1000000,0 --out-root CHUNKS/$root --out-format SS \
                    --min-informative 1000 --between-blocks 5000 
    This will cause each MAF file ($root.maf) and the corresponding reference sequence ($root.fa), to be read in, assembled in memory into a kind of pseudo-global alignment (with local alignments tiled beneath the human sequence), and then split into non-overlapping chunks of about 1,000,000 sites each; these chunks will be written in SS format to files with a prefix of $root in the CHUNKS directory. The --min-informative option causes fragments with fewer than 1000 "informative" sites (i.e., with at least two aligned bases) to be discarded, and the --between-blocks option tells the program to try to split between alignment blocks if possible, e.g., in recent transposon insertions in the human genome. (It will adjust the split point by at most 5000 sites.) The file $root.fa must be in FASTA format and must use the same coordinate system as the multiple alignment, as will be true if the FASTA files and MAFs from the same version (assembly) of the UCSC Genome Browser are used. See the msa_split help page for additional details.

    If desired, this step can be done in parallel, with one process per MAF file. Beware of the fact that each process will be quite I/O intensive.

  2. Estimate parameters for each fragment.

    In this step, we will estimate free parameters using phastCons for each alignment fragment. Because the number of species is relatively small, we can get away with full estimation of the conserved and nonconserved models (option 3 from Section 2.2). Suppose we already have a starting model, init.mod. We might use a command like:

            mkdir -p TREES     # put estimated tree models here
            rm -f TREES/*      # in case old versions left over
            for file in chr*.*.ss ; do 
                root=`basename $file .ss` 
                phastCons --target-coverage 0.125 --expected-length 20 \
                    --gc 0.4 --estimate-trees TREES/$root \
                    $file init.mod --no-post-probs
    Here, the --gc option is used to ensure that the same G+C content is assumed for all fragments, since the same G+C content will be assumed during prediction. Note that the --log and --quiet options may be useful here as well.

    If you have a compute cluster at your disposal, you will probably want to execute these individual processes in parallel.

  3. Combine the separately estimated parameters by averaging A program in PHAST called phyloBoot, which is primarily designed for bootstrapping, supports averaging of model parameters. It can be used to obtain average conserved and nonconserved models as follows:
            ls TREES/*.cons.mod > cons.txt
            phyloBoot --read-mods '*cons.txt' --output-average ave.cons.mod 
            ls TREES/*.noncons.mod > noncons.txt
            phyloBoot --read-mods '*noncons.txt' --output-average ave.noncons.mod 
    PhyloBoot will output a summary of the means, standard deviations, medians, etc. of each parameter. You may want to scan this summary to convince yourself that the standard deviations are not too large and that no outliers have severely skewed the means. The means and medians should be roughly equal and the standard deviations should be fairly small compared to the means. (Some larger standard deviations for branches near the root of the tree are normal.) There are sometimes a few zeroes in the 'min' column, especially for short branches; this is okay, but beware of very large values in the 'max' column, which could result from a failure to converge properly, and might skew the mean. If you see any bad outliers, you should re-estimate with different starting parameters or track down and discard the responsible .mod files. I've never had to do this; the estimates generally are well behaved.

  4. Predict conserved elements and conservation scores globally using the combined estimates.

    This is simply a matter of running phastCons on each fragment, using the averaged tree models.

            mkdir -p ELEMENTS SCORES
            rm -f ELEMENTS/* SCORES/*
            for file in chr*.*.ss ; do 
                root=`basename $file .ss` 
                phastCons --target-coverage 0.125 --expected-length 20 \
                    --most-conserved ELEMENTS/$root.bed --score \
                    $file ave.cons.mod,ave.noncons.mod > SCORES/$root.wig
    Note that the same --target-coverage and --expected-length arguments are used as in step 2, that this time we have asked the program to predict both discrete elements and base-by-base conservation scores, and that the tree models from step 3 have been used. The --score option tells phastCons to compute log odds scores for predicted elements. The --gc option is no longer necessary (ave.cons.mod and ave.noncons.mod contain the background distribution), but the --seqname and --idpref options could be useful in this step. (The default names and ids are obtained from the filenames, and in this case should be okay).

    The *.bed and *.wig files can now be combined across the whole genome or across each chromosomes, if desired, e.g.,

            cat ELEMENTS/*.bed | sort -k1,1 -k2,2n > most-conserved.bed
  5. Adjust tuning parameters and return to step 2, if necessary. Repeat until coverage and smoothing targets are met.

    Suppose our targets are 65% coverage of coding exons and a PIT of 10 bits. At UCSC, we would test the first constraint using the featureBits program, e.g.,

            featureBits -enrichment hg17 knownGene:cds most-conserved.bed
    The second constraint could be tested with consEntropy, e.g.,
            consEntropy .125 20 ave.cons.mod ave.noncons.mod
    As in Section 3, we adjust the tuning parameters as needed, only now we repeat steps 2, 3, and 4 after each adjustment, until convergence.

4.1.1 Random Samples and Local Parameter Estimates

With a large data set, e.g., genome-wide vertebrate alignments, little is gained by performing step 2 on the whole data set. The quality of the estimates will be affected little, and compute time may be reduced substantially, by estimating the free parameters from a random sample of, say, 100 to 200 alignment fragments. This can be accomplished as follows. The first time you execute step 2, define a random sample of fragments using a command such as
    ls chr*.*.ss | chooseLines -k 100 - > random-sample.txt
where chooseLines is a utility in PHAST that will randomly select k lines from a given input file or stream. Now each time you estimate the parameters, use a command such as
    mkdir -p TREES     # put estimated tree models here
    rm -f TREES/*
    for file in `cat random-sample.txt` ; do 
        root=`basename $file .ss` 
        phastCons --target-coverage 0.125 --expected-length 20 \
            --gc 0.4 --estimate-trees TREES/$root \
            $file init.mod --no-post-probs
Another thing to note is that one could make predictions based on locally estimated parameters rather than global averages, if desired, by skipping the averaging step (step 3), and substituting the following for the call to phastCons in step 4.
    phastCons --target-coverage 0.125 --expected-length 20 \
        --most-conserved ELEMENTS/$root.bed --score \
        $file TREES/$root.cons.mod,TREES/$root.noncons.mod \
        > SCORES/$root.wig
(Actually, in this case, steps 3 and 4 could be combined if desired.) In some ways this might be preferable to the approach described above -- e.g., predicted elements and conservation scores would reflect local deviations from the average G+C content or neutral substitution rate. However, it also potentially makes the program's output for different regions of the genome more difficult to compare. For this reason, I prefer the use of global averages. This issue is discussed in the Supplementary Material to the phastCons paper.

4.2 Many Sequences, Not Too Many Sites

Now let's consider the case of a set of alignments containing a fairly large number of sequences but a modest number of sites. The example I have in mind here is the ENCODE data set (,, which consists of 44 separate alignments (for the 44 ENCODE regions) with between 18 and 23 sequences of 500kb to 1.9Mb in length (the number of columns in the alignments is somewhat larger, because of gaps). PhastCons can actually compute conserved elements and conservation scores for any one of these alignments quite rapidly, and even if the data is pooled for the purposes of parameter estimation, it can handle the aggregate alignment (with approximately 35 million sites) without too much trouble, on a machine with sufficient RAM. The main problem here is the number of free parameters. If phastCons were run with the --estimate-trees option, 43 branch length parameters would have to be estimated, in addition to the scaling parameter rho and a few parameters describing the substitution process. The EM algorithm will converge very slowly with such a large number of free parameters.

In this case, I recommend making the estimation problem easier. The best way to do that here is probably to estimate the nonconserved model from fourfold degenerate sites, as described in Section 2.2, and then to let phastCons estimate only the parameter rho (option 2 from section 2.2). We'll work with an aggregate alignment constructed from the original alignments for the purposes of parameter estimation. Suppose we start with a set of FASTA-formatted files named for ENCODE regions, ENm001.fa, ..., ENr334.fa. We can concatenate these into a single file using msa_view with a command like:

    msa_view --aggregate {species-list} EN*.fa > all-encode.fa
where {species-list} is a comma-separated list of all sequence names in the alignments, e.g., "human,chimp,baboon,macaque,..." It will also save some time during re-estimation to convert this file to an ordered SS file:
    msa_view all-encode.fa --out-format SS >
Note that this aggregate alignment contains some adjacent pairs of columns that are not really adjacent, i.e., in the 43 places where two ENCODE regions have arbitrarily been joined together. The number of such columns is very small compared to the overall size of the data set, however, and its impact on the parameter estimates should be negligible.

For parameter estimation, we can use a command like:

    phastCons --target-coverage 0.05 --expected-length 20 \
        --gc 0.4 --most-conserved all-encode.bed --no-post-probs \
        --estimate-rho newtree nonconserved-4d.mod 
where nonconserved-4d.mod is the previously estimated model for 4d sites and the --gc option has been used to force the background distribution to be closer to the genome-wide average than the G+C-rich distribution estimated from 4d sites. Let's suppose in this case we are aiming for a total coverage by conserved elements of 5% of all bases in the reference (human) sequence. (Our initial guess for --target-coverage is 0.05 because in this case, essentially the entire human sequence is covered by alignments with other species.) We can easily measure the coverage of all-encode.bed with a command such as:
    awk '{total += $3 - $2} END {print total/29909540}' all-encode.bed
where 29,909,540 is the total number of bases in the data set in the coordinate system of the human sequence. (Note that phastCons will automatically report the conserved elements in this coordinate system.)

ConsEntropy cannot be used in this case to test the smoothness constraint, because of the large number of sequences. Instead, I recommend plotting the conservation scores and judging the smoothness by eye, as discussed in Section 3.2.

When the coverage and smoothness constraints have been met, predictions for the individual targets can be obtained with commands such as:

    for reg in EN*.fa ; do
        root=`basename reg .fa`
        phastCons --target-coverage  --expected-length  \
            --most-conserved $root.bed \
            newtree.cons.mod newtree.noncons.mod > $root.wig
where and are the final estimates of target-coverage and expected-length, respectively. These commands can be executed in parallel, if desired.



This section will cover the use of the Felsenstein/Churchill model, the indel models, the discrete gamma model for rate variation, the coding-potential feature, and other obscure and experimental features.



J. Felsenstein and G. Churchill, 1996. A hidden Markov model approach to variation among sites in rate of evolution. Mol Biol Evol, 13:93-104.

E. H. Margulies, M. Blanchette, NISC Comparative Sequencing Program, D. Haussler, and E. D. Green, 2003. Identification and characterization of multi-species conserved sequences. Genome Res, 13:2507-2518.

C. Mayor, M. Brudno, J. R. Schwartz, et al., 2000. VISTA: visualizing global DNA sequence alignments of arbitrary length. Bioinformatics, 16:1046-1047.

A. Siepel, G. Bejerano, J. S. Pedersen, et al. Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res (in press)

A. Siepel and D. Haussler, 2005. Phylogenetic hidden Markov models. In R. Nielsen, ed., Statistical Methods in Molecular Evolution, pp. 325-351. Springer, New York.

Z. Yang, 1995. A space-time process model for the evolution of DNA sequences. Genetics, 139:993-1005.


If this document doesn't include information you were looking for or if you found parts of it (or all of it!) confusing please send me feedback ( so I can improve it. You can even let me know if you found it helpful :)


Thanks to several patient users for their feedback on early versions of the software and documentation, especially Kate Rosenbloom, Hiram Clawson, Angie Hinrichs, Elliott Margulies, Daryl Thomas, David King, and James Taylor.