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## Troubleshooting LAMARC

This article lists some common sources of trouble, and suggestions on how to fix them.

### LIST OF FAQS:

1. The program will not compile on my machine.
2. The program says it can't find my data file, but it's right here.
3. My data file can't be read at all.
4. The data converter mangles my file.
5. The program crashes early or complains about lack of memory.
6. The program runs much too slowly.
7. How can I tell if I've run the program long enough?
8. Some of my parameter estimates are ridiculously high--ten or twenty digits. This can't be right.
9. My estimates have enormous error bars.
10. What does Theta mean if I have mtDNA (mitochondrial DNA) instead of nuclear DNA? Do I need to divide it by four?
11. The program works when I use a small or low-polymorphism data set, but crashes on a larger or higher-polymorphism data set.
12. I get a long warning message stating that my 'data may be difficult to model'--what does this mean?
13. My profile likelihood tables look ragged rather than smoothly curved--is something wrong?
14. The program stops with a message 'Unable to create initial tree. Starting parameter values may be too extreme;'--what does this mean?
15. Which microsatellite model should I use?
16. How can I do a likelihood ratio test using LAMARC?
17. Why can't the program use other data types, other data models, or other evolutionary forces?
18. What happened to the 'Normalize' option in previous versions of LAMARC?
19. Does LAMARC use 'site 0'? Do I?
20. How can I report a bug or inadequacy of the program or documentation?

1. "The program will not compile on my machine."

This is covered in a separate article, "Compiling LAMARC". You may also want to see if one of our pre-made executables will work for you.

2. "The program says it can't find my data file, but it's right here."

Check to see if your filename has an invisible extension. LAMARC does not think that "infile.xml" and "infile" are the same. Also check to make sure your file is in the folder or directory you think it is.

3. "My data file can't be read at all; the program crashes immediately or prints errors that have nothing to do with anything in my file."

Did you save your input file as a Word document, RTF, or some other fancy format? It needs to be plain unformatted text.

An early crash may also be a symptom of lack of memory; see the out of memory section.

4. "The data converter mangles my file."

Check to see if you are using the correct option for "interleaved" versus "sequential" data in conversion. Interleaved data presents the first line of sequence 1, then the first line of sequence 2... and eventually the second line of sequence 1, sequence 2, etc. Sequential data presents all of sequence 1, then all of sequence 2, and so forth. Misrepresenting one as the other will cause your sequence names to be treated as nucleotides and vice versa, with disastrous results.

5. "The program crashes early or complains about lack of memory."

On many Macintosh systems you can use the Finder to allocate more memory to a specific program, and you'll probably need to do this for LAMARC; the defaults are too low.

In general, if you suspect that there's not enough memory, try a smaller subset of your data for a trial run. Important: if you decide that you need to produce your final results based on a subsample of your data, the subsample must be random. It is allowable to leave out whole genetic regions or populations, but if you decide instead to leave out individual sequences or sites, choose them randomly. Leaving out the "boring" invariant sites or identical sequences will severely distort the results. Similarly, if you leave out genetic regions, choose them at random; don't preferentially choose the least polymorphic ones.

Decreasing the number of sampled genealogies will reduce memory demands somewhat, at a cost in accuracy. You will want to increase the interval between samples at the same time, so as to make each sample as independent (and thus informative) as possible.

LAMARC is a large program and realistic cases will require a computer with generous memory. Our development machines have about 2 gigabytes of RAM. Probably under about 500 megabytes the program will not work except for toy cases.

You may also want to consider whether you are asking for too many populations and parameters; see below.

6. "The program runs much too slowly."

If you compiled LAMARC yourself from source code, optimization may help (though some optimizers produce buggy code, so use at your own risk). The executables we supply are optimized to the best of our ability.

Running a smaller case may help. Please note that you cannot safely leave out "boring" data such as invariant sites or identical individuals (details here). We find that the information value of additional individuals is quite low beyond twenty individuals, so if you are running 50 individuals per population you can probably cut them randomly down to 20 and get good results.

If the program has barely enough memory it may "thrash", wasting a lot of time on memory management. (You can often tell if thrashing is occurring by listening to your computer; many will whirr or rattle from the constant hard disc access.) Adding more memory may help.

If you are estimating recombination, and the program runs well at first but then slows down, it may be adding more and more recombinations to the genealogies. You can set the "maximum number of events" parameter lower, but doing so risks a downward bias in your estimate. It's a good solution to rare slow-down periods, but not a good solution to a whole run full of highly recombinant trees. The latter may indicate that your sequence spans too long a region and the ends are essentially unlinked. LAMARC is not able to meaningfully estimate the recombination rate across unlinked sequences, and will bog down if asked to try. You can diagnose this problem by noticing high "dropped" counts in the runtime reports. (The "runtime reports" are given at the very end of your output file. These contain information about possibly interesting things that happened while the program was running.)

Similarly, if you are estimating migration and the program bogs down, you may have identified two groups as separate populations which are really one panmictic population. LAMARC cannot usefully estimate the migration rate in this situation, and will bog down trying. Consolidating the problematic populations together may get better results. The program STRUCTURE can be useful for detecting non-differentiated populations.

Profiling is expensive, and switching to fixed-point rather than percentile profiles, or eliminating profiles for some or all parameters, will help considerably. (But be sure you aren't eliminating information that you really need.) You should also be aware that some profiles take longer than others, and the estimate of time to finish profiling is very rough--it is not unusual for profiling to take two or three times as long as predicted, if the prediction happens to come from an easy profile and there are several hard profiles in the set.

Setting the output file verbosity to "concise" should drastically reduce the amount of time profiling takes, since the number of profiles calculated for each parameter is two instead of eleven. If you are writing a tree summary file, you will be able to re-load that file and run with different profiling options later.

LAMARC is a computationally intensive approach and simply won't succeed with really complex problems. For example, if you have twenty populations all exchanging migrants, you are trying to estimate 400 parameters. The amount of data required to do this would be very high; the amount of computation would be staggering. Try breaking your problem into subproblems. Constraining sets of these parameters to be zero, or to be identical, can greatly reduce the complexity of the problem and increase your chance of a good solution.

Finally, it's worth asking yourself how long the data took to collect. If they took several years to collect, an analysis which takes several weeks shouldn't seem too long. Run small pilot cases first to get an idea of the scale of the problem.

Some useful rules of thumb:

Adding more sequence length slows the program down, but less than linearly with the amount of sequence. This is the best way to refine an estimate of recombination rate in a single region.

Adding more individuals slows the program down linearly with the number of individuals, and you will also need to run more steps in your chains to get equivalently refined results, as the search space is bigger. We find that 20 individuals per population is usually enough, and we have never seen a use for more than 100.

Adding more genetic regions (loci) slows the program down linearly with the number of regions. This is far and away the most effective at improving estimation of Theta or migration. If you can choose between adding more individuals or adding more regions, always add more regions once you have 20 individuals per population.

If you have microsatellite data, the Brownian-motion approximation is much faster than the stepwise model. It is also a very good approximation except when population size is low. The usual symptom of breakdown in the Brownian model is data log-likelihood estimates of 0.0. If you see many of these, especially in the final chains of your search, the Brownian approximation is not safe for your data and will produce an upwards bias. In all other cases, however, we recommend it.

7. "How can I tell if I've run the program long enough?"

This is covered in a separate article, "Search Strategy."

8. "Some of my parameter estimates are ridiculously high--ten or twenty digits. This can't be right."

It is possible for a data set to be so uninformative with regard to migration (or, more rarely, recombination) that the likelihood surface is flat, or almost flat. This can lead to an almost infinite estimate of the parameter.

This is particularly common in migration cases where you are trying to estimate too many parameters from a small amount of data. Consider a case where you have only 1 individual from a certain population, and he turns out to have been a recent migrant. How big is that population? What are its migration rates to other populations? LAMARC really can't tell, and this is reflected by a flat likelihood surface. You can verify this by examining the profiling results.

If you think that some parameter really cannot be estimated, holding it fixed at a reasonable value can rescue your ability to estimate other parameters.

A second possible explanation is that you've run too few chains or chains that are too short. You can try running longer ones.

A third explanation, particularly for huge estimates of Theta, is that your data aren't correctly aligned and so appear much more variable than they should. It can be helpful to ask the program to echo back the input data, and examine it for alignment problems.

If some of your estimates are huge, the rest may be all right, but it is not wise to rely on this. It's better to reduce the problem until all estimates are reasonable.

LAMARC's strange behavior with inadequate data is not a program bug; if the likelihood surface for the given data really is flat, there's nothing the program can do to get an intelligent estimate. Running LAMARC in Bayesian analysis mode will produce text files containing portraits of the likelihood surface; these files can confirm whether the surface is flat.

9. "My estimates have enormous error bars."

While this might possibly improve with a longer run, it is usually an accurate reflection of your data. (In fact, a too-short run more often produces error bars that are narrower than they should be.) You might also try re-running with multiple replicates or heating.

If possible, add more genetic regions. If you can't do that, add additional data to the regions (longer sequences) or more individuals. In some cases (e.g. HIV sequences) additional individuals are the only possible way to improve your data set, and you'll have to be aware that you may never be able to get a really tight estimate.

Please do not ignore the error bars. They are there for a reason.

10. "What does Theta mean if I have mtDNA (mitochondrial DNA) instead of nuclear DNA? Do I need to divide it by four?"

Theta is always "number of heritable copies in the population * 2 * mu". If you put in mtDNA, the value that comes out will be 2Nf * mu, where Nf is the effective number of females. You do not need to divide it by four. A similar argument applies to Y chromosome DNA.

If you have both mtDNA and nuclear DNA, be sure to indicate to the program that they have different effective population sizes, either by setting the effective population size of the mtDNA region to 1 and of the nuclear DNA region(s) to 4, or by setting the effective population size of the mtDNA region to .25 and of the nuclear DNA region(s) to 1.

Also note that if you collected data from different sections of the mitochondrion, all data should be put in the same genomic region. If the relative mutation rates are different, you can put them in different segments, but then put both segments together in the same region. You will seriously underestimate your support intervals if you claim that each section is its own region.

11. "The program works when I use a small or low-polymorphism data set, but crashes on a larger or higher-polymorphism data set."

This may be a symptom of running out of memory (see previous questions). You should also check whether your data are aligned correctly; improperly aligned data will look like excessive polymorphism.

12. "I get a long warning message stating that my 'data may be difficult to model'--what does this mean?"

Some of the above items have discussed consequences of telling LAMARC that your data comes from two populations when it really comes from one, providing LAMARC with an inadequate amount of data, and analyzing highly polymorphic data. These high-level, big-concept problems can trigger low-level numerical problems which the program cannot relate to the big picture; the best it can do is describe the low-level problem.

When performing a maximum-likelihood analysis, LAMARC searches the likelihood surface for its maximum height. It does this once after each Markov chain, and several times more if parameter profiles are turned on. In rare cases, two shapes of surface can arise that are intractable and lead to warning messages.

One problem case is a flat surface (discussed above), or a surface that continues to rise beyond a reasonable value for one or more parameters. This implies that your data has insufficient power to accurately estimate the population parameters. The following warning message may appear:

    Warning!  Encountered a region of the log-likelihood surface in which the
    log-likelihood increases steadily, seemingly without an upper bound.
    This implies that your data is difficult to model.  The problematic
    parameter is parameter <your parameter name>; it has been increased
    or decreased to a value of  <some number>, and the maximum lnL,
    if one exists, seems to lie beyond this value.  The maximization routine
    is terminating....

Another type of problem surface is very spiky with multiple peaks and valleys. This can result when combinations of parameter values exceed some machine-specific threshold; for example, their product can become too large to store in the allotted amount of memory, or their quotient or difference can become too small to be distinguishable from zero. The following warning message may appear:

    Warning!  Calculated a log-likelihood of <some number> for the
    parameter vector p = (<some numbers...>), and determined that
    a greater log-likelihood could be found in a certain direction, but
    no greater log-likelihood was found in that direction.  This implies
    that your data may be difficult to model, or that there is a problem
    with lamarc.  The maximization routine is terminating....

(Those interested in the math may like to know that the problem is detected when the surface's gradient becomes inconsistent with the surface's height.)

If you receive either of these warning messages, or a message very similar to these, you may be able to ignore it if it only within one or two of the earlier Markov chains in your series of chains. The more reasonable the ultimate results are, the safer it is to ignore warnings appearing early or infrequently in your run. If you receive this type of message late or frequently in a run, then the ultimate results should be considered dubious.

If you receive the "... increases steadily, seemingly without an upper bound ..." warning, then you may be able to achieve better results by reducing the number of parameters you are estimating, or analyzing a subset of your data. If you receive the "... no greater log-likelihood was found in that direction ..." warning, then you can try proceeding in the same manner, but troubleshooting is much more difficult in this case. We encourage you to contact us and provide us with a copy of your data if you encounter this latter warning: doing so would help us as we continue to research ways of cleanly coping with these computational challenges.

13. "My profile likelihood tables look ragged rather than smoothly curved--is something wrong?"

Occasionally LAMARC, run in likelihood mode, encounters a likelihood surface it simply can't maximize reliably, often because it has more than one maximum. One symptom of this is ragged profile tables where the values of the parameters jump around from line to line rather than increasing or decreasing smoothly. When you see this, none of your estimates, even the MLE, are completely reliable. Ideas for improving the situation include running the program longer (more chains, longer chains or both) or reducing the number of parameters you are trying to estimate.

The Bayesian mode of LAMARC, which maximizes its parameters one at a time rather than jointly, is less prone to this but you may see the very similiar symptom of curvefiles with multiple spikes in them. Again, collecting more samples by running LAMARC longer, or simplifying the problem so that fewer samples are needed, are your best bets.

14. "The program stops with a message 'Unable to create initial tree. Starting parameter values may be too extreme; try using more conservative ones.' --what does this mean?"

The initial tree for the search (also called the "de novo tree") is created based on the starting parameters (either calculated from the data or provided by the user). Attempts to make a de novo tree may fail because too many migrations or recombinations are put in. The program will try 100 times to make a de novo tree, but if every one of them has too many events it will give up in order to avoid an infinite loop.

This error suggests that the current starting values for recombination or migration are far too high, given the currently specified upper limits on recombination or migration events. A common cause is breakdown of the FST calculation for migration rate. Check the starting values and make sure they are reasonable. When in doubt, try a slightly lower value; the program can adjust it upwards if necessary. Don't use extremely low values for migration (below 0.001) however; these can cause the program to become stuck at low values for a very long time.

15. "Which microsatellite model should I use?"

Try the Brownian-motion model first, since it is much faster. Consider switching to the stepwise-mutation model if you see signs, in the runtime reports, of failure of the Brownian approximation. These take the form of data log-likelihoods of 0.0. If many of these appear, or any appear in the final chains, switch to the stepwise model. You may want to start with the stepwise model if you know that your population size(s) are very small, since this is the weak point of the Brownian approximation.

16. "How can I do a likelihood ratio test using LAMARC?"

The short answer is that you can't. The "likelihoods" produced by the program are relative likelihoods, and they are meaningful only within one run--there is no way to compare them across runs. (They represent the answer to the question "How much better do the sampled trees fit the maximum likelihood values than the values they were sampled from?")

However, approximate confidence intervals based on the shape of the curve are possible. LAMARC presents these in two ways: as the percentile profiling in the MLE tables, and as full profile-likelihood tables (if requested). These should enable you to get a picture of the uncertainty in your analysis.

17. "Why can't the program use other data types, other data models, or other evolutionary forces?"

For version 2.0 we have included almost all of the commonly available mutational models. We do not have provision for RFLP or protein sequence data, because the existing maximum likelihood algorithms for these are agonizingly slow, or for AFLP data, because no one has yet developed an AFLP maximum likelihood algorithm. (If you succeed in doing so, and it runs at a reasonable speed, we will be happy to add it to LAMARC.) Most other data types can be accomodated with the K-Allele model.

New evolutionary forces are more difficult, but we will be slowly increasing the number of forces supported. Our next major project is natural selection.

If you are a programmer, you may also want to consider adding new data types or models yourself. We have tried to write LAMARC in a modular fashion that will accommodate additions fairly well. Only time will tell if we've succeeded. Feel free to write and ask questions about possible additions.

18. "What happened to the 'Normalize' option in previous versions of LAMARC?"

The program now automatically checks to see if normalization is needed, and turns it on if so. Normalization will not be needed for the majority of data sets, and since it causes a significant decrease in speed if on (and because the option was confusing to many of our users), we made control of this option automatic. If "verbose" runtime reports are selected, LAMARC will note when this occurs. If you feel that normalization is necessary for your data, the option remains to turn it on in the XML.

19. "Does LAMARC use 'site 0'? Do I?"

To our consternation, we recently discovered that the common biological naming convention is to call the site that's to the left of site 1, "site -1" instead of site 0. All versions of LAMARC prior to v2.1 do *not* follow this convention, so if you claimed that one of your SNPs was at site -5, and another SNP was at site 5, LAMARC would assume those SNPs were 11 nucleotides apart, and not 10. This probably doesn't make a huge difference, but it's probably worth fixing once you know.

As of version 2.1, the converter program lam_conv examines your data, and if you never use a '0' for a 'map position' or a 'first position scanned' (aka 'offset'), it assumes that you fall in the majority case, and that all your negative sites are one base closer to the positive ones than we previously believed. When it creates a LAMARC input file, it adds one to all your negative numbers, so if you tell the converter you have a SNP at site -5, and then examine the LAMARC input file, you will see '-4' in the list instead.

Because LAMARC usually doesn't report its results in terms of actual sites, this change will likely be invisible to you, and the only difference will be that LAMARC will now be a bit more precise.

However, if you're using our 2.1-introduced mapping feature, these results are reported in terms of the sites where the trait has been mapped. As such, it's more important to know whether there is a 'site 0' or not, assuming you have any negative map positions. Here, we let you have it both ways: in the XML, under the 'format' tag, the converter writes a 'convert-output-to-eliminate-zero' tag, which is set to 'true' unless (as noted) you ever used a '0' for a map position or first-position-scanned. When this is set 'true', LAMARC will assume you are following traditional biologist convention, and convert its values to the 'non-zero' scale before displaying them. This means that if it tells you that your trait might be mapped to sites "-1:1", it is talking about two sites, and not three. It also means that the final list of sites in the output file will skip directly from -1 to 1:

    -3         0.00079395
    -2         0.00079395
    -1         0.00078690
    1          0.00078688
    2          0.00078688

So, how can you tell if you yourself are using a system that includes a 0 or not? If all you have are positive numbers, it makes no difference, and you can safely ignore it. If you got your numbers from a genome browser or the like, it probably does not include a 0. In fact, you probably only have 0's in your site lists if a) you made a mistake, b) you made up your own system, or c) you are a tireless crusader for the forces of justice, with a penchant for attaching yourself to Sisyphean challenges. If you fall in the latter category, we'd love to hear from you, if only to commiserate. Which brings us to...

20. "How can I report a bug or inadequacy of the program or documentation?"

The easiest method is email to lamarc@u.washington.edu. Please tell us the exact symptoms of the bug, the operating system you're using, and if possible, send a copy of the data file that produces the problem. We also appreciate questions that the documentation doesn't adequately address or is unclear or hard to find, as this allows us to improve the documentation for the next release.

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