version 3.6

# SEQBOOT -- Bootstrap, Jackknife, or Permutation Resampling
of Molecular Sequence, Restriction Site,
Gene Frequency or Character Data

© Copyright 1991-2005 by the University of Washington. Written by Joseph Felsenstein. Permission is granted to copy this document provided that no fee is charged for it and that this copyright notice is not removed.

SEQBOOT is a general bootstrapping and data set translation tool. It is intended to allow you to generate multiple data sets that are resampled versions of the input data set. Since almost all programs in the package can analyze these multiple data sets, this allows almost anything in this package to be bootstrapped, jackknifed, or permuted. SEQBOOT can handle molecular sequences, binary characters, restriction sites, or gene frequencies. It can also convert data sets between Sequential and Interleaved format, and into the NEXUS format or into a new XML sequence alignment format.

To carry out a bootstrap (or jackknife, or permutation test) with some method in the package, you may need to use three programs. First, you need to run SEQBOOT to take the original data set and produce a large number of bootstrapped or jackknifed data sets (somewhere between 100 and 1000 is usually adequate). Then you need to find the phylogeny estimate for each of these, using the particular method of interest. For example, if you were using DNAPARS you would first run SEQBOOT and make a file with 100 bootstrapped data sets. Then you would give this file the proper name to have it be the input file for DNAPARS. Running DNAPARS with the M (Multiple Data Sets) menu choice and informing it to expect 100 data sets, you would generate a big output file as well as a treefile with the trees from the 100 data sets. This treefile could be renamed so that it would serve as the input for CONSENSE. When CONSENSE is run the majority rule consensus tree will result, showing the outcome of the analysis.

This may sound tedious, but the run of CONSENSE is fast, and that of SEQBOOT is fairly fast, so that it will not actually take any longer than a run of a single bootstrap program with the same original data and the same number of replicates. This is not very hard and allows bootstrapping or jackknifing on many of the methods in this package. The same steps are necessary with all of them. Doing things this way some of the intermediate files (the tree file from the DNAPARS run, for example) can be used to summarize the results of the bootstrap in other ways than the majority rule consensus method does.

If you are using the Distance Matrix programs, you will have to add one extra step to this, calculating distance matrices from each of the replicate data sets, using DNADIST or GENDIST. So (for example) you would run SEQBOOT, then run DNADIST using the output of SEQBOOT as its input, then run (say) NEIGHBOR using the output of DNADIST as its input, and then run CONSENSE using the tree file from NEIGHBOR as its input.

The resampling methods available are:

- The bootstrap. Bootstrapping was invented by Bradley Efron in 1979, and its use in phylogeny estimation was introduced by me (Felsenstein, 1985b; see also Penny and Hendy, 1985). It involves creating a new data set by sampling N characters randomly with replacement, so that the resulting data set has the same size as the original, but some characters have been left out and others are duplicated. The random variation of the results from analyzing these bootstrapped data sets can be shown statistically to be typical of the variation that you would get from collecting new data sets. The method assumes that the characters evolve independently, an assumption that may not be realistic for many kinds of data.

- The partial bootstrap.. This is the bootstrap where fewer than the full number of characters are sampled. The user is asked for the fraction of characters to be sampled. It is primarily useful in carrying out Zharkikh and Li's (1995) Complete And Partial Bootstrap method, and Shimodaira's (2002) AU method, both of which correct the bias of bootstrap P values.

- Block-bootstrapping. One pattern of departure from indeopendence of character evolution is correlation of evolution in adjacent characters. When this is thought to have occurred, we can correct for it by samopling, not individual characters, but blocks of adjacent characters. This is called a block bootstrap and was introduced by Künsch (1989). If the correlations are believed to extend over some number of characters, you choose a block size, B, that is larger than this, and choose N/B blocks of size B. In its implementation here the block bootstrap "wraps around" at the end of the characters (so that if a block starts in the last B-1 characters, it continues by wrapping around to the first character after it reaches the last character). Note also that if you have a DNA sequence data set of an exon of a coding region, you can ensure that equal numbers of first, second, and third coding positions are sampled by using the block bootstrap with B = 3.

- Partial block-bootstrapping. Similar to partial bootstrapping except sampling blocks rather than single characters.

- Delete-half-jackknifing.. This alternative to the bootstrap involves sampling a random half of the characters, and including them in the data but dropping the others. The resulting data sets are half the size of the original, and no characters are duplicated. The random variation from doing this should be very similar to that obtained from the bootstrap. The method is advocated by Wu (1986). It was mentioned by me in my bootstrapping paper (Felsenstein, 1985b), and has been available for many years in this program as an option. Note that, for the present, block-jackknifing is not available, because I cannot figure out how to do it straightforwardly when the block size is not a divisor of the number of characters.

- Delete-fraction jackknifing. Jackknifing is advocated by Farris et. al. (1996) but as deleting a fraction 1/e (1/2.71828). This retains too many characters and will lead to overconfidence in the resulting groups when there are conflicting characters. However it is made available here as an option, with the user asked to supply the fraction of characters that are to be retained.

- Permuting species within characters. This method of resampling (well, OK, it may not be best to call it resampling) was introduced by Archie (1989) and Faith (1990; see also Faith and Cranston, 1991). It involves permuting the columns of the data matrix separately. This produces data matrices that have the same number and kinds of characters but no taxonomic structure. It is used for different purposes than the bootstrap, as it tests not the variation around an estimated tree but the hypothesis that there is no taxonomic structure in the data: if a statistic such as number of steps is significantly smaller in the actual data than it is in replicates that are permuted, then we can argue that there is some taxonomic structure in the data (though perhaps it might be just the presence of aa pair of sibling species).

- Permuting characters. This simply permutes the order of the characters, the same reordering being applied to all species. For many methods of tree inference this will make no difference to the outcome (unless one has rates of evolution correlated among adjacent sites). It is included as a possible step in carrying out a permutation test of homogeneity of characters (such as the Incongruence Length Difference test).

- Permuting characters separately for each species. This is a method introduced by Steel, Lockhart, and Penny (1993) to permute data so as to destroy all phylogenetic structure, while keeping the base composition of each species the same as before. It shuffles the character order separately for each species.

- Rewriting. This is not a resampling or permutation method -- it simply rewrites the data set into a different format. That format can be the PHYLIP format. For molecular sequences and discrete morphological character it can also be the NEXUS format. For molecular sequences one other format is available, a new (and nonstandard) XML format of our own devising. When the PHYLIP format is chosen the data set is coverted between Interleaved and Sequential format. If it was read in as Interleaved sequences, it will be written out as Sequential format, and vice versa. The NEXUS format for molecular sequences is always written as interleaved sequences. The XML format is different from (though similar to) a number of other XML sequence alignment formats. An example will be found below. Here is a table to links to those other XML alignment formats:

  Andrew Rambaut's BEAST XML format | `http://evolve.zoo.ox.ac.uk/beast/introXML.html` and `http://evolve.zoo.ox.ac.uk/beast/reference/index.html` | A format for alignments. There is also a format for phylogenies described there.
  MSAML | `http://xml.coverpages.org/msaml-desc-dec.html` | Defined by Paul Gordon of University of Calgary. See his big list of molecular biology XML projects.
  BSML | `http://www.bsml.org/resources/default.asp` | Bioinformatic Sequence Markup Language includes a multiple sequence alignment XML format

The data input file is of standard form for molecular sequences (either in interleaved or sequential form), restriction sites, gene frequencies, or binary morphological characters.

When the program runs it first asks you for a random number seed. This should be an integer greater than zero (and probably less than 32767) and which is of the form 4n+1, that is, it leaves a remainder of 1 when divided by 4. This can be judged by looking at the last two digits of the integer (for instance 7651 is not of form 4n+1 as 51, when divided by 4, leaves the remainder 3). The random number seed is used to start the random number generator. If the randum number seed is not odd, the program will request it again. Any odd number can be used, but may result in a random number sequence that repeats itself after less than the full one billion numbers. Usually this is not a problem. As the random numbers appear to be unpredictable, there is no such thing as a "good" seed -- the numbers produced from one seed are statistically indistinguishable from those produced by another, and it is not true that the numbers produced from one seed (say 4533) are similar to those produced from a nearby seed (say 4537).

Then the program shows you a menu to allow you to choose options. The menu looks like this:

    Bootstrapping algorithm, version 3.6

    Settings for this run:
      D      Sequence, Morph, Rest., Gene Freqs?  Molecular sequences
      J  Bootstrap, Jackknife, Permute, Rewrite?  Bootstrap
      %    Regular or altered sampling fraction?  regular
      B      Block size for block-bootstrapping?  1 (regular bootstrap)
      R                     How many replicates?  100
      W              Read weights of characters?  No
      C                Read categories of sites?  No
      S     Write out data sets or just weights?  Data sets
      I             Input sequences interleaved?  Yes
      0      Terminal type (IBM PC, ANSI, none)?  ANSI
      1       Print out the data at start of run  No
      2     Print indications of progress of run  Yes

      Y to accept these or type the letter for one to change

The user selects options by typing one of the letters in the left column, and continues to do so until all options are correctly set. Then the program can be run by typing Y.

It is important to select the correct data type (the D selection). Each time D is typed the program will change data type, proceeding successively through Molecular Sequences, Discrete Morphological Characters, Restriction Sites, and Gene Frequencies. Some of these will cause additional entries to appear in the menu. If Molecular Sequences or Restriction Sites settings and chosen the I (Interleaved) option appears in the menu (and as Molecular Sequences are also the default, it therefore appears in the first menu). It is the usual I option discussed in the Molecular Sequences document file and in the main documentation files for the package, and is on by default.

If the Restriction Sites option is chosen the menu option E appears, which asks whether the input file contains a third number on the first line of the file, for the number of restriction enzymes used to detect these sites. This is necessary because data sets for RESTML need this third number, but other programs do not, and SEQBOOT needs to know what to expect.

If the Gene Frequencies option is chosen an menu option A appears which allows the user to specify that all alleles at each locus are in the input file. The default setting is that one allele is absent at each locus. Note that for sampling methods such as the bootstrap and jackknife, whole loci are sampled, not individual alleles.

The J option allows the user to select Bootstrapping, Delete-Half-Jackknifing, the Archie-Faith permutation of species within characters, permutation of character order, shuffling character order separately within each species, or Rewriting. It changes successively among these each time J is typed.

The P menu option appears if the data are molecular sequences and the J option is used to choose the Rewrite option. It gives you the choice between our normal PHYLIP format or a new (and nonstandard) XML sequence alignment format. This encloses the alignment between <ALIGNMENT> ... </ALIGNMENT> tags. Each sequence between <SEQUENCE> ... </SEQUENCE> tags, has a TYPE attribute of the sequence which is either "dna", "rna" or "protein". This is set by default to "dna" but can be changed by the user in an S Sequence type menu option. Each sequence has its name, enclosed between <NAME> ... </NAME> tags, and the data itself, enclosed between <DATA> ... </DATA> tags. The XML option is not available unless the data are molecular sequences. It is a new format -- no programs yet read it. In other cases the P menu option does not appear and the PHYLIP output format is assumed. Here is a simple example of this XML sequence alignment format, for the (silly) data set used in our main documentation file:

    <alignment>
       <sequence type="dna">
          <name>Archaeopt</name>
          <data>CGATGCTTAC CGC</data>
       </sequence>

       <sequence type="dna">
          <name>Hesperorni</name>
          <data>CGTTACTCGT TGT</data>
       </sequence>

       <sequence type="dna">
          <name>Baluchithe</name>
          <data>TAATGTTAAT TGT</data>
       </sequence>

       <sequence type="dna">
          <name>B. virgini</name>
          <data>TAATGTTCGT TGT</data>
       </sequence>

       <sequence type="dna">
          <name>Brontosaur</name>
          <data>CAAAACCCAT CAT</data>
       </sequence>

       <sequence type="dna">
          <name>B.subtilis</name>
          <data>GGCAGCCAAT CAC</data>
       </sequence>

    </alignment>

For the gene frequencies and restriction sites data types, this Rewrite option does not change the data set. The option will be useful mostly to write the data out in a standard format, in cases where the input file is messy-looking.

The B option selects the Block Bootstrap. When you select option B the program will ask you to enter the block length. When the block length is 1, this means that we are doing regular bootstrapping rather than block-bootstrapping.

The % option allows the user control over what fraction of the characters are sampled in the bootstrap and jackknife methods. Normally the bootstrap samples a number of times equal to the number of characters, and the jackknife samples half that number. This option permits you to specify a smaller fraction of characters to be sampled. Note that doing so is "statistically incorrect", but it is available here for whatever other purposes you may have in mind. Note that the fraction you will be asked to enter is the fraction of characters sampled, not the fraction left out. If you specify 100 as the fraction of sites retained and are using the jackknife, the data set will simply be rewritten. Note (as mentioned below) that this can be used together with the W (Weights) option to rewrite a data set while omitting a particular set of sites.

The R option allows the user to set the number of replicate data sets. This defaults to 100. Most statisticians would be happiest with 1000 to 10,000 replicates in a bootstrap, but 100 gives a rough picture. You will have to decide this based on how long a running time of the tree programs you are willing to tolerate. (The time needed to do the sampling in this program is not much of an issue).

The W (Weights) option allows weights to be read from a file whose default name is "weights". The weights follow the format described in the main documentation file. Weights can only be 0 or 1, and act to select the characters (or sites) that will be used in the resampling, the others being ignored and always omitted from the output data sets. If you use W together with the S (just weights) option, you write a file of weights (whose default name is "outweights"). In that file, any character whose original weight is 0 will have weight 0, the other weights varying according to the resampling. Note that if you write out data sets rather than weights (not using the S option), this output weights file is not written, as the characters are written different numbers of times in the data output file Note that with restriction sites, the weights are not used by some of the programs. Writing out files of weights will not be helpful with those programs. For the moment, with all gene frequencies programs the weights are also not used.

Note that it is possible to use SEQBOOT to rewrite a data set while omitting certain sites. This can be done, not with the rewrite choice in option J, but with its jackknife choice. Choose the delete-half jackknife, but then use the % option to set the fraction of sites sampled to 100%. Also use the W option to read a set of weights that select which sites to retain (those with weights 1 instead of 0). Use the R option to set the number of replicates to 1. The program will write one data set, with all the sites that have weights 1, in order.

The C (Categories) option can be used with molecular sequence programs to allow assignment of sites or amino acid positions to user-defined rate categories. The assignment of rates to sites is then made by reading a file whose default name is "categories". It should contain a string of digits 1 through 9. A new line or a blank can occur after any character in this string. Thus the categories file might look like this:

    122231111122411155
    1155333333444

The only use of the Categories information in SEQBOOT is that they are sampled along with the sites (or amino acid positions) and are written out onto a file whose default name is "outcategories", which has one set of categories information for each bootstrap or jackknife replicate.

In the discrete characters data type, three more options appear in the menu. These are N (aNcestors), X (miXture of methods), and F (Factors) options. They may be useful with the program MIX, which allows input of ancestors information and information specifying the mixture of parsimony methods to be used. Factors information is also read and used by programs MOVE, DOLMOVE, and CLIQUE, in calculating how many multistate characters are compatible with a tree. The mixture, ancestors, and factors information for the characters are specified in input files whose default names are "ancestors", "mixture", and "factors". Seqboot produces output files that properly reflect what the resampling implies for these files. The corresponding output files have default file names "outancestors", "outmixture", and "outfactors".

For futher description of the mixture, ancestors, and factors file formats and contents see the Discrete Characters Programs documentation file.

The S option is a particularly important one. It is used whether to produce multiple output files or multiple weights. If your data set is large, a file with (say) 1000 such data sets can be very large and may use up too much space on your system. If you choose the S option, the program will instead produce a weights file with multiple sets of weights. The default name of this file is "outweights". Except for some programs that cannot handle multiple sets of weights, PHYLIP programs have an M (multiple data sets) option that asks the user whether to use multiple data sets or multiple sets of weights. If the latter is selected when running those programs, they read one data set, but analyze it multiple times, each time reading a new set of weights. As both bootstrapping and jackknifing can be thought of as reweighting the characters, this accomplishes the same thing (the multiple weights option is not available for the various kinds of permutation). As the file with multiple sets of weights is much smaller than a file with multiple data sets, this can be an attractive way to save file space. When multiple sets of weights is chosen, they reflect the sampling as well as any set of weights that was read in, so that you can use SEQBOOT's W option as well.

The 0 (Terminal type) option is the usual one.

## Saving time by combining results of separate runs

Often runs of distance programs, or of phylogeny programs, on large numbers of bootstrap replicates are very time-consuming. If you have multiple computers, you can save time by splitting up these runs among multiple machines. For example, if you have 1000 replicate data sets (or weights) from bootstrapping, you could divide these into ten files of 100 data sets (or you could simply use Seqboot ten times with different random number seeds). If these are run on ten separate computers, the execution time is speeded up by as much as a factor of 10. Each input file of 100 data sets results in an output tree file. These can be concatenated end-to-end using a word processor program or using a command such as the Unix/Linux `cat` command. Make sure that these files are not turned into Microsoft Word format when this is done. The consensus tree program CONSENSE will hande the concatenated tree file properly.

If a distance matrix method is being used, you can also produce the distance matrices on different machines, and concatenate them end-to-end to produce an input file of distance matrices for FITCH, KITSCH, or NEIGHBOR. This is particularly relevant for NEIGHBOR, which in most cases makes trees more quickly than the distance matrices can be produced.

## Input File

The data files read by SEQBOOT are the standard ones for the various kinds of data. For molecular sequences the sequences may be either interleaved or sequential, and similarly for restriction sites. Restriction sites data may either have or not have the third argument, the number of restriction enzymes used. Discrete morphological characters are always assumed to be in sequential format. Gene frequencies data start with the number of species and the number of loci, and then follow that by a line with the number of alleles at each locus. The data for each locus may either have one entry for each allele, or omit one allele at each locus. The details of the formats are given in the main documentation file, and in the documentation files for the groups of programs.

## Output

The output file will contain the data sets generated by the resampling process. Note that, when Gene Frequencies data is used or when Discrete Morphological characters with the Factors option are used, the number of characters in each data set may vary. It may also vary if there are an odd number of characters or sites and the Delete-Half-Jackknife resampling method is used, for then there will be a 50% chance of choosing (n+1)/2 characters and a 50% chance of choosing (n-1)/2 characters.

The Factors option causes the characters to be resampled together. If (say) three adjacent characters all have the same factors characters, so that they all are understood to be recoding one multistate character, they will be resampled together as a group. WARNING: at the moment the Factors option does not work properly together with the Permute Characters option, and I urge you not to try that combination of options.

The order of species in the data sets in the output file will vary randomly. This is a precaution to help the programs that analyze these data avoid any result which is sensitive to the input order of species from showing up repeatedly and thus appearing to have evidence in its favor.

The numerical options 1 and 2 in the menu also affect the output file. If 1 is chosen (it is off by default) the program will print the original input data set on the output file before the resampled data sets. I cannot actually see why anyone would want to do this. Option 2 toggles the feature (on by default) that prints out up to 20 times during the resampling process a notification that the program has completed a certain number of data sets. Thus if 100 resampled data sets are being produced, every 5 data sets a line is printed saying which data set has just been completed. This option should be turned off if the program is running in background and silence is desirable. At the end of execution the program will always (whatever the setting of option 2) print a couple of lines saying that output has been written to the output file.

## Size and Speed

The program runs moderately quickly, though more slowly when the Permutation resampling method is used than with the others.

### TEST DATA SET

        5    6
    Alpha     AACAAC
    Beta      AACCCC
    Gamma     ACCAAC
    Delta     CCACCA
    Epsilon   CCAAAC

### CONTENTS OF OUTPUT FILE

(If Replicates are set to 10 and seed to 4333)

        5     6
    Alpha     ACAAAC
    Beta      ACCCCC
    Gamma     ACAAAC
    Delta     CACCCA
    Epsilon   CAAAAC
        5     6
    Alpha     AAAACC
    Beta      AACCCC
    Gamma     CCAACC
    Delta     CCCCAA
    Epsilon   CCAACC
        5     6
    Alpha     ACAAAC
    Beta      ACCCCC
    Gamma     CCAAAC
    Delta     CACCCA
    Epsilon   CAAAAC
        5     6
    Alpha     ACCAAA
    Beta      ACCCCC
    Gamma     ACCAAA
    Delta     CAACCC
    Epsilon   CAAAAA
        5     6
    Alpha     ACAAAC
    Beta      ACCCCC
    Gamma     ACAAAC
    Delta     CACCCA
    Epsilon   CAAAAC
        5     6
    Alpha     AAAACA
    Beta      AAAACC
    Gamma     AAACCA
    Delta     CCCCAC
    Epsilon   CCCCAA
        5     6
    Alpha     AAACCC
    Beta      CCCCCC
    Gamma     AAACCC
    Delta     CCCAAA
    Epsilon   AAACCC
        5     6
    Alpha     AAAACC
    Beta      AACCCC
    Gamma     AAAACC
    Delta     CCCCAA
    Epsilon   CCAACC
        5     6
    Alpha     AAAAAC
    Beta      AACCCC
    Gamma     CCAAAC
    Delta     CCCCCA
    Epsilon   CCAAAC
        5     6
    Alpha     AACCAC
    Beta      AACCCC
    Gamma     AACCAC
    Delta     CCAACA
    Epsilon   CCAAAC