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LASTZ

TABLE OF CONTENTS

• Introduction
• Installation
• Examples
• Command-Line Options
• Inferring Score Sets
• File Formats
  • FASTA
  • Nib
  • Two-bit
  • Quantum DNA
  • Scores Files
  • Inference Control Files
  • LAV
  • GFA
  • MAF
  • AXT
  • Textual Alignment Output
  • Other Formats
• Case Studies

Introduction

This document describes installation and usage of the LASTZ sequence alignment program. LASTZ is a replacement for BLASTZ, and is command-line compatible with BLASTZ's options. [Backward compatible but adds more.]

LASTZ -- Tool for (1) Pairwise DNA sequence alignment and (2) alignment scores inference.

What's Changed

Since this is the first release of LASTZ, we refer here to how LASTZ is different from BLASTZ

The short-by-two error has been corrected in LASTZ.

You can produce textual alignment output by including the command line option --format=text. Similarly, LASTZ can also create files in maf and axt format.

[Other stuff-- generic seeds, scoring inference etc. etc.]

Installation

If you have received the distribution as a packed archive, unpack the archive by whatever means are appropriate for your computer. The result should be a directory <somepath>/lastz-distrib-X.XX.XX that contains a src subdirectories (and some others). You may find it convenient to remove the revision number (-X.XX.XX) from the directory name. Before building or installing any of the programs, you will need to do one of two things. Either create the shell variable

    $LASTZ = <somepath>/lastz-distrib-X.XX.XX

    <somepath>/lastz-distrib-X.XX.XX/make-include.mak

Then to build the LASTZ executable, from bash (or a similar command line shell), do the commands below. This will build two executables (lastz and lastz_D) and copy them into your installDir.

    cd <somepath>/lastz-distrib-X.XX.XX/src
    make
    make install

A simple self test is included so you can test that the build succeeded. To run it, do this command:

    make test

    make: *** [test] Error 1

Examples

[Discuss a few simple command lines here.] [--nogfextend --nogapped produces only the seed hits]

Aligning a sequence to itself

[move this to the end of the document.] When a sequence is being aligned to itself, the full alignment result will contain mirror-image copies of each alignment block. It is computationally wasteful to process both copies. LASTZ can address this problem in three different ways.

The first way is to simply give LASTZ the same sequence as target and query. In this case, LASTZ does not know that it is aligning a sequence to itself, and performs computation on both copies. A typical result would look like this:

The second way is to replace the query by the --self option. LASTZ will save computation by only computing on one block of each mirror-image pair. It still reports both copies in the output, as shown below. Note that it leaves out the trivial self-alignment block along the diagonal.

The third way is to replace the query by --self and add the --nomirror option. In this case LASTZ only reports one copy of each each mirror-image pair, as shown here:

Command-Line Options

If you are familiar with BLASTZ, you can run LASTZ the same as you ran BLASTZ, with the same options and input files. In addition to BLASTZ compatibility, LASTZ provides other options.

The general format of the lastz command line is

    lastz target_specifier [query_specifier] [options]

The target_specifier and query_specifier are usually just the names of files containing the two sequences to be aligned, either in FASTA or nib format. They can also specify subsequences; details are given below. The general format for options is --<name>[=<value>]. For BLASTZ compatibility some options can be set with <letter>=<number>. [How to prevent line break inside the double hyphen?]

Running the command lastz without specifiers or options gives a list of the most commonly used options. Running

    lastz --all

Sequence Specifiers

A sequence specifier normally just indicates the file to be used in the alignment. However, a file's subrange and strand can be specified, as well as a masking file, a sequence-selector and a nickname for the sequence. the alignment. The format is

    [nickname::]file_name[/select_name][{mask_file}][[start,end]][-]

<select_name> is only valid for the 2BIT file format, and specifies the single sequence from that file to use, rather than all sequences.

<nickname> is a name to use for this sequence in any output files.

{mask_file} specifies a file to use to mask bases. [Need to discuss masking file format in formats section.]

The --self option can be used, in place of the query specifier, to perform self-alignment of the target sequence. Using --self, rather than specifying the same file as target and query, lets LASTZ know not to report duplicate copies of the same alignment blocks.

Seeds

LASTZ gives the “user” many seeding choices. The intent is that these will be selected by some program (those the quote marks around “user”), but they are available from the command line for any user.

N-mer match: A space-free seed can be specified by the length of the N-mer match required.

--seed=match(<length>)

General seed patterns: Any spaced seed pattern can be specified. The pattern is a string of 1s, 0s and Ts, where a 1 indicates that a match is required in that position, a 0 indicates a mismatch is allowed, and a T indicates a transition mismatch is allowed.

--seed=<pattern>

The default seed is --seed=1110100110010101111 which is the 12 of 19 seed used as the default in BLASTZ.

Half-weight seed patterns: If a seed pattern consists of only 0s and Ts, it is implemented internally as a half-weight seed. These seeds can be used in conjuction with filtering on the number of matches or transitions in a seed hit. For example,

--seed=TTT0T00TT00T0T0TTTT --filter=2:15 implements the default seed pattern, requiring the twelve T positions to be matches or transitions, requiring ten matches total (among the 19 positions), and allowing not more than two transversions.

Additionally, --seed=half(<length>) can be used to specify a space-free half-weight seed.

Half-weight seeds use much less memory internally. A half-weight seed uses the same amount of memory as a normal seed pattern half as long.

Single, double or no transitions: By default, any single match position (a 1 in a spaced seed, or any position in an N-mer match) is allowed to be a transition. --notransition disables this. Further, --transition=2 allows any two match positions to be transitions.

Twin hit seeds: The sensitivity of the seed can be decreased by ignoring seed hits that don't have a second hit nearby.

--twins=[<min>:]<maxgap> requires two seed hits on the same diagonal. The distance between the hits (the number of bases between the end of the first hit and the beginning of the second) must be between <min> and <maxgap>. If <min> is absent, zero is used (which means the twin seed hits may be adjacent but not overlap). Negative values can be used. For example --twins=-5:10 means the twins can overlap by as much as 5 bases or can have as much as 10 bases between them.

[Discuss CL options for seeds here.] [Add subsections for other option groups.]

All Options

[This is probably not up-to-date.]

Inferring Score Sets

[need more here.] Log-odds scores can be inferred from the sequences given. The resulting scoring set can be saved to a file or immediately used to align the sequences.

To infer scores, use the --infer or --inferonly option. The latter will stop after inferring scores. The --infscores[=<file>] option causes the inferred scoring set to be written to a file. Otherwise, the scoring set is written to the header of the alignment output file (as a comment). As a last resort, if alignment is not performed the scoring set is written to the console. The file format is the same used to input scoring sets.

Inference is achieved by computing the probability of each of the 18 different alignment events (gap open, gap extend and 16 substitution events). The probabilities are estimated from alignments of the sequences. Of course, we don't have alignments, so we start by using a generic scoring set to generate alignments, infer scores, then realign, and so on, until scores “converge”. We perform ungapped alignments until the substitution scores converge, then perform gapped alignments (holding substitution scores constant) until gap scores converge.

Control options for the inference process can be provided in a control file specified as part of the --infer or --inferonly options.

Usually it is undesirable to use all alignment blocks during inference. Blocks with a high rate of subsitutions (low identity) are likely to be false positives. On the other hand, blocks with few subsitutions (high identity) will be found regardless of what scores we use. Thus it is desirable to base inference only on statistics from a mid range of identity. By default, we use the middle 50% (that is, the 25th through 75th percentile of identity found in the alignment), but this can be changed in the control file.

File Formats

[check this.] LASTZ usually receives two sequences and a scores file as inputs, and produces an alignment file as output.

DNA sequences can be provided in FASTA, NIB or 2BIT format. These sequences can contain a series of A, C, G, T and N in upper or lower case. Lower case indicates repeat-masked bases, while N indicates unknown sequence (and is also often used to separator). Additionally, a quantum DNA sequence can be provided as the query.

FASTA and 2BIT formats support more than one sequence within the same file. Files containing multiple sequences can only be used as the query file, not the target. However, an exception is made for 2BIT targets (see the select_name field above).

FASTA (sequence input)

FASTA format stores DNA sequences as plain text. The first line should begin with a “>” followed by the name of the sequence. Remaining lines should contain DNA. They can be of any length.

If the file contains multiple sequences, each should start with the “>” header line.

Nib (sequence input)

NIB format stores a single unnamed DNA sequence, packed as two bases per byte. As of January, 2008, a spec for NIB files can be found at http://genome.ucsc.edu/FAQ/FAQformat#format8.

Two-bit (sequence input)

2BIT format stores multiple DNA sequences encoded as four bases per byte with with some additional information describing runs of masked bases or Ns. As of Jan/2008, a spec for 2BIT files can be found at http://genome.ucsc.edu/FAQ/FAQformat#format7.

Quantum DNA (sequence input)

[Discuss Quantum DNA and quantum code files.]

Scores Files

[Discuss Scores Files.] [blastz score matrix files] [quantum] Here's an example:

    # This matches the default scoring set for blastz
    
    bad_score          = X:-1000  # used for sub['X'][*] and sub[*]['X']
    fill_score         = -100     # used when sub[*][*] not defined
    gap_open_penalty   =   30
    gap_extend_penalty =  400
    
         A     C     G     T
    A   91  -114   -31  -123
    C -114   100  -125   -31
    G  -31  -125   100  -114
    T -123   -31  -114    91

       A     C     G     T
      91  -114   -31  -123
    -114   100  -125   -31
     -31  -125   100  -114
    -123   -31  -114    91

Inference Control Files

[Need more information here.]

Here's an example:

    # base inference on alignments in the middle 50 percentile
    # by percent-identity
    min_identity       = 25.0%    # 25th percentile
    max_identity       = 75.0%    # 75th percentile

    # scale scores so max substitution will be 100 and only use
    # alignments scoring as well as 20 substitutions
    inference_scale    = 100      # score for max substitution
    hsp_threshold      = 20*inference_scale
    gapped_threshold   = hsp_threshold

    # allow substitution score inference to iterate at most
    # 20 times;  don't perform gap score inference-- instead
    # hardwire gap scores relative to max substitution
    max_sub_iterations = 20
    max_gap_iterations = 0
    gap_open_penalty   = 4*inference_scale
    gap_extend_penalty = 0.3*inference_scale

min_identity and max_identity specify the range of sequence identity upon which inference is based. Only alignment blocks within this range contribute to inference. If the value ends with a percent sign, the range is a percentile of the values found in the overall alignment. Otherwise it is a fixed percentage. For example, min_identity=70 and max_identity=90 indicates that blocks with identity ranging from 70 to 90 percent will be used, while min_identity=25% and max_identity=75% indicates that 50 percent of the blocks will be used (the middle 50 percent).

inference_scale specifies a value for the largest substitution score (i.e. the score for the best match). All other scores are scaled accordingly. If this is set to none, scores are log-odds using base 2 logs.

hsp_threshold and gapped_threshold correspond to the command line --hspthresh and --gappedthresh options (also known as K and L in BLASTZ lingo).

max_sub_iterations and max_gap_iterations specify limits on the number of iterations that will be performed. For example, if you only want a substitution scoring matrix, you can set max_gap_iterations=0.

gap_open_penalty and gap_extend_penalty correspond to the command line --gap=<[open,]extend> option (also known as O and E in BLASTZ lingo). These are the values used for the first iteration of gap-scoring inference.

step (which is not shown in the example above) corresponds to the command line --step option (also known as Z in BLASTZ lingo). A large step, e.g. step=100, could potentially speed up the inference process. Ideally, this would base inference on a subsample of only one percent of the whole. However, the subsample actually ends up larger than that and is biased toward HSPs that are either longer or have a lower substitution rate. This happens because subsampling occurs at the seed-hit level, and such HSPs generally have more seed hits. Future versions of LASTZ may include a means to compensate for this bias.

entropy (which is not shown in the example above) corresponds to the command line --entropy option (also known as P in BLASTZ lingo). Legal values are “on” or “off”. If on, sequence entropy is incorporated in the filtering of high scoring pairs.

LAV

[Discuss LAV.] Here's a typical lav file:

    #:lav
    d {
      "lastz.v0.3 malus.fa aurantium.fa C=2 W=8 T=0 
         A    C    G    T
        91 -114  -31 -123
      -114  100 -125  -31
       -31 -125  100 -114
      -123  -31 -114   91
      O = 400, E = 30, K = 3000, L = 3000, M = 0"
    }
    #:lav
    s {
      "malus.fa" 1 191411218 0 1
      "aurantium.fa" 1 90634903 0 1
    }
    h {
      "> apple"
      "> orange"
    }
    a {
      s 20643
      b 46566766 2083211
      e 46567353 2083795
      l 46566766 2083211 46566796 2083241 61
      l 46566797 2083245 46566814 2083262 78
      l 46566821 2083263 46567353 2083795 65
    }
    a {
      s 4233
      b 47246530 10635696
      e 47246660 10635826
      l 47246530 10635696 47246660 10635826 63
    }
    ... many more a-stanzas ...
    #:lav
    s {
      "malus.fa" 1 191411218 0 1
      "aurantium.fa-" 1 90634903 1 1
    }
    h {
      "> apple"
      "> orange (reverse complement)"
    }
    a {
      s 13897
      b 1005819 5352698
      e 1006099 5352978
      l 1005819 5352698 1006099 5352978 74
    }
    ... many more a-stanzas ...
    #:eof

    <filename> <start> <stop> <rev_comp_flag> <contig>

    a {
      s 13916
      b 4886 21292
      e 5171 21537
      l 4886 21292 4899 21305 79
      l 4900 21308 4924 21332 92
      l 4925 21334 5024 21433 88
      l 5027 21434 5040 21447 100
      l 5086 21448 5117 21479 84
      l 5118 21484 5171 21537 87
    }

    s {
      "malus.fa" 1001 2000 0 1
      "aurantium.fa" 2001 5000 0 1
    }
    ...
    a {
      s 7321
      b 301 501
      e 400 600
      l 301 501 400 600 82
    }

    s {
      "malus.fa" 1001 2000 0 1
      "aurantium.fa-" 2001 5000 0 1
    }
    ...
    a {
      s 7321
      b 301 501
      e 400 600
      l 301 501 400 600 82
    }

    x {
      n <count>
    }

    m {
      x <start> <end>
      x <start> <end>
      x <start> <end>
      x <start> <end>
       ...
    }

    Census {
      1 <count>
      2 <count>
       ...
    }

GFA

[Discuss GFA.]

MAF

[Discuss MAF.]

AXT

[Discuss AXT.]

Textual Alignment Output

[Discuss Textual alignments.] [ Warn that we may change the textual format in future versions of LASTZ, it is only provided for eyeballing alignment blocks, not for programs to read. Programs are better off reading Lav, Maf, or Axt formats.]

Other Formats

LASTZ source code includes support for other output formats which are intended mainly for the convenience of the developers. If you have specific questions, please contact us.

Case Studies

[Provide guidance on what params to use when.]