File: README.txt

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tantan
======

Introduction
------------

tantan is a tool for masking simple regions (low complexity and
short-period tandem repeats) in biological sequences.

The aim of tantan is to prevent false predictions when searching for
homologous regions between two sequences.  Simple repeats often align
strongly to each other, causing false homology predictions.

Setup
-----

You need to have a C++ compiler.  On Linux, you might need to install
a package called "g++".  On Mac, you might need to install
command-line developer tools.  On Windows, you might need to install
Cygwin.

Using the command line, go into the tantan directory.  To compile it,
type::

  make

Optionally, copy tantan to a standard "bin" directory (using "sudo" to
request administrator permissions)::

  sudo make install

Or copy it to your personal bin directory::

  make install prefix=~

You might need to log out and back in for your computer to recognize
the new program.

Normal usage
------------

* Suppose you have some nucleotide sequences (DNA or RNA) in a
  FASTA-format file called "ntseqs.fa".  You can identify simple
  repeats like this::

    tantan ntseqs.fa > masked.fa

  This will create a new FASTA file called "masked.fa" that replaces
  all masked regions with lowercase letters.  (tantan also works on
  FASTQ-format, though it does not use the quality data.)

* To mask proteins effectively, tantan needs to use different
  algorithm parameters than for nucleotides.  You have to tell it when
  you have proteins, using the "-p" option::

    tantan -p aaseqs.fa > masked.fa

  If you omit "-p" and the sequences look proteinaceous, tantan will
  print a warning message.

Advanced usage
--------------

* By default, tantan indicates repetitive regions with lowercase
  letters.  You can make it replace repetitive letters with (say) "N"
  by using the "-x" option::

    tantan -x N ntseqs.fa > masked.fa

* By default, tantan does not preserve lowercase letters in the input
  sequences.  You can tell it to preserve them by using the "-c"
  option.  So the output will have the union of the lowercase in the
  input and the lowercase assigned by tantan::

    tantan -c ntseqs.fa > masked.fa

* tantan's masking rate is usually OK, but you can alter it by
  changing the "-r" parameter from its default value of 0.005.  Higher
  values increase the amount of masking, and lower values decrease it.
  This increases the masking rate::

    tantan -r 0.02 ntseqs.fa > masked.fa

* Finally, to mask extremely AT-rich DNA, you should change tantan's
  scoring matrix.  The "test" directory contains a matrix "atMask.mat"
  that works well for DNA with ~80% A+T, such as Plasmodium and
  Dictyostelium genomes.  We recommend masking such DNA like this::

    tantan -m atMask.mat -r 0.01 atrich.fa > masked.fa

The preceding examples cover all of tantan's options that you should
ever need.

Recommendations for homology search
-----------------------------------

1) Mask *both* (sets of) sequences.

2) If for some reason you wish to mask only one (set of) sequence(s),
   increase "-r" to 0.02 (0.05 for AT-rich DNA).

3) For DNA-versus-protein alignment, increase "-r" for the proteins to
   0.02.  If for some reason you mask only one (set of) sequence(s),
   make sure it's the proteins.

4) Some alignment tools exclude lowercase from their initial seeding
   phase, but treat lowercase identically to uppercase during their
   subsequent extension phase.  Unfortunately, this does not reliably
   prevent false homology predictions.  It is OK to re-align without
   masking after homology has been decided: FASTA and LAST can do
   this.

For more information, please read the tantan publication (see below).

FAQ
---

Why does tantan sometimes mask isolated bases?
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

This happens when a region is borderline repetitive, and a single base
creeps just over the threshold.  Don't worry about it, it's not a
problem (at least for tantan's aim of preventing false homology).

Does tantan mask functional sequence?
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Of course.  Proteins and protein-coding exons can contain simple
repeats.  Repeats can be functional.  If we want to avoid false
homology we have to mask them.  Remember that tantan merely lowercases
repeats, so it's easy to lift the masking after determining homology.

Options
-------

-p  interpret the sequences as proteins
-x  letter to use for masking, instead of lowercase
-c  preserve uppercase/lowercase in non-masked regions
-m  file for letter-pair score matrix
-r  probability of a repeat starting per position
-e  probability of a repeat ending per position
-w  maximum tandem repeat period to consider
-d  probability decay per period (period-(i+1) / period-i)
-i  match score
-j  mismatch cost
-a  gap existence cost
-b  gap extension cost
-s  minimum repeat probability for masking
-n  minimum copy number, affects -f4 only
-f  output type: 0=masked sequence, 1=repeat probabilities,
                 2=repeat counts, 3=BED, 4=tandem repeats
-h, --help  show help message, then exit
--version   show version information, then exit

Advanced issues
---------------

When tantan masks tandem repeats, it tends to leave the first
(left-most) repeat unit unmasked.  This sometimes allows us to find
homologs we would otherwise miss::

  TGCAAGCTA TTAGGCTTAGGTCAGTGC ttaagcttaggtcagtgc AACATA
  ||| ||| | |||||||||||||||||| ||| |||||||||||||| ||| ||
  TGCTAGCAA TTAGGCTTAGGTCAGTGC ttaggcttaggtcagtgc AACGTA

However, there is a danger of non-equivalent repeat units being
unmasked.  This happens especially if we mask DNA on one strand but
align it on the other strand::

                     TGCAAGCTA TTAGGCTTAGGTCAGTGC ttaagcttaggtcagtgc AACATA
                               ||||||||||||||||||
  TGCTAGCAA ttaggcttaggtcagtgc TTAGGCTTAGGTCAGTGC AACGTA

(My thanks to Junko Tsuji and Paul Horton for finding these issues.)

Finding straightforward tandem repeats
--------------------------------------

Option -f4 runs tantan in a different mode, where it finds
straightforward tandem repeats only.  (Technically, it uses a Viterbi
algorithm instead of a Forward-Backward algorithm.)  This is *not*
recommended for avoiding false homologs!  But it might be useful for
studying tandem repeats.  The output looks like this::

  mySeq   14765   14780   6       2.5     GTCATG  GTCATG,GTCATG,GTC
  mySeq   632362  632377  2       6       GC      GC,GC,GC,GCt,GCT,GCT
  mySeq   1278353 1278369 3       6.5     TCA     TCA,TCA,TCA,TC-,TC,TC,T
  mySeq   3616084 3616100 3       5.33333 TGG     TGA,TGA,TGG,TGG,TGG,T

The first 3 columns show the start and end coordinates of the
repetitive region, in `BED
<https://genome.ucsc.edu/FAQ/FAQformat.html#format1>`_ format.  Column
4 shows the length of the repeating unit (which might vary due to
insertions and deletions, so this column shows the most common
length).  Column 5 shows the number of repeat units.  Column 6 shows
the repeating unit (which again might vary, so this is just a
representative).  Column 7 shows the whole repeat: lowercase letters
are insertions relative to the previous repeat unit, and dashes are
deletions relative to the previous repeat unit.

Miscellaneous
-------------

tantan is distributed under the GNU General Public License, either
version 3 of the License, or (at your option) any later version.  For
details, see COPYING.txt.

If you use tantan in your research, please cite:
"A new repeat-masking method enables specific detection of homologous
sequences", MC Frith, Nucleic Acids Research 2011 39(4):e23.

tantan's website is: http://www.cbrc.jp/tantan/

If you have any questions, comments, or problems concerning tantan,
please email: tantan (ATmark) cbrc (dot) jp.