last-split
==========

This program estimates "split alignments" (typically for DNA) or
"spliced alignments" (typically for RNA).

It reads candidate alignments of query sequences to a genome, and
looks for a unique best alignment for each part of each query.  It
allows different parts of one query to match different parts of the
genome.  This is useful for DNA queries that cross rearrangement
breakpoints, or RNA queries that cross splice junctions.

Examples
--------

Split alignment of DNA reads to a genome
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Assume the DNA reads are in a file called "q.fastq" (in fastq-sanger
format), and the genome is in "genome.fasta" (in fasta format).  We
can do the alignment like this::

  lastdb -m1111110 db genome.fasta
  lastal -Q1 -e120 db q.fastq | last-split > out.maf

Spliced alignment of RNA reads to a genome
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

This time, we provide the genome information to last-split, which
causes it to do spliced instead of split alignment, and also tells it
where the splice signals are (GT, AG, etc)::

  lastdb -m1111110 db genome.fasta
  lastal -Q1 -e120 db q.fastq | last-split -g db > out.maf

This will favour splices starting at GT (and to a lesser extent GC and
AT), and ending at AG (and to a lesser extent AC).  However, it allows
splices starting and ending anywhere.  It also favours splices with
introns of typical length, specified by a log-normal distribution
(i.e. cis-splices).  However, it allows arbitrary trans-splices
between any two places in the genome.  If you wish to turn off
trans-splicing, add -t0 to the last-split options.

Unfortunately, the cis-splicing calculations can be slow, depending on
various factors such as query sequence length.  If it is too slow,
please try the next recipe.

Faster trans-spliced alignment
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Here we use -c0 to turn off cis-splicing, and -t0.004 to specify a
higher probability of trans-splicing::

  lastdb -m1111110 db genome.fasta
  lastal -Q1 -e120 db q.fastq | last-split -c0 -t0.004 -g db > out.maf

Output
------

The output is in MAF(-like) format::

  a score=150 mismap=0.000413
  s chr21  15963638 25 + 48129895 TCAGATGAGGACCTAATTTATTACT
  s query7       50 25 +       75 TCAGATGAGGACCTAATTTATTACT
  q query7                        EBEEC@CE=EEE?FEDAED5?@@D@
  p                               !#$'BBBBBBBBBBBBBBBBBBBBB

The "mismap" is the estimated probability that this part of the query
should be aligned to a different part of the genome.  The line
starting with "p" indicates the probability that each base should be
aligned to a different part of the genome.  It uses a compact code:

  ======  =================   ======  =================
  Symbol  Error probability   Symbol  Error probability
  ------  -----------------   ------  -----------------
  ``!``   0.79 -- 1           ``0``   0.025 -- 0.032
  ``"``   0.63 -- 0.79        ``1``   0.02  -- 0.025
  ``#``   0.5  -- 0.63        ``2``   0.016 -- 0.02
  ``$``   0.4  -- 0.5         ``3``   0.013 -- 0.016
  ``%``   0.32 -- 0.4         ``4``   0.01  -- 0.013
  ``&``   0.25 -- 0.32        ``5``   0.0079 -- 0.01
  ``'``   0.2  -- 0.25        ``6``   0.0063 -- 0.0079
  ``(``   0.16 -- 0.2         ``7``   0.005  -- 0.0063
  ``)``   0.13 -- 0.16        ``8``   0.004  -- 0.005
  ``*``   0.1  -- 0.13        ``9``   0.0032 -- 0.004
  ``+``   0.079 -- 0.1        ``:``   0.0025 -- 0.0032
  ``,``   0.063 -- 0.079      ``;``   0.002  -- 0.0025
  ``-``   0.05  -- 0.063      ``<``   0.0016 -- 0.002
  ``.``   0.04  -- 0.05       ``=``   0.0013 -- 0.0016
  ``/``   0.032 -- 0.04       ``>``   0.001  -- 0.0013
  ======  =================   ======  =================

Other symbols indicate lower error probabilities, and "~" is the
lowest possible.  In general::

  Error probability <= 10 ^ -((ASCII value - 33) / 10)

The "mismap" is simply the lowest probability from the "p" line.

Split versus spliced alignment
------------------------------

Here is a split alignment::

  Query         ttctttgat--gctagtcctgatgttatggtattttttatcgaatgataa
                  |||||||--||||||                |||x||||||||||||
  Genome chrA  ...ctttgatatgctagt...             |||x||||||||||||
  Genome chrB                                 ...tttatatcgaatgata...

And here is a spliced alignment::

  Query        ctagtcgatatt--gctgtacgtctgttagctat-tttttcctctgtttg
                  |||x|||||--|||||||||----|||||||-|||||x|||||
  Genome chrA  ...gtctatattatgctgtacgt... |||||||-|||||x|||||
  Genome chrB                          ...tagctatattttttctctg...

Split alignment allows arbitrarily large unaligned parts in the middle
of the query, whereas spliced alignment applies a standard gap
penalty.  (Both allow arbitrarily large unaligned parts at the edges
of the query.)

For DNA queries, you might wish to try "trans-spliced" alignment
without considering splice signals::

  lastdb -m1111110 db genome.fasta
  lastal -Q1 -e120 db q.fastq | last-split -c0 > out.maf

Going faster by parallelization
-------------------------------

With large datasets, it's important to go faster by using multiple
CPUs.  One way to do that is by using GNU parallel
(http://www.gnu.org/software/parallel/)::

  parallel --pipe -L4 "lastal -Q1 -e120 db - | last-split" < q.fastq > out.maf

Beware that older versions of GNU parallel were slow when using --pipe
-L, so be sure to use a recent version.

Options
-------

  -h, --help
         Show a help message, with default option values, and exit.

  -g, --genome=NAME
         Do spliced alignment, and read splice signals (GT, AG, etc)
         from the named genome.  NAME should be the name of a lastdb
         database.

  -c, --cis=PROB
         Do spliced alignment, and set the average probability per
         base of cis-splicing.  The default value roughly fits human
         RNA.

  -t, --trans=PROB
         Do spliced alignment, and set the average probability per
         base of trans-splicing.

  -M, --mean=MEAN
         Do spliced alignment, and set the mean of ln(intron length).
         The default value fits human RNA.

  -S, --sdev=SDEV
         Do spliced alignment, and set the standard deviation of
         ln(intron length).  The default value fits human RNA.

  -m, --mismap=PROB
         Don't write alignments with mismap probability > PROB.
         Low-confidence alignments will be discarded unless you
         increase this value!

  -s, --score=INT
         Don't write alignments with score < INT.  The default value
         is somewhat higher than the lastal score threshold.
         Specifically, it is e + t * ln(1000), where e is the score
         threshold, and t is a scale factor that is written in the
         lastal header.  This roughly means that, for every alignment
         it writes, it has considered alternative alignments with
         one-thousandth the probability.

  -n, --no-split
         Do probability calculations as usual, but write the
         *original* alignments, annotated with "p" lines and mismap
         probabilities.  Note that the mismap and score limits still
         apply.

  -v, --verbose
         Show progress information on the screen.

  -V, --version
         Show version information and exit.

Details
-------

* The input must be in MAF format, and it must include header lines
  (of the kind produced by lastal) describing the alignment score
  parameters.

* The program reads one batch of alignments at a time (by looking for
  lines starting with "# batch").  If the batches are huge
  (e.g. because there are no lines starting with "# batch"), it might
  need too much memory.

* lastal can optionally write "p" lines, indicating the probability
  that each base is misaligned due to wrong gap placement.
  last-split, on the other hand, writes "p" lines indicating the
  probability that each base is aligned to the wrong genomic locus.
  You can combine both sources of error (roughly) by taking the
  maximum of the two error probabilities for each base.

* The program assumes that the same score parameters were used for all
  alignments.  So it is incompatible with the recipe in the bisulfite
  document.

To do
-----

* An option to specify splice signals and their strengths.
