View source for Lastz/chain/net/multiz considerations/caveats/restrictions/limitations

== Introduction ==
The lastz/chain/net/multiz processing pipeline is the primary alignment procedure used at
the U.C. Santa Cruz Genomics Institute to produce multiple alignments.  There are a number
of considerations that should be taken into account by consumers of the resulting data
that could certainly affect conclusions drawn from such analysis.

== lastz ==
As with any alignment algorithm, the choice of parameters for '''lastz''' is critical to the
results produced by this alignment program.  Typically the parameters chosen fall into three
categories based on the phylogenetic distance and/or clade relationship of target and query
sequences.

# primate to primate alignments  (e.g. human<->chimp)
# closer phylogenetic relationship (e.g. human<->mouse)
# more distant phylgenetic relationship  (e.g. human<->fish)

Parameters used for lastz are recorded in the [[UCSC Multiple Alignments]] page.
See also: [http://www.bx.psu.edu/~rsharris/lastz/README.lastz-1.04.00.html lastz documentation.]

One specific parameter that is interesting is the '''--masking=<count>  (aka M=<count>)'''
ususlly set at a count of 254.  From the lastz documentation:
<pre>
 Dynamically mask the target sequence by excluding any positions that appear in too many alignments from further consideration for seeds.

 Specifically, a cumulative count is maintained of the number of times each target location is aligned. After each query sequence and strand is
 processed, any locations that have been output in at least <count> alignment blocks are masked, so they will be excluded from the seeding stage
 for subsequent query sequences. Since repetition discovered while processing one sequence strand is only masked for subsequent sequence
 strands, this option has no effect on the first strand of the first sequence in the query file.
</pre>

Therefore, any target sequence that has more than 254 matching alignments to the query sequence will output only
those first 254 alignments.  Query matches beyond that number will not be included in the output.

== chain ==

== net ==

== multiz ==
The first important point to consider for '''multiz''' results is that these alignments are
classified as '''reference based''' alignments.  Only alignments between the query sequences
and the target sequence are included in the final result of the multiple alignment.  The inputs
to '''multiz''' are only the pair-wise alignments of the query to target sequence alignments.
There is no indication of potential relationships between the query sequences with the other
query sequences.  There could be important alignment relationships between query sequences
which will not be seen in the '''multiz''' result.

The second important point about the '''multiz''' alignments is the choice of chain/net
used as input to '''multiz'''.  The choice is one of three types of chain/net results:

# syntenic net - used for higher quality genome assemblies, not too phylogentically distant
# reciprocal best net - used when the query genome is poor quality, high contig count, lower N50 size
# net - the fundamental net alignment, used for phylogenetically distant species

The type of chain/net used in the alignment is captured in the [[UCSC Multiple Alignments]] page.

* The '''syntenic net''' and '''reciprocal best net''' are subsets of the fundamental '''net''' alignment.
* The '''syntenic net''' eliminates alignments that would be interruptions to synteny in the query to target alignment.
* The '''reciprocal best net''' selects only those alignments between target and query that are reciprocal.  Only the single best alignment between query and target is included in the result.  This eliminates '''many to many''' alignments from query to target.