An Integrated Data Analysis Suite and Programming ... - TOBIAS-lib

42 CHAPTER 2. A HIGH-THROUGHPUT DNA SEQUENCING DATA ANALYSIS SUITE
While alignment scores become available after the initial stage, ltering is delayed until after
backtracing for reasons of threshold calculation.
The end alignment mode for the left end of the alignment determines the mode of alignment
matrix initialization and alignment score calculation. With left end alignment mode local
initialization and score calculation proceeds according to standard local alignment, with rst
row and column initialized to zero and the alignment score at each eld of the matrix truncated
at zero from below. Left end mode dangling_any utilizes the same matrix initialization, but
does not clip alignment scores at zero. With dangling_ref and dangling_qry, only the rst row
or column is zero-initialized, respectively, given the width of the matrix is determined by the
size of the reference sequence. Sequence overhangs for the respective other sequence are valued
with the regular gap penalty. Mode global does not perform pre-initialization of the matrix, as
in standard global alignment.
For selection of the best out of multiple permitted pairs of end alignment mode for a reference
oligomer, multiple passes of dynamic programming alignment must be performed due to the
distinct requirements of matrix initialization. For each distinct left end alignment mode a corresponding
right end alignment mode may be dened. However, alignment is only performed if the
conguration is not included by a dierent pair of modes specied, i. e. if the constraint on the
right end of the alignment is weaker than that specied by the next left end mode that includes the
current one. Due to the hierarchical nature of tolerated sequence overhang congurations there
is nonetheless a chance that the same alignment will be produced multiple times by dierent keyword
pairs. For example, (dangling_ref;dangling_qry) and (dangling_qry;dangling_ref)
both dene supersets of the (global;global) conguration. Such redundancies can be eliminated
prior to backtracing by determining the subset of the respective end alignment mode that
has already been covered by previous alignments and excluding it by assigning corresponding
elds of the alignment matrix the maximum penalty.
For backtracing initialization and alignment score calculation, the alignment algorithm
keeps track of values and locations of last row, last column and global score maximums for the
matrix. Mirroring matrix initialization, backtracing starts at global score maximums for right
end alignment mode local, at last row and last column maximums for modes dangling_ref
and dangling_qry, respectively, at the combined last row and last column maximums for
dangling_any, and at the lower right corner for global.
Whenever the backtracing algorithm encounters multiple possible elds of origin for an alignment
score, alternative paths are stacked for later completion, unless retrieval of only a single
representative alignment was requested. While exhaustive tracing of all possible alignment paths
can be combinatorially unfavorable, generating a representative for all distinct pairs of mapping
end coordinates can be performed eciently. For this purpose, each eld of the matrix that has
been reached by the backtracing algorithm from the same right end coordinates is marked as
visited. If a eld marked as visited is reached from one of the stacked alternative paths, the
respective path is aborted and the algorithm proceeds to assessing the next path.
For read clipping purposes it is typically only relevant whether an oligomer can be assigned
to unique coordinates on the sequencing read. For assessment of coordinate uniqueness, the
backtracing algorithm proceeds only until alternative end coordinates have been encountered, and
the alternative trace is not emitted. If the backtrace completes without encountering coordinates
dierent from the primary trace, its end coordinates are marked as unique.
Reliability of a sequence match is determined by both the length of the match and its relative
amount and type of edit operations. Alignment score ltering is therefore congured by setting
slope and oset of a linear function. By default, the function's parameter is the length within the
shorter sequence out of reference and query that is spanned by the match. Alignments with a
score below the threshold thus calculated are not propagated to the list of results and sequencing