To identify positions with a 'significant' portion of 'consistent' unaligned end reads we first estimate 'null-distributions' of the fractions of left and right unaligned end reads at each position in the read mapping, and subsequently use these distributions to identify positions with an 'excess' of unaligned end reads. In these positions we create a Left (LB) or Right (RB) breakpoint signature. To estimate the null-distributions we:
- Calculate the coverage, , in each position, of all uniquely mapped reads (Non-specifically mapped reads are ignored. Furthermore, for paired read data sets, only intact paired reads pairs are considered -- broken paired reads are ignored).
- Calculate the coverage in each position of 'valid' reads with a starting left unaligned end, (of minimum consensus length 3bp).
- Calculate the coverage in each position of 'valid' reads with a starting right unaligned end, (of minimum consensus length 3bp).
The two user-specified settings 'The P-value threshold' and the 'Maximum number of mismatches' determine which breakpoint signatures the algorithm will detect (see here and figure 28.39). The p-value is used as a cutoff in the binomial distributions estimated above: if the probability of obtaining the observed number of left (or right) unaligned ends in a position with the observed coverage, is smaller than the user-specified cut-off, a Left breakpoint signature (LB), respectively Right breakpoint signature (RB), is created. The 'Maximum number of mis-matches' parameter is used to determine which reads are considered 'valid' unaligned end reads. Only reads that have at most this number of mis-matches in their aligned parts are counted. The higher these two values are set, the more breakpoints will be called. The more breakpoints are called, the larger the search space for the Structural variation detection algorithm, and thus the longer the computation time.
In figure 28.42, three unaligned end signatures are shown. The left-most LB signature is called only when the p-value cut-off is chosen high (0.01 as opposed to 0.0001).