Figure 2.

Calculation of features for the donor site

(A) SQUIRLS calculates seven features to evaluate variant impact on the donor site. The individual information content $\left(R_i\right)$ of the reference and alternate canonical splice site and of the donor site in the following exon (Exon $k+1$) are calculated and used to determine the difference in information content between the reference and alternate canonical splice site $\left(\Delta R_{ican}\right)$, the difference between the best candidate cryptic splice site and the alternate sequence of the canonical splice site $\left(\Delta R_{icrypt}\right)$, and the difference between the donor site at exon k and k+1$\left(\Delta R_{inext}\right)$, because differences in splice site strength can be predictive of exon skipping.¹⁴ See Table 1 for information about other features.

(B) In this example, a variant in intron k creates a cryptic splice site with 8 bits, which is greater than the individual information of the canonical splice site (4.5 bits), so $\Delta R_{icrypt}$ = 3.5 bits. The variant does not change the sequence of the canonical splice site, so $\Delta R_{ican}$ = 0. The individual information of the donor site of the next exon has 0.5 bits more than that of exon k, so $\Delta R_{inext}$ = −0.5 bits.

(C) In this example, a variant in the canonical splice site (e.g., the +5 position) reduces the strength of the canonical splice site from 7.5 to 3.0 bits and simultaneously creates a novel cryptic site with an individual information content of 8.3 bits. An example of this is the variant GenBank: NM_000314.7; c.253+2T>C (PTEN [MIM: 601728]), which alters the canonical splice site and simultaneously changes the sequence of a cryptic splice site located 3 nucleotides downstream, resulting in the inclusion of 4 intronic nucleotides in the variant mRNA.⁴⁷