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Fourth Step. VIRGO verifies whether mismatches occur into double-stranded regions by creating a short reverse complementary sequences centered on the mismatch. VIRGO aligns the created sequence with a region of maximum 4001 nucleotides centered on the A-G mismatch. If the percentage of the alignment is greater than or equal to 85%, VIRGO annotates that mismatch as occurring into a double-strand region.
