Originally posted by polsum
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Alignments involving one or more ambiguous reference characters (N, -, R, Y, etc.) are considered invalid by Bowtie. This is true only for ambiguous characters in the reference; alignments involving ambiguous characters in the read are legal, subject to the alignment policy. Ambiguous characters in the read mismatch all other characters. Alignments that "fall off" the reference sequence are not considered valid.
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