Fig. 4.

Miscoding lesions and the 454 process. During preparation of templates for 454 sequencing, the ends of DNA fragments are first repaired by T4 DNA polymerase (A), and in a later step linkers are filled in by Bst DNA polymerase (B). During blunt-end repair by T4 DNA polymerase (A), miscoding lesions (black circles) on 3′-overhanging ends are removed, whereas miscoding lesions on 5′-overhangs result in complementary misincorporations (white circles) in the resultant 454 sequences. Similarly, extension by the strand-displacing Bst DNA polymerase (B) causes miscoding lesions in the template DNA downstream of nicks or gaps to result in complementary misincorporations in the sequences generated.