Figure 3

Maximum Depth Sequencing (MDS). The chromosomal DNA strand containing the sequence of interest, such as G11 of bbr_0113, is digested 3’ by a restriction enzyme (AatII). A synthetic oligonucleotide containing 5’ the NGS adapter P5 sequence (blue line), 14nt random sequence “family tag” (orange rectangle) and a sequence (green line) being reverse and complementary to the sequence of the newly produced 3’end of the chromosome strand, is used as template to incorporate the family tag and adapter sequence 3’ to the sequence of interest. Un-incorporated oligos are digested by Exonuclease I. Then, 12 cycles of linear amplification are performed with a specific “primer amplifier” complementary to the newly neo-synthesized DNA 3’end. Corresponding products are then used as template in exponential amplification involving the addition of a second primer. It contains 5’ the NGS adapter P7 sequence (in blue), an 8nt random sequence but can be facultative (in grey rectangle), and a sequence (in green) being reverse and complementary to a specific sequence of the sequence of interest. The purified PCR product is then subjected to NGS sequencing with the family tag reflecting a unique chromosomal DNA strand molecule.