Table 4.

Effect of the order of releasing enzymes on cDNA-AFLP performance

RE 1	RE 2	RE 3	Coverage	Redundancy	PCR samples	Ø Fragment length (nucleotides)
L	M	H	65 %	38 %	713	226
L	H	M	65 %	38 %	624	219
M	L	H	65 %	38 %	672	226
M	H	L	65 %	38 %	648	226
H	L	M	64 %	38 %	552	217
H	M	L	64 %	38 %	552	217

Simulation of cDNA-AFLP with sequential digestion protocol on Arabidopsis RefSeq data was performed using marking enzymes MboI and FatI and six releasing enzymes with different frequencies of occurrence: frequently cutting enzyms H (TasI and MseI), enzymes with a medium frequency of occurrence M (BsaWI and TatI) and infrequently cutting enzymes L (AcyI and CfrI). For each permutation of H, L and M enzyme classes, cDNA-AFLP were simulated for 8 permutations of the releasing enzymes (RE). Average values of coverage, redundancy, PCR effort and fragment length for each permutation of H, L and M were calculated.