Vu et al. BMC Genomics 2010, 11:301 http://www.biomedcentral.com/1471-2164/11/301 Page 6 of 8 Table 1: Comparison of screening BAC library by conventional PCR and by MT-PCR-HRM. Compared features Screening by conventional Screening by MT-PCR-HRM PCR using 3D pooling strategy using 2D pooling strategy Maximum number of 384-well plates to be pooled in one super pool 10 384-well plates 25 384-well plates Number of super pools to be tested to identify a positive superpool (N: The total plate number of the BAC library) N/10 PCR reactions (PCR/PCR-HRM) needed to identify a positive super pool N/10 Reactions needed to identify the plate ID for one positive super pool Reactions needed to identify the clone ID from one positive plate Total number of reactions needed to get positive BAC clone ID from whole N/10 + 50 library Multiplexing possibility Checking on agarose gel Cost Procedure duration to anchor 1 marker Scoring data Organism Brachypodium distachyon Potato Barley Wheat Swine Cattle 300 850 5500 16000 2600 3100 24 66 430 1250 203 242 1 3 17 50 8 10 10 30 30 30 30 30 10 40 No Needed 12 h 54* 54 54 54 54 54 64 87 101 134 92 94 0.16 £/1 PCR reaction + cost for agarose gel electrophoresis 0.17 £/1 PCR-HRM reaction Manually from gel photos Semi-Automatic (figures and summary tables can be exported from HRM rotor system) N/25 N/25 10 18 Yes NO 3 h N/25 + 28 22 29 35 45 31 32 Table 2: MT-PCR-HRM pooling strategy to accommodate for variable genome size. 1Cgenome No 384 No super (Mb) well plates pools No matrix pools No single pools Total required simplex Total required triplex MT-PCR-HRM MT-PCR-HRM Estimated number of pools and MT-PCR-HRM reactions required to find a single marker (or 3 markers simultaneously) within the denoted genomes, assuming a 3× coverage and 100 Kb average insert size for the corresponding BAC libraries. Note: *1× coverage = 18 single pools (see Figure 1); 3× coverage = 54 single pools