Optimal experiment design

The goal for this part of discussion is how to obtain as much information as possible from a single polony slide. One obvious way to achieve this goal is to improve polony density, which is equivalent to reducing polony size. Polony size depends on the concentration of polyacrylamide gel, as well as the size of PCR amplicons. We intially started with 3kb amplicons in 6% gel, and were able to obtain >5,000 polonies with the size of 20~50 microns in one slide. Big amplicons cover more SNPs than smaller amplicons do, so less amplification primers need to be used for a given set of SNPs. This provides a cost advantage because the acrydite-modified primers account for a large percentage of cost in polony haplotyping experiments. However, the plan with big amplicons failed to work because of two technical reasons: (1) SBE gives extremely weak signals when the SNP is >800bp away from the anchor primer. (2) The amplification efficiency on large amplicons is very low, so that even one can get many polonies within a slide, the number of overlapping polonies is very small. In fact, only those overlapping polonies are informative. We then switched to small amplicons. We did experiments on CD36 gene and NOS3 gene with two set of parameters. For the CD36 gene, the average amplicon size is 840-bp, and the amplification were done in 10% gel. We had on average 1,397 polonies on each SNP, and 275 two-locus haplotype reads on each pair of SNPs. We used larger amplicons (1,045-bp) but lower gel concentration (7%) in the NOS3 gene. As expected, the polony density is slightly higher (1,812 polonies/slide), but the average number of two-locus haplotype reads is three-fold lower (97/slide) because of the difference in amplicon size. These results suggest that  an average amplicon size of 800~850-bp probably provides the best efficiency.