The efficiency could probably be greatly improved by sequencing a few additional strains in each cluster, for example, 1197 and 408 in Cluster 3 and 1198, 88, and 939 in Cluster 1.
This seems like an extreme, difficult case, yet our results show that even it can be solved by sequencing a practical number of mutant strains (see Fig. 2C).
The data collected as part of this study are relative to Haemophilus sp, but the same strategy could be used to design and produce supragenome arrays for any species for which the majority of the supragenome has been sampled by sequencing a reasonable sized subset of strains.
This issue could be circumvented either by sequencing all strains and eliminating aberrant genotypes or by constructing additional replicate strains.
With only two exceptions all genes containing mutations in the isolated monoclonal strains were confirmed by sequencing the strain mix after prolonged selection, further validating the results.
Second, only 10 high-quality variants were identified by sequencing another lab strain, MAP7, an Rd derivative resistant to seven antibiotics (Catlin et al. 1972), and only 3 of these are not accounted for by its antibiotic resistances (Table 6).
However, it should be noted that published results [ 21], and our own data from an ongoing re-sequencing study focussed on selected loci, show that a significant percentage of the identified changes can be verified by re-sequencing a different strain from the same evolutionary lineage.
Massively parallel DNA sequencing technologies are becoming affordable for genotyping and have been applied to map recombination events by sequencing progeny strains and their parents across a wide range of different taxa.
Given the design of the tools we have developed, further increasing their power by sequencing additional strains and measuring additional phenotypes of interest should be straightforward.
