We left you with assemblies numbering tens of contigs. The next step was for our students to order them. We chose to go ahead only for strains 705 and 743. For strain 705 we had an optical map to help ordering contigs, and for 743 we combined assemblies, as well as similarity to closely related bacteria, since it was anyway the assembly with the less contigs.
The students then designed PCR primers at the extremities of these contigs, at the end of the autumn semester. These primers were ordered, and at the begining of the spring semester the students performed all the combinatorial PCRs:
For 705: 92 contigs, 95 PCRs, 75 positive PCRs, 42 gaps closed sequencing these PCR products. Thus we were left with 50 contigs to annotate, which were mapped to 3 replicons plus “unmapped”.
For 743: 50 contigs, 46 PCRs, 24 positive PCRs, 16 gaps closed sequencing these PCR products. Thus we were left with 30 contigs to annotate, which were mapped to 2 replicons plus “unmapped” (but probably a third replicon).