bisulphite-converted, adaptor-ligated DNA molecules were enriched by 4–8 cycles of PCR with the following reaction composition: 2.5 U of uracil-insensitive PfuTurboCx Hotstart DNA polymerase (Stratagene), 5 μl 10× PfuTurbo reaction buffer, 31 μM dNTPs, 1 μl Primer 1, 1 μl Primer 2 (50 μl final). The thermocycling parameters were: 95 °C for 2 min, 98 °C for 30 s, then 4–8 cycles of 98 °C for 15 s, 60 °C for 30 s and 72 °C for 4 min, ending with one 72 °C for 10 min step. The reaction products were purified using AMPure XP beads. Up to two separate PCR reactions were performed on subsets of the adaptor-ligated, bisulphite-converted DNA, yielding up to two independent libraries from the same biological sample. Final sequence coverage was obtained by sequencing all libraries for a sample separately, thus reducing the incidence of ‘clonal’ reads that share the same alignment position and probably originate from the same template molecule in each PCR. The sodium bisulphite non-conversion rate was calculated as the percentage of cytosines sequenced at cytosine reference positions in the Lambda genome.
