Sequence logo and entropy analyses of mapped 3’reads. (a) We also compared sequence logo data for mapped 3’ reads of Ctl, Heat, and Heat + OptiK. (b) After generating 35 bp computational control reads for the 3’ reads of the three libraries, sequence logo results were compared. Ctl also showed strong biases at the beginning of 3’ reads. Even though the biases of Heat were smaller, there were persistent low level biases at all nucleotide positions. Because the biases of Heat were not the same sequence bias patterns as Ctl, RNaseIII also generates sequence biases at 3’ ends. The 3’ end sequence bias, A or T, indicated the sequence bias of T4PNK phosphatase activity. Like 5’ reads, Heat + OptiK had a similar sequence bias pattern as Heat, but it was much smaller. Thus, OptiKinase weakens T4PNK sequence biases, and Heat + OptiK is also the best method for 3’ reads. (c) shows their entropy results including computational control data (Com. ctl). In (d) entropy data were compared.