Sequence logo and entropy analyses of mapped 5’reads. (a) We compared sequence logo results for 10,000 randomly selected mapped 5’ reads of Ctl, Heat, and Heat + OptiK. (b) After generating computational control reads, sequence logo results were also compared. Ctl showed strong biases near the beginning of 5’ reads. Heat had less biases at 5’ but showed persistent low level biases at all nucleotide positions. The biases did not have the same sequence bias patterns as Ctl. Therefore, RNaseIII generates sequence biases at 5’. Though Heat + OptiK had a similar sequence bias pattern as Heat, it was much smaller throughout the entire nucleotide positions. Thus, OptiKinase weakens T4PNK sequence biases, and Heat + OptiK is the best method. (c) shows entropy results of the three libraries including the entropy data of their computational control reads (Com. ctl) for 5’ end reads. (d) The entropy results of the three libraries were compared. Lower entropy values correspond to greater sequence bias.