AbstractSequencing by hybridization (SBH) is a proposed approach to DNA sequencing. The SBH-spectrum of the target sequence is a list of all k-mers occurring at least once in the sequence. Sequencing is successful if the SBH-spectrum is a result of only that sequence and ambiguous otherwise. Unfortunately, the expected number of sequences consistent with a given spectrum increases exponentially with the target sequence length.In this paper, we extend previous work of [S. Snir, E. Yeger-Lotem, B. Chor, Z. Yakhini, SBH+RE—restriction enzymes dramatically enhance SBH, Technical Report, Department of Computer Science, The Technion, Haifa, Israel, 2002] to increase the resolving power of SBH by including information from enzymatic digestion assays. In addition to the hybridization assay, we conduct a small number of complete digestion assays using different restriction enzymes. The computational phase of identifying consistent sequences then combines the hybridization and digestion information. This combination of SBH and digestion assays significantly increases the length of sequences that can be uniquely determined. We give procedures for selecting the best enzymes for the job, prove that a variant of the reconstruction problem which includes an extra free parameter is hard, and give effective heuristics to improve search-based reconstruction algorithms. We also give a lower bound on the number of restriction enzymes required for unique reconstruction.