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Careful design in the primary steps of a next-generation sequencing study is critical for obtaining successful results in downstream analysis. In this study, a framework is proposed to evaluate and improve the sequence mapping in targeted regions of the reference genome. In this regard, simulated short reads were produced from the coding regions of the human genome and mapped to a Customized Target-Based Reference (CTBR) by the alignment tools that have been introduced recently. The short reads produced by different sequencing technolog