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Next-generation sequencing (NGS) has revolutionized the genetic landscape. It is a lengthy, labor-intensive process that yields results never before achieved. As a result, it is imperative that the quality of the RNA or DNA starting material as well as DNA libraries be evaluated from the start. Difficulties can often arise when only fragmented or low concentrated starting material is available. Furthermore, most NGS sample preparation protocols require PCR amplification to generate DNA libraries prior to sequencing, the likelihood of artifact generation could contribute to bias, affecting the potential results. Here we discuss several critical steps in the next-generation sequencing workflow which require the implementation of quality control procedures to increase workflow efficiency, which in turn helps to reduce downstream sequencing costs.