Recently, with the decreasing cost of DNA sequencing and improving strategy of data analysis, whole-genome sequencing (WGS) has been used for detection of pathogenic genes in cancer, Mendelian diseases and other complex diseases and gradually become applicable for clinical diagnosis. WGS enables not only the identification of single nucleotide variants (SNVs), insertions and deletions (InDels) both in coding region and non-coding regions, but also the detection of copy number variations (CNVs) and structural variations (SVs) on a genome scale. In this review, we discuss the canonical workflow and methods of bioinformatics analysis in WGS, and its application in disease research and clinical diagnosis. In addition, this review provides an overview in application, progress and challenge on WGS in medical genetics