Base Calling, Read Mapping, and Coverage Analysis

Although patient genotyping has been performed for many years, it has been limited to relatively short loci. Next-generation sequencing (NGS) has enabled genotyping at an unprecedented scale, but the clinical utility of the approach places a premium on accuracy at all four steps that lead to variant detection including library preparation and template amplification, base calling, alignment/mapping of sequence reads, and coverage analysis to assess the overall quality and completeness of the targeted genotype region. A quality score is assigned to each base call that indicates the confidence of the call, and is dependent on a number of factors intrinsic to the quality of a sequencing run. Since current NGS technologies produce sequence reads that are relatively short, the reads must be aligned (or mapped) onto the human reference genome so the aligned reads can be used to make variant calls; sequence alignment is computationally the most difficult and expensive step of variant analysis, and is a major source of error. Genotype quality based on NGS data varies greatly from position to position within a targeted region of the genome, so variant calls themselves are normally associated with a quality score derived from various metrics in order to judge the reliability of the variant call within a specific position of the targeted region.