Analysis of DNA sequence variants detected by high-throughput sequencing

Abstract

The Undiagnosed Diseases Program at the National Institutes of Health uses high-throughput sequencing (HTS) to diagnose rare and novel diseases. HTS techniques generate large numbers of DNA sequence variants, which must be analyzed and filtered to find candidates for disease causation. Despite the publication of an increasing number of successful exome-based projects, there has been little formal discussion of the analytic steps applied to HTS variant lists. We present the results of our experience with over 30 families for whom HTS sequencing was used in an attempt to find clinical diagnoses. For each family, exome sequence was augmented with high-density SNP-array data. We present a discussion of the theory and practical application of each analytic step and provide example data to illustrate our approach. The article is designed to provide an analytic roadmap for variant analysis, thereby enabling a wide range of researchers and clinical genetics practitioners to perform direct analysis of HTS data for their patients and projects.

Figure 1

Cumulative Filtration of Exome Variant Lists from 22 Families. A set of 22 exome projects is displayed using two different analytical approaches: one uses all available family data (black) and the other uses only data from the proband (red). The y-axis is the log₁₀ of the number of cumulatively filtered, residual variants. The x-axis shows filtration steps, which are sequential from left to right. The last two steps (homozygotes and heterozygotes) use the post-quality-filtration variants to filter and are not sequential. Note that the implementation of homozygote and heterozygote filters differs between single exome analyses and family based analyses. Mendelian segregation and phase information is not available in the case of single exome analysis. Homozygotes are not checked for inheritance from both parents. The “heterozygote” count is a tabulation of all pair-wise combinations of variants for those cases where more than one heterozygous variant is found in the same gene. Single exome projects start with fewer variants and end with a larger number of candidates for further study. See the text for a further explanation of the various filtration steps.