Methods to Study Genomic DNA Sequence Variation

Abstract

In this chapter, we describe the advances in molecular biology methods and discuss how current methods address the highly heterogeneous nature of human genomic variation. We start presenting a historical perspective of early developments that culminated in our knowledge of human genomes’ enormous diversity. Next, we propose a classification of variants according to these criteria: variant’s length, frequency, and consequence. Variant’s length is critical when choosing between two main groups of methods: fragment-based or sequence-based. We explore these two groups in Sect. 3.3. Two other divisions were considered to describe the methods: if they are targeted or genome-wide approaches and if they focus on common or rare variants. Fluorescence in-situ hybridization (FISH), array comparative genomic hybridization (array-CGH), multiplex ligation-dependent probe amplification (MLPA), and triplet repeat primed PCR (TP-PCR) are discussed in the section of fragment-based methods. Sanger sequencing, genotyping microarrays, and a detailed description of next-generation sequencing are covered in the sequence-based section. In Sect. 3.4, we discuss current applications, including a brief workflow for molecular diagnosis, rare-variant association testing, and polygenic risk scores. Finally, Sect. 3.5 explores perspectives and presents a glimpse of promising methods, such as Cell-free DNA, long-read sequencing, and integrative approaches with other ‘omics’. Although it is still difficult to interrogate the full spectrum of human genome variation using a single method, advances might soon overcome these challenges.