Identification of Nucleotide Variation in Genomes Using Next-Generation Sequencing

Discovery of genome-wide variation has taken a huge leap forward with the introduction of next-generation sequencing (NGS) technology. Variant discovery requires sampling of a number of haplotypes. This can be either the two haplotypes of a diploid organism or multiple haplotypes in a population. Variant discovery can be done by sequencing pooled DNA and NGS makes it cost-effective to sample many haplotypes. In this chapter, we discuss various sequencing strategies for variation discovery, focusing mainly on single nucleotide polymorphisms, and to a lesser extent on short insertion/deletions (Indels). We discuss different options, such as specific library construction and the amount of sequencing required to meet a certain objective. While the benefits of NGS to their own research may be obvious to many researchers, the main obstacle for applying NGS is often practical - how to manipulate and analyze the amount of data from a typical NGS run. The methods therefore focus on practical considerations dealing with large NGS datasets, such as sequence processing and filtering, mapping to a reference genome, and variant calling. In addition, we focus on data standards and tools to manipulate and analyze data from such standardized datasets. By providing examples and links to easy-to-implement scripts and software, we hope to lower the threshold for biologists to further explore the wealth of information that can be obtained by these new molecular resources