89 Stella K. Kantartzi (ed.), Microsatellites: Methods and Protocols, Methods in Molecular Biology, vol. 1006, DOI 10.1007/978-1-62703-389-3_6, © Springer Science+Business Media, LLC 2013 Chapter 6 Next-Generation Sequencing for High-Throughput Molecular Ecology: A Step-by-Step Protocol for Targeted Multilocus Genotyping by Pyrosequencing Jonathan B. Puritz and Robert J. Toonen Abstract Next-generation sequencing technology can now provide population biologists and phylogeographers with information at the genomic scale; however, many pertinent questions in population genetics and phylogeography can be answered effectively with modest levels of genomic information. For the past two decades, most population-level studies have lacked nuclear DNA (nDNA) sequence data due to the com- plications and cost of amplifying and sequencing diploid loci. However, pyrosequencing of emulsion PCR reactions, amplifying from only one molecule at a time, can generate megabases of clonally amplified loci at high coverage, thereby greatly simplifying allelic sequence determination. Here, we present a step-by-step methodology for utilizing the 454 GS FLX Titanium pyrosequencing platform to simultaneously sequence 16 populations (at 20 individuals per population) at 10 different nDNA loci (3,200 loci in total) in one plate of sequencing for less than the cost of traditional Sanger sequencing. Key words 454, NGS, nDNA, Population genetics, Phylogeography , Sequencing cost, Sanger 1 Introduction For 20 years, mitochondrial DNA (mtDNA) has been the standard molecular sequence marker for population geneticists and phylo- geographers (1, 2). The mitochondrial genome, with a rapid muta- tion rate and small effective population size, contains a wealth of information for recent population histories. Moreover, mtDNA loci are easy to prime, amplify, and sequence due to uniparental inheritance and a lack of recombination. However, the mtDNA genome is essentially a single marker and cannot alone represent all of the evolutionary processes acting upon a population (3, 4). Thus, the need to incorporate nDNA sequence data into analyses has been widely recognized by the field over the last 10 years (5–8). The use of nDNA sequence loci in phylogeography and popu- lation genetics has been limited by laboratory and analytical difficulties (reviewed in 7, 9, 10). nDNA loci that are polymorphic at the