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 
complications 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.