In recent years the amount of biological sequence data available for research has increased significantly and complete genome sequences have become commonplace. Next-generation sequencing (NGS) is expected to revolutionize biomedical research. However, the analysis of millions of DNA/RNA sequences is becoming a real challenge as the capabilities of standard computers have been far exceeded. This book guides readers in the processing and analyzing of genome-wide data with minimum computer RAM requirements. It focuses on practical solutions to a wide range of next-generation sequencing bioinformatic problems such as data import, read demultiplexing, trimming, quality checks, alignment, transformations, normalization, annotation, motif search, and differential expression. No prior programming knowledge is required.