Next generation sequencing (NGS) has become a powerful approach in the field of infectious diseases and has revolutionized clinical microbiology and virology. However, despite the advances in these technologies, the use of NGS remains challenging for complex samples, such as ultra-low copy samples, samples with poor integrity of nucleic acids, and single cells. NGS is currently being adapted to the analysis of such complex samples through the development and standardization of robust methodologies for enriching and amplifying low copy genomes, for restoring and amplifying degraded nucleic acids, and for developing instruments for nanotechnology miniaturization of sample preparation. We discuss these innovative methodologies and the substantial progress in sample preparation methods.