Next-generation sequencing (NGS) in the microbiological world: How to make the most of your money

The Sanger sequencing method produces relatively long DNA sequences of unmatched quality and has been considered for long time as the gold standard for sequencing DNA. Many improvements of the Sanger method that culminated with fluorescent dyes coupled with automated capillary electrophoresis enabled the sequencing of the first genomes. Nevertheless, using this technology to sequence whole genomes was costly, laborious and time consuming even for genomes that are relatively small in size. A major technological advance was the introduction of next-generation sequencing (NGS) pioneered by 454 Life Sciences in the early part of the 21th century. NGS allowed scientists to sequence thousands to millions of DNA molecules in a single machine run. Since then, new NGS technologies have emerged and existing NGS platforms have been improved, enabling the production of genome sequences at an unprecedented rate as well as broadening the spectrum of NGS applications. The current affordability of generating genomic information, especially with microbial samples, has resulted in a false sense of simplicity that belies the fact that many researchers still consider these technologies a black box. In this review, our objective is to identify and discuss four steps that we consider crucial to the success of any NGS-related project. These steps are: (1) the definition of the research objectives beyond sequencing and appropriate experimental planning, (2) library preparation, (3) sequencing and (4) data analysis. The goal of this review is to give an overview of the process, from sample to analysis, and discuss how to optimize your resources to achieve the most from your NGS-based research. Regardless of the evolution and improvement of the sequencing technologies, these four steps will remain relevant.