Fungal Genomics Methods and Protocols
Having experienced unprecedented growth since the turn of the millennium, the dramatic expansion of resources and techniques in fungal genomics is poised to fundamentally redefine the study of fungal biology. In Fungal Genomics: Methods and Protocol
- PDF / 102,110 Bytes
- 9 Pages / 504 x 720 pts Page_size
- 6 Downloads / 209 Views
1. Introduction Deoxyribonucleic acid (DNA) molecules are the bearers of information needed for the development and functioning of all cells in a living organism. The DNA molecules of each organism are composed of chains of monomeric nucleotides (the four bases adenine, “A”; cytosine, “C”; guanine “G”; and thymine, “T”) and are organized into chromosomes, called genome. To understand the mechanisms that govern the complex biological processes of an individual and to unveil the genetic differences among different organisms, it is necessary to know the composition of the DNA molecules through the process of genome sequencing. To date, no technologies are available to read the entire sequence of a single molecule directly. Instead, various methods have been developed to decode small snippets of DNA, one piece at a time. In principle, these small sequence reads can be pieced together into longer pieces by bioinformatics analysis, ultimately to reach the completing of the genome, as long as there are sufficient amount of information. In reality, however, each Jin-Rong Xu and Burton H. Bluhm (eds.), Fungal Genomics: Methods and Protocols, Methods in Molecular Biology, vol. 722, DOI 10.1007/978-1-61779-040-9_1, © Springer Science+Business Media, LLC 2011
1
2
Grabherr, Mauceli, and Ma
genome project has to face multiple challenges, including to (a) obtain accurate sequence for each fragment, (b) get readings of sequences from the entire genome equally, (c) get readings that are long enough so that repetitive regions can be resolved, and (d) produce massive amounts of data quickly and inexpensively. In summary, the determining factors for each genome project are accuracy, completeness, cost, and time. Earlier, most genomic projects were focused on the accuracy and completeness of issues. Since the early 2000s, the focus of genomic projects shifted, as a variety of sequencing technologies that emphasize on the cost and time issues have been established. Here, we briefly introduce the main sequence technologies, including the Sanger method and the emerging next-generation sequencing (NGS) technologies, and compare their strengths and weaknesses for genome sequencing applications.
2. Sanger Sequencing In the 1970s, two pioneer sequencing technologies, the Maxam– Gilbert (1) and the Sanger method (2), were developed almost simultaneously, and the inventors of these technologies, Sanger, Maxam, and Gilbert were awarded the Nobel Prize. While the Maxam–Gilbert sequencing method was widely used initially, the development of automated high-throughput DNA sequence analyzers made Sanger sequencing the method of choice for all genome sequencing projects. Frederick Sanger and colleagues introduced the chain termination method of sequencing in 1977 (3), which remained the standard method of DNA sequencing for the next 30 years. Chain termination sequencing starts with a preparation of identical single-stranded DNA (ssDNA) molecules (clonal templates). A short oligonucleotide is annealed to the same position in each ssDNA molecule
Data Loading...
