of restriction sites contained in each clone. Once the overlap between the clones has been found and their order relative to the genome known, a scaffold of a minimal subset of these contigs that covers the entire genome is shotgun-sequenced. Because it involves first creating a low-resolution map of the genome, hierarchical shotgun sequencing is slower than whole-genome shotgun sequencing, but relies less heavily on computer algorithms than whole-genome shotgun sequencing. The process of extensive BAC library creation and tiling path selection, however, make hierarchical shotgun sequencing slow and labor-intensive. Now that the technology is available and the reliability of the data demonstrated, the speed and cost efficiency of whole-genome shotgun sequencing has made it the primary method for genome sequencing. Newer sequencing technologies The classical shotgun sequencing was based on the Sanger sequencing method: this was the most advanced technique for sequencing genomes from about 1995–2005. The shotgun strategy is still applied today, however using other sequencing technologies, such as short-read sequencing and long-read sequencing. Short-read or "next-gen" sequencing produces shorter reads (anywhere from 25–500bp) but many hundreds of thousands or millions of reads in a relatively short time (on the order of a day). This results in high coverage, but the assembly process is much more computationally intensive. These technologies are vastly superior to Sanger sequencing due to the high volume of data and the relatively short time it takes to sequence a whole genome. Metagenomic shotgun sequencing Having reads of 400-500 base pairs length is sufficient to determine the species or strain of the organism where the DNA comes from, provided its genome is already known, by using for example a k-mer based taxonomic classifier software. With millions of reads from next generation sequencing of an environmental sample, it is possible to get a complete overview of any complex microbiome with thousands of species, like the gut flora. Advantages over 16S rRNA amplicon sequencing are: not being limited to bacteria; strain-level classification where amplicon sequencing only gets the genus; and the possibility to extract whole genes and specify their function as part of the metagenome. The sensitivity of metagenomic sequencing makes it an attractive choice for clinical use. It however emphasizes the problem of contamination of the sample or the sequencing pipeline. See also Clinical metagenomic sequencing DNA sequencing theory References Further reading External links Molecular biology DNA sequencing 1981 in biotechnology Metagenomics Bioinformatics