Sequencing Whole Genomes

There are two general strategies for sequencing a complete genome. The method preferred by the Human Genome Project is the hierarchical shotgun sequencing method. In this approach, genomic DNA is cut into pieces of about 150 Mb and inserted into BAC vectors, transformed into E. coli where they are replicated and stored. The BAC inserts are isolated and mapped to determine the order of each cloned 150 Mb fragment. This is referred to as the Golden Tiling Path. Each BAC fragment in the Golden Path is fragmented randomly into smaller pieces and each piece is cloned into a plasmid and sequenced on both strands. These sequences are aligned so that identical sequences are overlapping. These contiguous pieces are then assembled into finished sequence once each strand has been sequenced about 4 times to produce 8X coverage of high quality data.