Shotgun Library Construction for DNA Sequencing

Abstract

Shotgun cloning is a method to generate the templates needed for DNA sequencing. This process entails breaking a large target DNA randomly into smaller fragments; end sequencing these smaller fragments; and from the overlapping sequences of the randomly generated fragments, reassembling the initial target sequence. Although this random strategy initially was described more than two decades ago (1–3), only a few years following the original reports describing the dideoxynucleotide method for DNA sequencing (4–7), it immediately did not gain wide-scale acceptance. It was not until the introduction of instrumentation (8–10) that was capable of collecting the required large quantity of data required for successfully implementing the shotgun method, that this approach began to be widely accepted. The shotgun cloning strategy presently is the method of choice for generating the major portion of the sequence data for sequencing projects. This holds true for target DNAs as small as a 4-kbp restriction digest fragment (1) or as large as an entire 3-Gbp mammalian or plant genome (11–13) since many of the methods used have been automated (14,15). These partially or fully automated methods include the shotgun DNA template clone isolation; DNA sequence reaction pipetting; DNA sequence data collection; DNA sequencing chemistry; and computer-based shotgun sequence data assembly, visualization, and editing (16–18). Therefore, it is reasonable to initially obtain from 6- to 10-fold shotgun sequence coverage and then proceed with more directed closure and finishing methods.