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Shotgun sequencing is a method used in molecular biology to determine the sequence of a long DNA molecule. It involves breaking the DNA into many smaller fragments, sequencing each fragment individually, and then using a computer to reassemble the sequences into the original order. This process is analogous to assembling a shredded document by first cutting it into many pieces, then reading each piece, and finally piecing them back together based on overlapping text.

The fragments are typically generated by randomly shearing the DNA using physical methods or enzymes. Once fragmented, each piece is then sequenced using a high-throughput sequencing technology, such as next-generation sequencing. These technologies can generate millions of short DNA sequences, known as reads, in a single run. The critical step that follows is bioinformatics. Specialized algorithms are employed to align and assemble these short reads. The assembly process relies on identifying overlapping regions between reads. Where two reads share a common sequence, they are likely to be adjacent in the original DNA molecule. By finding enough overlapping segments, the computer can reconstruct the order of the fragments and thus the complete sequence of the original DNA.

Shotgun sequencing has been instrumental in sequencing entire genomes, including the human genome. Its ability to handle large amounts of DNA and its relative efficiency have made it a cornerstone of genomics research, enabling studies in areas such as gene discovery, evolutionary biology, and personalized medicine. Variations of shotgun sequencing exist, such as paired-end sequencing, where both ends of a DNA fragment are sequenced, providing additional information for assembly.