- Shotgun sequencing is a powerful and widely used approach to DNA sequencing that involves randomly breaking a genome into numerous small fragments, sequencing each fragment individually, and then reassembling the complete sequence using computational algorithms based on overlapping regions.
- This method revolutionized genomics by enabling the sequencing of entire genomes without the need for prior knowledge of their structure. It played a pivotal role in major scientific efforts, most notably the Human Genome Project and the sequencing of complex organisms such as fruit flies, mice, and humans.
- The term “shotgun” is derived from the method’s random fragmentation process, akin to how a shotgun blast scatters pellets unpredictably. In shotgun sequencing, high-molecular-weight DNA is first sheared into thousands or millions of small fragments using mechanical or enzymatic methods. These fragments are then cloned into vectors or directly sequenced using high-throughput sequencing technologies. Each fragment is sequenced independently, generating a large collection of short reads. These reads are then analyzed using bioinformatics tools to identify regions of overlap, which are used to reconstruct the original DNA sequence in a process known as sequence assembly.
- There are two main variants of this method: whole-genome shotgun sequencing (WGS) and hierarchical shotgun sequencing. In WGS, the entire genome is fragmented and sequenced at once, which offers speed and efficiency. However, this approach can be computationally intensive and challenging, especially for large or repetitive genomes. Hierarchical shotgun sequencing, by contrast, involves first mapping the genome into larger, ordered fragments (such as bacterial artificial chromosomes), sequencing each one separately using shotgun strategies, and then assembling them based on their known positions. This approach was originally favored in the Human Genome Project due to its greater reliability, though WGS ultimately proved faster and became the standard for subsequent projects.
- The success of shotgun sequencing depends heavily on sequencing depth (coverage)—the number of times a given region of DNA is read. High coverage ensures that overlaps between fragments can be reliably identified and helps detect sequencing errors. In the early days of shotgun sequencing, Sanger sequencing was used to read the individual fragments, which made the process time-consuming and expensive. The emergence of next-generation sequencing (NGS) technologies greatly accelerated shotgun sequencing by enabling massively parallel sequencing of millions of fragments at once, at a fraction of the cost and time.
- Despite its transformative impact, shotgun sequencing has limitations. Genomes with extensive repetitive sequences or high structural complexity pose challenges for accurate assembly, often resulting in gaps or misassembled regions. To address these issues, advanced algorithms, paired-end sequencing, and long-read technologies (such as PacBio and Oxford Nanopore) are now often used to complement or enhance traditional shotgun strategies.
