MCB 182 Lecture 2.3 - Shotgun sequencing

Summary

This video script delves into the principles of shotgun sequencing used to decode large genomes like the human genome. It compares whole genome shotgun sequencing with hierarchical shotgun sequencing, explaining how each approach fragments and reassembles DNA sequences. The role of bacterial artificial chromosomes (BACs) in storing DNA fragments and the importance of physical mapping to reconstruct the genome are emphasized. Additionally, the script introduces advanced sequencing technologies such as Illumina sequencing, PacBio, and Nanopore sequencing, which have revolutionized genomic analysis beyond traditional methods.

Takeaways

• 😀 Sequencing an entire genome, like the human genome, is not feasible in one go due to its vast size (around 3 billion nucleotides).
• 😀 Shotgun sequencing involves randomly fragmenting the genome into smaller pieces, sequencing them, and then reassembling the original genome from these fragments.
• 😀 Whole Genome Shotgun Sequencing (WGSS) fragments the entire genome and directly sequences the pieces, then uses computational methods to assemble them.
• 😀 Hierarchical Shotgun Sequencing divides the genome into larger fragments first, and then sequences these smaller fragments, organizing them hierarchically.
• 😀 The debate between WGSS and hierarchical shotgun sequencing was significant during the Human Genome Project, with WGSS ultimately becoming the standard.
• 😀 Bacterial Artificial Chromosomes (BACs) are used to clone large fragments of DNA into bacterial cells for sequencing in hierarchical shotgun sequencing.
• 😀 Physical mapping, using restriction enzymes to cut DNA at specific sites, helps identify overlapping regions of DNA, facilitating the assembly of the genome.
• 😀 The physical map shows the exact locations of DNA sequence elements, whereas genetic maps show the relative distances between genetic landmarks.
• 😀 BAC libraries are highly redundant, meaning multiple copies of genome segments are created to increase sequencing coverage, but careful selection reduces overlap.
• 😀 Advances in sequencing technologies, such as Illumina sequencing, PacBio Smart Cells, and nanopore sequencing, have made genome sequencing faster and more efficient compared to traditional Sanger sequencing.

Q & A

What is shotgun sequencing?

Shotgun sequencing is a method used to sequence entire genomes by randomly breaking them into smaller fragments, sequencing these fragments, and then assembling them to reconstruct the original genome sequence.

Why can't a whole genome be sequenced all at once using traditional sequencing methods?

Sequencing an entire genome all at once is not feasible because the genome can be extremely large (e.g., the human genome is around 3 billion nucleotides long), and current sequencing technologies cannot handle such a large number of sequences in a single experiment.

What is the main difference between whole genome shotgun sequencing and hierarchical shotgun sequencing?

Whole genome shotgun sequencing involves directly fragmenting the entire genome into small pieces and sequencing them randomly, while hierarchical shotgun sequencing first involves breaking the genome into larger fragments, sequencing those, and then progressively sequencing smaller pieces within them.

What are bacterial artificial chromosomes (BACs) used for in hierarchical shotgun sequencing?

Bacterial artificial chromosomes (BACs) are used to clone large fragments of DNA, typically between 150 to 350 kb, into bacterial cells for easier handling and sequencing in hierarchical shotgun sequencing.

How do BACs help in the assembly of a genome during sequencing?

BACs help in genome assembly by providing large DNA fragments that overlap with each other. By sequencing these fragments and using restriction enzymes to identify overlapping regions, researchers can piece together the original genome sequence.

What role do restriction enzymes play in hierarchical shotgun sequencing?

Restriction enzymes are used to cut DNA fragments at specific sites, generating unique patterns that help identify overlaps between different BACs. This information is essential for assembling the larger genome sequence from smaller fragments.

What is a physical map in the context of genome sequencing?

A physical map refers to the precise positioning of DNA sequence elements in the genome. It helps researchers understand the relative locations of specific genes, sequences, and markers, which is crucial for assembling fragmented DNA sequences into a complete genome.

How does whole genome shotgun sequencing differ from hierarchical shotgun sequencing in terms of speed and organization?

Whole genome shotgun sequencing is faster and more efficient because it bypasses the need for creating large, organized libraries of BACs. In contrast, hierarchical shotgun sequencing involves more time-consuming steps, including organizing larger BACs into libraries before sequencing smaller fragments.

Why is redundancy important in a BAC library for genome sequencing?

Redundancy in a BAC library is crucial because it ensures that multiple copies of each genome segment are available, increasing the likelihood of successfully sequencing and assembling the entire genome. Overlapping BACs help ensure complete coverage of the genome.

What are some modern sequencing technologies that have higher throughput than traditional Sanger sequencing?

Modern sequencing technologies that offer higher throughput than traditional Sanger sequencing include sequencing by synthesis (Illumina), PacBio smart cells, and nanopore sequencing.