The key difference between clone by clone sequencing and shotgun sequencing lies in their method of conduct. Clone by clone sequencing method involves mapping of chromosomes and cloning prior to sequencing while clone by shotgun sequencing omits both chromosome mapping and cloning steps during the sequencing.
Clone by clone sequencing and clone by shotgun sequencing are two methods of modern-day genome sequencing. Clone by clone sequencing is a more reliable method of sequencing. However, the method of clone by shotgun sequencing is faster and cheaper. The article focuses on the subtle difference between clone by clone sequencing and shotgun sequencing.
CONTENTS
1. Overview and Key Difference
2. What is Clone by Clone Sequencing
3. What is Clone by Shotgun Sequencing
4. Similarities Between Clone by Clone Sequencing and Shotgun Sequencing
5. Side by Side Comparison – Clone by Clone Sequencing vs Shotgun Sequencing in Tabular Form
6. Summary
What is Clone by Clone Sequencing?
Clone by clone sequencing is a method of genome sequencing. It requires the mapping of each chromosome prior to DNA splitting. After mapping, the DNA should be broken down into fragments of 150 kilobases long. These fragments are then ready for sequencing. Next step is the insertion of DNA fragments into Bacterial Artificial Chromosomes (BACs) and then into bacterial cells. Since the fragments are now inside the bacterial cells, each time when the bacteria divide, the inserted DNA fragments also divide and produce many identical copies. Then, the individual bacterial clone DNA is fragmented into 500 base pair long fragments. They are smaller and overlapping fragments. Next, the sequencing takes place with the insertion of these fragments into a vector with a known DNA sequence. Starting from the known sequence of the vector, sequencing continues up to unknown sequence.
After finishing the sequencing, it is necessary to identify the areas of overlapping sequences. Thereafter, joining of the fragments takes place to form the larger fragments initially presented in BACs. Next, the assembly of larger fragments into the chromosome takes place according to the genome map.
Figure 01: Clone by Clone Sequencing
The significant advantage of clone by clone sequencing is that the pre-prepared genome map helps a reliable assembly of larger fragments. But, the disadvantage of this sequencing method is that it is time-consuming to generate genome maps and making clones. However, this was the method preferred during the ‘Human Genome Project’.
What is Clone by Shotgun Sequencing?
Clone by shotgun sequencing is a sequencing method which randomly breaks up DNA sequences into many small fragments and reassembles the sequence by observing the overlapping regions. Larger mammalian genomes are difficult to clone sequence and assemble. It is due to their structural complexity and size. Even though the clone by clone sequencing method is reliable, it takes a long time to sequence genomes of complex organisms. Therefore, clone by shotgun sequencing has become a reliable cheaper sequencing method that can be performed faster. Hence, modern-day scientists rely on this sequencing method to tackle complex genomes.
Figure 02: Clone by Shotgun Sequencing
During clone by shotgun sequencing method, no conventional genome mapping and cloning steps take place. Initially, the entire genome is fragmented into varying sizes from 20 kilobases to 300 kilobases. Next, sequencing takes place. Then, using sophisticated computer software, it is necessary to assemble the fragments by looking at the overlapping regions.
Clone by shotgun sequencing helps to improve the accuracy of existing genome sequences. The main advantage of this method is that it is a much faster and less expensive than clone by clone sequencing method. However, this process doesn’t involve the use of a genetic map. Hence, errors during the assemblage are more likely to occur. Thus, this is a major disadvantage in clone by shotgun sequencing method.
What are the Similarities Between Clone by Clone Sequencing and Shotgun Sequencing?
- Both clone by clone sequencing and shotgun sequencing are two methods of genome sequencing.
- During both sequencing techniques, assembly of broken-down fragments takes place by identifying overlapped regions.
- Also, breakdown of DNA into smaller fragments is necessary for both clone and shotgun sequencing.
What is the Difference Between Clone by Clone Sequencing and Shotgun Sequencing?
Clone by clone sequencing technique involves two major steps: mapping of chromosomes and cloning. In contrast, clone by shotgun sequencing does not follow these two steps; it randomly breaks up DNA sequences into many small fragments and reassembles the sequence by observing the overlapping regions. Hence, this is the key difference between clone by clone sequencing and shotgun sequencing. Due to this factor, clone by clone sequencing is an expensive and time-consuming process while clone by shotgun sequencing is faster and cheaper.
However, errors during assembly are less likely to occur during clone by clone sequencing. Hence, it has high reliability. However, clone by shotgun sequencing is comparatively a less reliable technique. So, this is also a significant difference between clone by clone sequencing and shotgun sequencing.
The below info-graphic presents more information regarding the difference between clone by clone sequencing and shotgun sequencing.
Summary – Clone by Clone Sequencing vs Shotgun Sequencing
In summarizing the difference between clone by clone sequencing and shotgun sequencing, the clone by clone sequencing and shotgun sequencing are two methods of genome sequencing. However, clone by clone sequencing technique involves both genome mapping and cloning processes while clone by shotgun sequencing does not. So, this is the key difference between clone by clone sequencing and shotgun sequencing. Since genome mapping takes place during clone by clone sequencing, errors are less likely to occur during the assembly of sequences. But, clone by shotgun sequencing is a much faster and cheaper process. Yet, it is comparatively less reliable. Clone by clone sequencing was the preferred sequencing method during the ‘Human Genome Project’. But, the modern-day molecular biologist relies more on clone by shotgun sequencing.