Sequencing

DNA Sequencing

 

 

What is Sequencing?

 

DNA sequencing is the process of determining the nucleotide order of a given DNA fragment. Thus far, most DNA sequencing has been performed using the chain termination method developed by Frederick Sanger. This technique uses sequence-specific termination of a DNA synthesis reaction using modified nucleotide substrates. However, new sequencing technologies such as Pyrosequencing are gaining an increasing share of the sequencing market. More genome data is being produced by pyrosequencing than Sanger DNA sequencing these days. Pyrosequencing has enabled rapid genome sequencing. Bacterial genome can be sequenced in a single run with several X coverage with this technique. This technique was also used to sequence the genome of James Watson recently.

 

Why sequencing is important?

 

Genome sequencing is figuring out the order of DNA nucleotides, or bases. When you read a sentence, the meaning is not just in the sequence of the letters. It is also in the words those letters make and in the grammar of the language. Similarly, the human genome is more than just its sequence. Sequencing the genome is an important step towards understanding it. Scientists also hope that being able to study the entire genome sequence will help them understand how the genome works—how genes work together to direct the growth, development and maintenance of an entire organism. Finally, genes account for less than 25 percent of the DNA in the genome, and so knowing the entire genome sequence will help scientists study the parts of the genome outside the genes. This includes the regulatory regions that control how genes are turned on an off, as well as long stretches of junk DNA.

 

How sequencing is operated?

 

The whole genome can't be sequenced all at once because available methods of DNA sequencing can only handle short stretches of DNA at a time. So instead, scientists must break the genome into small pieces, sequence the pieces, and then reassemble them in the proper order. An automatic sequencing machine spits out what genome scientists call "raw" sequence. In raw sequence, the reads or short DNA sequences are all jumbled together. The process of transforming the fragmented rough draft into a long, continuous final product without breaks or errors is called finishing. Finishing often takes longer than the sequencing itself.

 

Which kinds of sequencing techniques are available?

 

To facilitate their full genome sequencing initiatives, “Illumina” licensed nanopore sequencing technology from Oxford Nanopore Technologies. Illumina sequencing system based on reversible terminators for sequence determination. It is an approach that generates several billion bases of accurate nucleotide sequence per experiment at low cost. Single molecules of DNA are attached to a flat surface, amplified in situ and used as templates for synthetic sequencing with fluorescent reversible terminator deoxyribonucleotides. Another possible way to accomplish cost-effective high-throughput sequencing is by utilizing fluorophore technology. Pacific Biosciences is currently using this approach in their SMRT (Single Molecule Real Time) DNA sequencing technology. SMRT sequencing is a harnesses the natural process of DNA replication and enables real-time observation of DNA synthesis by using phosphorlinked nucleotides.

 

References

 

1. N/A. (2008). Accurate Whole Human Genome Sequencing using Reversible Terminator Chemistry. Nature. 456(7218): 53–59.
2. Xiaoge, G., Kevin, L., Karen, O., Jenny Z., Sandeep S. D., & Sue J.R. (2015). SMRT Sequencing for Parallel Analysis of Multiple Targets and Accurate SNP Phasing. G3. g3.115.023317