Question

Please answer all questions please

1.Explain how the NGS approach has been able to bring the cost of sequencing down.

2.In a nutshell, what are the major steps performed during next generation sequencing of DNA? How do these steps differ in the Illumina platform as compared to the Roche 454 platform?

3. Now that the human genome has been sequenced, what types of information still need further research?

Answer 1

1. NGS approach has been able to bring the cost of sequencing down in the following way:

NGS technologies also known as high-throughput sequencing and have an ability to sequence DNA at very fast speed. It include modern sequencing technologies like Illumina , Roche 454 .

NGS technologies applies a different methodology from the Sanger chain-termination method that was used earlier. It works on the sequencing by synthesis technology that is SBS, pyro-sequencing and sequencing by ligation. SBS sequencing method is founded on reversible dye-terminators that helps in the single bases identification as soon as they are cooperated into DNA strands. In this way, this process of sequencing is very fast and is also used to identify the complexity that is present in genome of human.

2. Major steps that is performed during next generation sequencing of DNA are:

a. Library preparation: include the preparation of DNA library using the DNA random fragmentation followed by ligation with the help of custom linkers.

b. Clonal amplification: The DNA library generated by first step, is amplified using PCR technology.

c. Sequencing: Amplification of the DNA library is then sequenced using pyrosequencing, sequencing by ligation, and reversible dye terminators.

Illumina used reversible dye terminators as a sequencing method, whereas Roche 454 uses pyrosequencing as a sequencing method.

3. Now that the human genome has been sequenced, annotation of the human genome is required like gene annotation, proteins annotation that still need further research. HapMap project is going on that aim is to validate SNPs that are several million in number and that were identified during the sequencing of genome followed by characterisation of the extent of their linkage patterns that is present in the populations of continents like European, Asian and African ancestry. Another project, The ENCODE project was also started to identify human genome functional and regulatory elements. .