There are two sequencing-by-synthesis methods (pyrosequencing and illumina rever

Question

There are two sequencing-by-synthesis methods (pyrosequencing and illumina reversible terminator methods) as well as the older chain termination method with fluorescently-tagged dideoxynucleotide terminators(sanger sequencing). During the individual sequencing reaction & data collection for these methods, there are fundamental differences in expectations because of the methods' properties, although the data analysis will produce the same final DNA sequence for the template if all goes well.

Compare and contrast in depth (evaluate) the 3 methods (pyro, illumina, and sanger) for the biochemical components/ reaction in template sequence determination (at the nucleotide level) and detection of that result. Do not just give an explanation of the three techniques. Point out what is similar or different between the techniques and explain those aspects.

For example: In the two sequence-by-synthesis methods, the oligonucleotide primer that is used as the starting point for the polymerization reaction in sequencing must be complementary to a sequence that was added as an adaptor to the template DNA because the template is generated from sheared DNAs and is itself generally unknown. In contrast, because the templates in Sanger sequencing are clones or PCR products, the sequencing primers are designed from prior sequence information about the input template DNA.

Explanation / Answer

Sanger is highly accurate over long lengths but expensive. Next Gen are usually high output but less accurate. There are two next-generation sequencing technologies:- illumina). The next-gen sequencing method is faster than Sanger sequencing,The Sanger sequencing sequence only a few thousand nucleotides in a week. The next-gen sequencing method can sequence about 200 billion nucleotides in a week, and it is effective. It is based on "sequencing by synthesis" principle, Sanger sequencing is the first-generation, pyrosequencing was second-generation, and Illumina sequencing is next-generation.

Sanger sequencing uses the chain terminators, ddNTPs, that are added to growing DNA strands and terminate synthesis at different points. It is a dideoxy chain termination method. 500,000,000 different sequencing reactions runs in the Illumina sequencing, on a single small slide. Illumina sequencing uses fluorescently-tagged nucleotides. It does not require the chain-terminating dideoxy nucleotides like sanger seqencing. Next-generation techniques(illumina, ) are based on a "sequencing by synthesis" principle. It genrates unique signal, through nucleotides incorporation into a strand of DNA, the unique signal is a fluorescent molecule.

Sanger sequencing workflow:- It involves the 5 step;

1. Isolation of DNA.,

2 PCR;- amplification of DNA,

3. Generate sequence:-by cycle sequencing; purify reactions to remove fluorescent ddNTP,

4. Run sequence:- to perform capillary electrophoresis, products of the sequencing reaction are injected into capillaries filled with polymer.

5. Analyze data:-By using downstream software applications, translate raw data into corresponding electropherograms .

Next-generation sequencing :-It is completed in as little as 1 day, or with in 1 week . It involves following step:-

1 Select Targets:-design your own with Ion AmpliSeq™ Designer or Select from our pre-designed gene panels.

2. Construct Library:-. Fully automated construction of DNA or RNA libraries with AB Library Builder™ 3. Template Preparation:- fully automated template preparation.

4. Run Sequence:- Use Ion PGM™ and Ion Proton™ Sequencer.

5. Analyze Data:- automated data analyzer are used for biological interpretation.

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