BIOL 2500 - Topic 10 (part 5)

Sanger DNA sequencing now

We use a more automated method of Sanger sequencing method, where it is still termination-based but now it only requires 1 reaction rather than 4

How can Sanger DNA sequencing happen in the same tube?

It uses an automated gel-electrophoresis system, and it labels the DNA strands with fluorescently labelled ddNTPs

Modern Sanger DNA sequencing (how it reads the sequence)

1.) The sample is electrophoresed through a capillary tube in a polyacrylamide gel

2.) As they move through the tube, they pass by a laser that excited the ddNTPs

3.) The frequency of the signal produced by the ddNTPs are picked up by the computer

4.) This sequences the DNA fragment, as the beginning of the fragment goes first and then the larger fragments come through last

NGS

Next-generation sequencing

Advantages of NGS

1.) It is faster and cheaper

2.) It has led to the field of bioinformatics (due to the large amount of data it produces)

Speed of NGS

It can sequence 50 human genomes a day

How is NGS so fast

It utilizes computational power to put the fragments together into a genome-wide consensus sequence

NGS uses the __________ sequencer

Illumina

Third-gen DNA sequences (TGS)

It is a similar and almost parallel process to NGS, just slightly more improved

Use of TGS

It is used in genome-wide association studies (GWAS) and other genomics studies, which compares genomes across multiple organisms

NGS process (synthesis part)

1.) The DNA is fragmented into pieces and tagged with adaptor molecules at both ends of the fragments

2.) The dsDNA is denatured

3.) The DNA is placed into a flow cell, where it is amplified to produce clusters of identical strands

Reaction mixture of NGS

It has similar components to a PCR reaction mixture, such as…

1.) Fluorescently tagged dNTPs

2.) Buffer

3.) Taq polymerase

4.) Primers

Use of adaptor molecules (for us)

It allows us to find and identify the fragments later

Use of adaptor molecule in the reaction

It anchors the strands to the flow cell, where the DNA is amplified

NGS is sequency by __________

Synthesis

NGS process (analysis part)

1.) A laser excites the fluorescent dNTPs and then a photoreceptor records the wavelength intensity

2.) The software records the information and converts the wavelengths to a known dNTP

The NGS process is ________ multiple times

Repeated

Sanger vs. Illumina NGS

Unlike Sanger, NGS determines sequences by synthesis, rather than termination

When is it good to use Sanger sequencing

For short DNA sequences

When is it good to use NGS

For large samples with long sequences