Introduction to Third Generational DNA Sequencing

What is DNA sequencing?

DNA sequencing is a technique used to find the sequence of nucleotides within a DNA molecule, giving information for the genetic make-up of an organism. This has many applications such as; identifying genetic disorders, learning more about the genome or to monitor genetic processes (DNA replication).

Third generational DNA sequencing is a form of DNA sequencing that analyzes long strands of DNA (mainly) to identify genetic disorders through applications such as Oxford nanopore and PacBio SMRT.

An example of of the use of third generational DNA sequencing is the use of this type of sequencing on newborns (also referred to newborn sequencing) to find the nucleotide combinations responsible for the genetic disease that may cause genetic disorders in earl childhood. Thus allowing doctors to act against the disorder before it becomes a malignant concern.

There are two main types of DNA sequencing: Sanger DNA sequencing method and next generation sequencing (may also be referred to as parallel sequencing).

How it works

Sanger sequencing 1. Prepare DNA molecules (by heating which separates them).

Add special nucleotides, a primer (to indicate the starting point for the polymerase) and dideoxynucleotides (ddNTPs) to stop DNA replication process.
Use a special type of copying mechanism (polymerase) to complete the pairs and once it lands on a terminal nucleotide, it will stop. This process will split the DNA molecule into multiple small fragments that differ in length.
Use a special gel (referred to as gel electrophoresis) to split the DNA fragments. They do this by creating an electric field that will attract the DNA fragments to the positive side and because the smaller molecules will move faster (due to their ability to easily navigate through the matrix), creating a gradient of increasing lengths of DNA fragments.
Use dyes such as bromide to stain DNA molecules, so that we can see them clearly.
Use computational technology such as Bioedit and SeqTrace to read the colors and identify DNA sequences.

Next Generation Sequencing (NGS)
1. Prepare DNA samples
2. Add adapters (fluorescent special tags used for DNA fragments) to DNA molecules.
3. Conduct a polymerase chain reaction. This reaction with copy these fragments to allow sufficient amounts of copies for DNA sequencing.
4. Insert these amplified fragments into systems like Illumina to add appropriate pairs and use the data produced for sequencing. (Note: only <600 base pairs can read at a time, though these systems will stitch the sequencing data from the shorter sequences into longer sequences).
5. Geneticists then program these computational technologies to look out for specific chains that would cause genetic disorders and the computational technology would look out and identify any of these chains.
Table comparing the two methods

Feature	Sanger	NGS
Sequencing Volume	One DNA fragment at a time	Scans millions of fragments simultaneously
Accuracy	Extremely High Accuracy (99.99%), often referred to as the “gold standard” for validation	Generally less accurate but is in development to increase accuracy.
Speed and Cost	Time consuming (up to a few days) and expensive for big projects	More cost-friendly with faster speeds (5 - 24 hours).
Read length (avg length of DNA fragments)	Up to 1000 base pairs	Up to 600 base pairs but platforms like Pacbio offer longer reads
Applications	Best for sequencing small regions, and validating NGS results.	Best for large-scale projects.

In summary, both these methods are crucial in changing the future of genetic medicine as these mechanism will allow doctors to fight off and identify genetic disease and tailor a utile treatment for the patients at hand.