Next-generation Sequencing: Deep Study Overview
Next-generation Sequencing: Overview, The overview of our article today will first cover traditional sequences and we will spend most of our time talking about Illumina sequencing by synthesis. Followed by other two competitive platforms from Oxford Nanopore and Pacific Biosciences at the end of the speech.
The Human Genome Project thus truly encouraged cheaper sequencing development. This has been a 20-year effort costing 3 billion dollars in 2001. And the cost of sequencing the human genome for a single genome was reduced to about $100 million. And traditional sequencing of Sanger is used. Well, I want to let you know something about DNA before we go into Sanger sequencing, just to get everyone to speed.
Therefore, a sequence of DNA is what I have described here. It consists of two strands, which run counter to parallel. So, one strand goes one way and the other goes the other way. Your C is G-hybrid, and your A is T-hybrid.
DNA can be re-natured/Denatured together or added on various sequences of DNA and hybridized on them, such as a piece of DNA strand, would now lie with the lower template strand. DNA can also be copied using polymers. The DNA is a dual-beam molecule, however, it can be denatured and separated into individual strands.
The DNA polymerases can bind on a template strand to short parts of DNA hybridising them. They can polymerize the DNA in a newly synthesized strand with building blocks to extend the DNA. The DNAs are referred to as deoxyribonucleotide triphosphates as these building blocks.
The 4 versions, the A’s, the C’s, the T’s and the G’s, are over here. And all of them share many common features. First, there is a group of triphosphates that can be attached to the building block by the increasing strand. You have a 3-inch hydroxyl group that is used to add additional DNA structural blocks.
They’ve been attached to four different bases. If you wanted to sequence a piece of DNA like this with conventional Sanger sequences, you would first have to denature it and add a primer so that the DNA polymerase can bind on it.
Now we are using fluorescent terminators instead of simply adding the traditional DNA bases, so that polymerase can extend this DNA molecule. Fluorescent terminators are very similar to the blocks of DNA shown here, but a few differences exist. First of all, you may notice that different colours exist.
So, each of four bases is joined to a different fluorescent group: yellow for A, blue for G, red for C and green for T. Additionally there is a termination or elimination of the 3 ‘hydroxyl groups on the building blocks that enable DNA to continue expanding.
Therefore, fluorescent terminators are called. They are fluorescent and have a terminator that prevents a further extension of the DNA strand by DNA polymerase. So we have trillions and trillions of copies of this template in a test tube. We put normal DNA components in building blocks and spike these fluorescent terminators at a low concentration.
What results is you have a set of newly synthesized fragments of DNA of all different sizes, because they have a fluorescent terminator randomly incorporated. And these molecules are then placed on a DNA sequencer in the test tube that separates them and allows the sequence to be determined.
What is occurring in the sequencer is the size of these molecules, from the biggest to the smallest. The smallest come from the sequencer and are first detected, followed by the next piece of DNA. A chromatogram traces that colour emerges from the DNA sequencer.
You can build your DNA sequence when you follow the colour changes when each of the various parts of the DNA comes out of the sequencer. These machines can execute up to three hundred samples at a time and produce about sequence bases for each sample. And in one day you can sequence up to 1 million DNA parts or 1 million DNA bases.
It might sound like a lot, but in fact, you sample the human genome many times, many times, sometimes 7 times on average, in order to sequence the human genome. This means that it would take approximately 100 years to try to sequence a single human genome on a single sequence machine.
So obviously in less than one hundred years, the Human Genome Project was finished, and the manufacturers of those instruments made that possible. In this huge factory, there’s only one sequencing device, where dozens or hundreds of machines run 24/7 365 days a year. Therefore, the human genome was a very big company. It produced much useful information. But it’s just from several people’s genetic material. So, the majority of what this genome does is still not understood. We’ve got the sequence, you know, but it’s only of a small subset again.
But in order to really understand the role of this genome, we must sequence the variety of genetic material present in the human population from thousands to millions of different persons. And it’s clear we can’t do that by sequencing the traditional sanger. Fortunately, the sequencing costs have fallen dramatically in the last twenty years.
A graph shows how much the 1 million bases of DNA cost in sequence. Once finalized in 2001, the human genome project will cost nearly $10,000. We now have about 1 cent of a million DNA bases, which is a 1 million times lower sequence costs.
Illumina Next-Generation Sequencing
Fall in cost of sequencing was largely driven by Illumina, the leading player on the next-gen sequencing market, introduced new sequencing systems. If we compare the machine output and the Sanger sequence platforms, we can see how much more sequences these instruments generate.
When you look at the number of reads you could get from a single run, the MiSeq will generate 30 million reads, and the HiSeq generates three billion reads, the Nova Seq generates 13 billion reads, while the Sanger sequencing system produces around 400 reads, so that is a huge difference. The dominant player on the market is light sequencing.
It is an imaging method and generates many readings and thousands of readings per run. From each of these readings, we can generate 300-600 sequence bases. It’s actually, very exact. The error rate is about 1 in 1,000 bases. And with our new machines, actually, we can sequence a human genome for $1,000 in fewer than 48 hours and the sequencing in the flow cell on these Illumina platform shapes.
And we can generate about 1-30 million reads per run in this MiSeq flow cell. Next up is the HiSeq flow cell. You see that the cell is much larger than the MiSeq flow celled, and this bigger immobilization allows us to generate more readings in one run, near 3 billion reads. And lastly is the NovaSeq flowcell, even larger.
If you have something similar to RNA, you can convert it into DNA with the enzymes. You take this DNA and then add the adapter sequences at the end. So, in blue are primer sequence binding sites that allow a Sequencing Reaction for a DNA template. This is similar to Sanger Sequencing.
An illustration of the HiSeQ flux cell. So, 8 chains and samples are actually going to the slide. There are 8 different channels inside there and a blender of short, orange and green DNA molécules within the slider. And those orange and green molecules will bind to the two ends. This is a double-stranded molecule that we denature and put in the cell flux.
And once it occurs, we add a newly synthesized strand to the base of the flow cell. We wash out the original template and then allow the newly synthesized strand to bind to another DNA sequence on the surface, in this example the orange piece. Once that happens we add a Polymerases DNA, we add the DNA composition block. Details can be read on Illumina website.
Application of Next-Generation Sequencing
A prenatal examination was used to perform a very invasive amniocentesis sample, which was associated with certain rates of complication. However, with next-generation sequencing, we can really take a blood sample from our mother and not anywhere in the fetus and actually sequence all the diabetic dynamics of the mom.
In cancer, cancer cells constantly released their DNA into the bloodstream. And because the cancer genome will be slightly different from the normal genome we can identify it through sequencing. And with transplanting the same thing. We can then use it in the detection of chromosomal abnormality in a body. And we can use it in cancer and transplantation patients.
The DNA sequences of those donors are very different than the DNA sequences of the recipient and we can detect it by sequencing this material. Another example is the use of next gene sequencing to detect pathogens. Pathogens have their own DNA and RNA set which is very different from human Sequences. And typically we can detect their sequence in this sample if a pathogen is present.
And finally, cancer is again driven by mutations in the context of cancer treatment, and there are a number of different cancer treatments that are very specific for certain cancers. And by following a cancer sample sequence generation, we can decide on the best treatment types for patients and improve the outcomes of cancer samples.
You must understand how to protect a patient’s genetic information if all this is there. However, when technology continues to develop and mature, it will be quite exciting to see where and what new applications are being developed for next-generation sequencing.
For more articles keep following us! Next-generation Sequencing: Overview