When Human Genome Project was running, and finally done in 2003, I was in school. The total thing was a sci-fi to me. As they are pouring a drop of blood or a string of hair, and getting the whole secret story of human written in ATGC ! There were several fun facts published that time, like the distance of sun and earth relative to the length of whole DNA, or the about the mass of it. Sequencing DNA seemed very easy to me until I entered in the Virology lab, where we answer about any unknown thing, like, ahh, do the PCR and sequence it!
In molecular Biology, this thing is common for all organisms, doing PCR and sequencing. The genome is consisted of RNA (only RNA for some viruses) and DNA, but all tends to DNA sequencing, as the RNA is converted into DNA by reverse transcription. DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. It includes any method or technology that is used to determine the order of the four bases—adenine, guanine, cytosine, and thymine—in a strand of DNA.
DNA sequencing is largely done by Sanger sequencing along with Maxam-Gilbert sequencing in some little cases. When we talk about sequencing, we usually refer to the chain termination or Sanger sequencing method. We use this in our lab too, for sequencing several parts of viruses to characterize them. I have never done any whole genome sequence, but some here do it by sequencing part by part and then aligning them.
When I came to know about Metagenomics and Next generation Sequencing that sci-fi feeling came back, but in a polished and I-can-do-it version 🙂 So, I decided to share them with my friends, who may know them already. Here I am skipping the whole thing about Sanger method, just adding a schematic picture to differentiate the two generations.
I collected some stuff from Wikipedia and technique types were from just one source, EMBL-EBI website, as anyone can take the online course at a glance. But there are many other methods, maybe I’ll discuss some if the mood comes back 🙂
Next-generation sequencing (NGS), also known as high-throughput sequencing, is the catch-all term used to describe a number of different modern sequencing technologies, which are given below-
Illumina dye sequencing was based on inventions of S Balasubramanian and D Klenerman of Cambridge University. Here, the slide is flooded with nucleotides and DNA polymerase. These nucleotides are fluorescently labelled, with the colour corresponding to the base. They also have a terminator, so that only one base is added at a time. An image is taken of the slide. In each read location, there will be a fluorescent signal indicating the base that has been added. The process is repeated, adding one nucleotide at a time and imaging in between. All of the sequence reads will be the same length, as the read length depends on the number of cycles carried out.
This technique offers a number of advantages over traditional sequencing methods. Due to the automated nature it is possible to sequence multiple strands at once and gain actual sequencing data quickly. Additionally, this method only uses DNA polymerase as opposed to multiple, expensive enzymes required by other sequencing techniques.
The system relies on fixing nebulized and adapter-ligated DNA fragments to small DNA-capture beads in a water-in-oil emulsion. The DNA fixed to these beads is then amplified by PCR. Each DNA-bound bead is placed into a ~29 μm well on a PicoTiterPlate, a fiber optic chip. A mix of enzymes such as DNA polymerase, ATP sulfurylase, and luciferase are also packed into the well. The PicoTiterPlate is then placed into the GS FLX System for sequencing.
Ion semiconductor sequencing
Unlike Illumina and 454, Ion torrent and Ion proton sequencing do not make use of optical signals. Instead, they exploit the fact that addition of a dNTP to a DNA polymer releases an H+ ion. Like 454, the slide is flooded with a single species of dNTP, along with buffers and polymerase, one NTP at a time. The pH is detected is each of the wells, as each H+ ion released will decrease the pH. The changes in pH allow us to determine if that base, and how many thereof, was added to the sequence read.
NGS is significantly cheaper, quicker and is more accurate and reliable than Sanger sequencing. It needs least amount of template DNA, as mainly works on the synthesis process, where Sanger methods depends on chain termination. only one read (maximum ~1kb) can be taken at a time in Sanger sequencing, whereas NGS is massively parallel, allowing 300Gb of DNA to be read on a single run on a single chip. It is also useful for shorter and repeated sequences. Today, Next Generation Sequencing are just outside our lab door, and tomorrow we will slide the door and let it in! 😀