Metagenomic virus detection in clinical specimens : a new era

As seen in this Chinese doll picture, the organisms in our nature is not only they look like, they live in an intermingling fashion, which calls for the whole study of them , or the Metagenomics. With the advancement of newer sequencing methods, the area of metagenomics has largely expanded, or on the other way, the broad-range studies of microorganisms in genetic level has geared up the newer and newer inventions of high-throughput sequencing systems. Okay, what it is, Metagenomics is a beautiful study, nothings less important here.

But when we come to the world of viruses, they are not the main ones in the environmental pool. Sure they are present there, but the main sphere of viruses are living cells as they only live well in intracellular condition. In many cases, viruses enter in a body, replicate here, fight with each other or live in peace, spread infections, and when they are shed, from the host body, they are not the same, they have mutated and changed by this time. This made the theory of Quasispecies. So, the environmental study of metagenomics can not be applied here.

Another matter is the studies of Zoonosis. Everyday, there is a new evidence coming out, about, this or that newly emerged disease has come from this or that animals. When a case is found, with no former case, its very difficult to find out the causative agent. So, the specific and effective study of Metagenomics from clinical sample is very important.

This study comes forward with a very new method, tissue-based unbiased virus detection for viral metagenomics (TUViD-VM). Formerly there were many methods available for viral metagenomic study as Hybridization, Sequence independent single primer amplification( SISPA), Arbitrary primed PCR (AP-PCR), Rolling circle amplification etc. This study group from Robert Koch Institute, Germany, not only established a new protocol, but also have shown the efficacy very well.

Schematic description of tissue-based universal virus detection for viral metagenomics protocol. Estimated durations of each step are shown in parentheses. The protocol takes 12 h to complete

This study compared the protocol in different virus groups, different hosts, different extraction, PCR and sequencing methods. The only limitation of this study might be the higher cost for sample processing. It also need high capacity computational analyses as both the viral and host genomes are mixed in a clinical sample gene pool. The researchers nullified the constrain, as sequence data for mammalian hosts are very limited.

This study opened a new era of viral study. Where a newly emerged disease call for an outbreak, and we find nothing in our hands to search the causative agent, as we saw for Ebola just a few days earlier, an established protocol of tissue based viral metagenomics can  help a lot!

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Next Generation Genome Sequencing – Today And Tommorrow

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.

Figure 1.

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 sequencing

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.

454 sequencing

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! 😀

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