BioBattalion Webinar Series Chapter 4 with Dr. Shikha Sharma

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BioBattalion
yeah, actually there’s going to be a lot of animations so you have to like we want the slides and just be there because yeah so if you haven’t like already subscribed to YouTube channels please do so to receive instant notifications on upcoming events and audience can pose the questions in the comments box below so I’ll be moderating each at the end of the presentation. Go live now. We’ll start with the presentation. So just start with the intro. Yeah. Hello, everyone. Welcome to the fourth chapter of buy battalion Webinar Series. Today it’s an absolute delight for us to host Dr. Shikha Sharma Project Scientist DBT National Theatre plant Genome Research New Delhi. Dr. Shikha has done her PhD in bacterial genomics and evolution from insect where she worked and whole genome sequencing and analysis of human associated bacterial human microbiome. Recently, she joined nit gr bioinformatics for Brock improvement group as project scientists, where she’s working on plants microbiome and planned bacterial interactions. She has been associated with bacterial omics research for more than seven years, and has in depth understanding of the creek and cursor bacteria research avenues. Now over to Dr. Shika.

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Dr. Shikha Sharma
Yeah, I can see that. All right. We can start Malmo and tell me when to switch the slides. I’ll be okay, fine. So hello, everyone who I will be talking on the topic today about bacterial searching genomic era. And what is basic? What is this basically precisely that we know that NGS is coming, a lot of things are happening in next generation sequencing genomics and a lot of things are happening each day. So there is a revolution kind of thing is happening in bacteria research. So for an example, I’m just showing you in the image above, I kind of, I will say made this image in graphics, that this is a bacterial genome of Mike, it can be any microbe or any other bacteria, in fact anything. So evolution have put a lot of things into words like Gene clusters, and all those things. And the NGS has got the ability to extract that important information. And this is what I’m basically going to share in the slides. And I think that these slides are basically going to tell us that what are the basic things that we can do invector research and this is going to be really important for any new PhD students or research people who are just new in vascular research. So let’s go on the next slide. Okay. So, first of all, I think that yes, for biological research and every other particle search is very important and but I think that bacteria as a whole is very significant in itself, because then the life started on earth around 3.8 billion years ago. So, bacteria was the first point of life, which was predicted over here. And since then, today’s bacteria is present all over the world, in each and different kinds of niches in human body, outside body and environments, different kinds of extreme conditions. The reason behind this is that when we see a bacteria, so, they are highly sustainable, they are highly adaptable, and the interesting thing is that when we saw on the technical part, so, they require a very few things or very few gene for their existence, and that was the reason they were there around 3.8 billion years ago, and they have got this ability to combat environmental pressure. So, these are the reasons that they have been going under continuous evolution and the ecological changes and CO evolving with the niches in which they are present which is happening continuously in their genome, like horizontal gene transfer three computation, Genome dynamics, etc. So, for the next time. So, besides that, I think that significance of accidents research is something that we need to discuss. Because there are two aspects of bacterial research. One is that a lot of pathogens which are there are initially right now, there is a lot of viral pathogens, which are there, but initially when the microbiology and whatever research part had started in the beginning, so, the main focus of human mankind basically not humans, was to search for different pathogens, which are there and the bacterial pathogens, they produces different types of virulence factors, they produces toxins and raw different structural as well as chemical mechanisms to evade the defense systems. And we read everyday about the antibiotic resistance, which is there, which is getting locked into the bacterial genomes, there are bacterial variants which are coming each day, etc. So I think that there is a continuous evolution in the pathogenicity of a single bacteria, for example, MTB, so each time you’re going to find a drug, so, there’s always going to be a better pathogen. So, we cannot just stop at one research or at one result that okay, this is the drama This is the mechanic of this particular pathogen. And besides the defense pathogen, the next important aspect of bacterial research is potential could have been just click More Yeah. So, the reason behind this is because most of the world right now is basically focused on this potential part, because there is

Dr. Shikha Sharma
a lot of things that we can extract a lot of important therapeutics that we can extract from bacteria, for example, of course, antibiotics and anti microbials especially when there is an increasing drug resistance, we have to look for a lot of different antibiotics. And the thing is that whatever bacteria we know right now, and there are not many of them, there are only 1% of the actual amount of bacteria or actual abundance of bacteria, which is present in the world are added to So, we need to extract a lot of other bacterias we have to study a lot of bacteria research we have to do a lot of it, so that we can extract all these informations like we need enzyme drugs, probiotics, they are completely dependent upon this potential part. And there are a lot of other studies that we can do in order to understand the life hormone that is not possible for do it directly on rodents or, you know, humans like experimental studies or they’re an evolution studies are there. So, these experimental parts are also done on bacteria, because they can we can grow them easily we can manipulate them genetically manipulate them easily. So this is the reason and for example, my institute from which I did my PhD, so they are highly Ambrose formed last, I think, 10 to 12 years in this clock cluster that is natural streptokinase which is there in the market, and they are improving it each year adding a new factor each year by extracting by this Unwin bacteria research on this one microbe and this is how they are improving it each day and it is this product is there. It’s a huge seller of my institute in which there in the market. So discipline about my association with this peculiar researches that my PhD was in the microbial genomics part and I want to on the pathogen in the non pathogenic, that is commensal, bacteria and a lot of other pathogenic as well as other kinds of bacteria as well. And after my PhD, I am presently where I’m working, that is an rpgr. There also, I’m working on studying the bacterial pathogen interactions, as well as the bacterial comments of bacterium and plant pathogen interactions, and the plant microbiome. So, all these things, I’m going to discuss a bit of all these things into my these slides. And I think that this is going to give you a brief information that what is happening and what kind of research we can do in this bacterial part in considering the NGS artifacts. So, go into the My next slide.

Dr. Shikha Sharma
Before we go into the details, and I will take some of the case studies that we have done so far, I would like to talk about the very important and very basic aspects of research, which is there in the context of NGS, culture omics and meta genomics, I think that this is highly discussed two topics. And for example, suppose this is niche, it can be any environment, it can be any body site, it can be anything. And of course, because bacteria is highly abundant in nature, there are a lot of going to be a lot of bacteria, different types of bacterial species, some of which we know some of 99% of those bacteria, we don’t know because we never cultured them, because there is no conditions we know to culture them. So they are uncontrollable we can say. So, in order to extract out that okay, what can we do different type of bacteria which is present there are different methods of doing it one is culture omics, in which we are going to take out those bacteria and they have to work a lot on different aspects. For example, location of isolation, if you are considering a niche, for example, gut, so, what kind of what area of gut you are going to use for the isolation and what is the temperature of the incubation or time of incubation that you are going to take and aerobic requirements and all these factors, what I’m discussing over here, they also depends upon what kind of bacterial want to isolate, because suppose, in this niche access to this niche is going to have different kinds of bacteria. For example, there is some bacteria is going to be very abundant in nature. If I talk about oral cavity, strep, Steptoe focus is very abundant if I talk about gut to lactobacillus is very abundant. So if I’m if I’m going to talk about skin staphylococcus epidermidis is very common. So, what happens that when this abundant bacteria is there, and along with that abundant bacteria, slow growing bacteria is also there. So, when we are going to do the culturing part, so, the slow growing bacteria is not going to come out very easily when abundant bacteria is there, because it is going to utilize all the nutrients so in order to extract the flow, growing bacteria, we need to focus and we need to standardize a lot of the factors. And one of the most important part of that is enrichment, that specific bacteria needs to be enriched awesome use of a selective media that will enhance the growth of that slow growing bacteria. So, once we have that single bacteria that we want, and we have single colonies and we prepare it population, and now we can do next generation sequencing. So, now we have a whole genome of a single bacteria or you can put bacterial species, we have all the information regarding that part. So that is known as basically search domain. Next, give it one click Yeah. But what happens is around since 99% of bacteria is on culturable, so we are going to handle this particular part by meta genomic

Dr. Shikha Sharma
engine X. So considering the same niche, because we can we don’t know what are the conditions, what are the different types of bacteria is there and we don’t know anything basically, and we seen that bacteria is uncontrollable. So, we are going to directly extract the DNA so that DNA consisting of all the bacteria, technically all the bacteria, which is present in niche, and then without knowing anything, we are just going to sequence all of it and then the pipe the most difficult and the crucial part in such a kind of such kind of studies, that the analysis part is a little bit more tedious. So but this is going to give us the information about those uncultured bacteria that what can we do what can go here. So this is the basic concept basic difference between them into the next slide. Okay, so, I want to talk about a few new concepts that has changed in the Bible MGS for example, technology. Initially when technology anomic started in 19th century though we Started used to you know classify bacteria depending upon their morphology, their chemical reaction, physiological properties etc. Then we came onto differential experimental methods like DNA DNA hybridization in this bacterial genomes are going to be extracted and they are cut into the pieces and then they’re hybridized over each other to see how much they are going to attach to each other. But these parts were not very informative most of the time the bacterial species were misclassified and because it was it was seen that two bacterial species which are different, these are two different bacterial species can have same biochemical physiological properties can have same morphology and chemical requirements and all those things. And as far as DNA DNA hybridization is considered, it was it is it still is very tedious experimentally, very difficult part two and not very fast. So, they started out with Okay, we will do the serial sequence the 16th sequencing and we will do the polyphonic phasic ekonomi and all those things. Then, they came onto rpbg analysis and mlsa Fine, because it was easier with printing things, but in the recent years, the gold standard of defining a new species has changed that is after next gen sequencing era, initially it used to be 15 is arguably MLST etc. But right now, they are AI that is average nucleotide identity and digital DNA DNA hybridization. In some, if you have to go by typical taxonomy concept, then you have to do in some of the cases you have to move polyphonic taxonomy, but if you want very fast and easy reverse, then these two parameters which are very easy to calculate, once you have complete genome of the bacterial species, you can define you can see you can check out for yourself that those bacterial species are new species or they are the old one by these two values, which is there and a lot of published articles are there on this particular topic. So, coming to the next part.

Dr. Shikha Sharma
So, I would like to study case, these are two different first of all, for Nia and ddH. These are two different tools, which are there and these are online tools. So you just have to submit your genome or and how you have to do the calculations. One thing that I would like to discuss here is that this AI and vision end and capitalization is always calculated between the query genome and with a genome of a type strain. Now, the question arises, what is the type strain type strain is basically a reference string, suppose if there is a bacterial species say lactobacillus roofrack, in this day going to be 1000s of strains. So we don’t know that what strain is going to be reference strain because there’s always a continuous evolution in bacterial genome, in fact, is much more faster than we anticipated. So, how do we know that this was the initial strain or this was the strain which has undergone the mutation. So, there is the first trend that is going to be founded in a particular species that is generally taken as a type strain and it is all it is always going to be referred as a reference. So we are going to calculate that ni and DDS for our query strain the genome of strain as well as the genome or considering the case study. So this was one isolate, m 31. And by 16, so we came to know that it is closer into lactobacillus, but we don’t know what is what fishing and by 16 as it came out somewhat near to try, but we did a whole genome sequencing and see as I told earlier, the morphologic growth pattern and biochemical tests etc, are exactly similar with roofrack. And if we are going to go by the initial standard, so, it is going to give us the idea that it is to try but by 16 as an RGB, you will feel that it is to try but if we take ni and digital DNA and hybridization, so this is this value shows that it is a new species. Next slide. So this was tectonic genomics, that is taxonomy on the basis of whole genome sequencing next to Phylogenomics that is called genetic analysis on the basis of genomic. So, there are a lot of things which happens in college and I just want to discuss three that AI three the AMI values that I discussed in the earlier slide, so a tree that is based on that AMI and then a conserved protein tree and SNP tree and what are their significance. So coming to the first part. Yeah. So, in this AMI tree, we just we just normal calculate average nucleotide identity as I told you in the earlier slide, with the type strain and we just among each other also and then we just create a new bitmap or whatever, we want to save them see them. So, this basically tells us that which strain is how much genetic genetic relatedness genomic relatedness that which strain is similar to the other strain and how much it is different to the other strains. For example, the all of them are different strains. And you will see that there are two clusters depending upon the color difference in colors or depending upon the AMI values. And these values are significant because these are whole genome based this is not based on say one gene or two gene or a five or seven gene. So, you can see that our two, what the pink one and the blue one. So, in this case, when we look out their AI values, so, we came to know that the AI values in the pink cluster was very low for the CSR, this means that all these things, which are there in NTBI, which are submitted in NCBI as Desiree or not Gary, but some other new species, and so, we this was a miss classification and this particular part is the goal to be resolved by AI values to come into the next part that is conserved protein tree. So,

Dr. Shikha Sharma
yeah, just just give give one more Yeah. Yeah, okay. So, ni is asked for species or classification suppose, are discussed in the last slide. But what happens when there is a genius when there are two or three geniuses, for example, in the last slide that is decided they were all the strange word from generic species only. But what happens when there are a lot of different geniuses as well, especially when they’re very close, like, when we talk about the physical or the morphological features and the chemical effects, so pseudos en una Astina, trichomonas, then una and all these above strange species, they all geniuses, they all have the same kind of ecological features. So how do we make a phylogenetic tree or how we distinguish between them. So, for this, we generally make a conserved gene conjunct protein tree or we can say, amino acid chain, for example, for species we took the nucleotide identity has to take the amino acid identity. So so this kind of information and the concept protein three is very important for deferring the evolution. So this is the three that we have enabled this this way, I think I’m I’m not cited the paper from which I’ve taken this image. So I’m sorry. So, but this particular tree is basically based upon the orthologues genes, which is present in 35 strains. So if you’re going to see a nice tree, so, this is going to give you a different picture, maybe K four and K and three, which are very close in this particular image are not completely different in the AMI tree, but, but we wanted to see that what is the what has the evolution put in the name of changes into this. So, in order to do so, we we wanted to we wanted to discover it, the evolution and evolution is always can be confirmed by studying the amino acid or the protein in a particular genome. So, this is why the conserved protein tree is important. So coming to the next part, yes, if we want to see the relatedness between strain, which are very close to each other, for an example, so that that SNP are needed to be studied, and for this, we make a SNP tree. So, for example, all of these isolates in human lineage in this this is my this image was from my work on lactobacillus Truetrac. So, this human lineage, all these isolates are completely clonal to each other, and if we are going to see fit to lineage, so in this also 20.236 and 5395 I’m not sure about Pictou but there are some strains in poultry humans, yeah, in poultry, human JC and LP 54 and wh WT they are completely clonal to each other. So we can find out they’re sewn into each other because they there is no branching lineage, all of them are coming into a single line, there is no much of the market demarcation between them. So we can say that there are less number of SNPs between them and hence, they are considered as clones of each other. So coming to the next part. Yes, one of the most important aspects that is being studied today is pro bio genomic. That is, we know that a lot of commensal bacteria is present throughout human body and they’re different. They’re present in different niches for example, guard down and oral cavity vaginal diversity etc. So, we study this diversity by either bicultural meats and meta genomic as we have discussed in the previous slide.

Dr. Shikha Sharma
So, the most there are different things that we can study in the way that we can know we can study we have to study and we should study and we always study in probiotic sciences in NGS that is providing for an example, considering the right species to formulate a particular probiotic or right combination of species or strains and a detailed view of actual mechanical action, then it can also tell us that a particular species is electronics or a Taconic in nature. What do you mean by this is that if a particular species is indigenious to a particular host, so, suppose if I’m taking a particular probiotic, if the bacteria that is present in that probiotic is not autonomous to me, that means, that is not indigenious to me, that is not from a well adapted human body as to the human body. After that, we always wanted to study and we should always see that whatever antibiotics are present in the commensal bacteria, of course, it’s the commensal bacteria and keep antibiotics present and if they go resistant over the time, so, it is not going to be a problem. But the problem is that if they are present some classmates or some area, which can be easily, you know, jumped by insertion sequences, and they can be introduced into some pathogenic practice, that is going to be a huge problem. So, we want our probiotic strains, three of those antibiotic gene. So, that is why we need NGS and we also do genomic and transcriptomic profiling, yes, and the most important part is study of bacteria. commensal bacteria infect any kind of bacteria is loaded with a lot of anti biotics and a lot of antimicrobial peptides and growth should always be studied. And that is why for genomic profile genomics, yes, coming to the next slide. So, I would like to discuss two part in this. One is skin microbiome and oral microbiome. So coming in. So, we know that staphylococcus epidermidis is a commensal. In bacteria commensal means that it is not pathogenic, it is just there it helps in the normal homeostasis of skin. And it shows, in fact shows activity against pathogenic bacteria like Staphylococcus aureus, and produces antimicrobial agent. But the thing is that in the recent year, it has emerged as the opportunistic pathogen of nosocomial infection. So initially, it started out its journey as abundant common sense, but in the recent years, it has emerged as an opportunistic pathogen, it is causing a lot of bloodstream infections, it has role in neonatal sepsis. And it shows it has also has a multi drug resistant form. And the problem between these two types of strains right now is that technically on genomic level and as well as in the biophysical or any other method, we don’t know that whether it is the commensal epidemic or it is the pathogenic one. So, the bed the doctors or the conditions are not able to differentiate differentiate between the two we do not have a specific marker to differentiate between the two and hence, we cannot treat them if you are if you are going to have a particular a drug or ointment against the epidermidis. So, it is going to harm the abundant commensal bacteria as well commensal epidermidis as well and again, because it has an important role in immunomodulation it produces antimicrobial agents. So it is going to affect our health. So, that is the reason that there is a need to differentiate between these two types of bacteria. So next. Next, so that is, in order to study that what we did is that we isolated around 28 epidemics, these isolates from the healthy individuals, that means the healthy, the commensal bacteria, and we did a whole genome sequencing and comparative analysis. So these other 28 isolates, which we isolated from 28 healthy individual, and then we wanted to study genomic relatedness next next okay. So cool. Can I you know, Stop my camera because the internet connection is low. So just for the presentation I’m going to stop my camera and just I will just talk Okay, I’ll do that

Unknown Speaker
yes

Dr. Shikha Sharma
yeah fine okay yeah under okay. So, we took those 28 isolate from healthy individuals and then we mixed them with 21 isolates, which are already sequences present in NCBI of western region. So, and we get formed this ni tree that we discussed in the previous slide. So to know that genomic relatedness between all these ICs So, the black, the one which are defined, which are designated in the black font, the Indian other Indian isolates and the one in the yellow one out of Western isomers I just want to remind that all these isolates are the summons. But what, but we just add pathogenic strain or physics to as well in this analysis. So, as you know, when we started out this study, we imagined that since all these isolates are commensals, and artistic we are pathogenic, so, they are going to form to Artistics, we’re going to be completely different on genomic level from all these commensal isolates, but before that, all these isolates are divided into two different lineages, that is region one and region and the pathogenic strain has clubbed with other commensal isolates in pgpool unit. So, this was very kind of, I will say not shocking, but very different kind of result we were not expecting this. So, next. Next, Next, yes, we did some other analysis among other analysis also. So, we studied all these isolates in the PG one lineage and we see that we saw that all these isolates impeachable image are highly clonal to each other, we discussed clonality in the previous slide. So, there was really this MLST that means, we extract out important closest important gene from small these isolates, and we studied that whether there is some SNPs present or not. So, when the SNPs are not present over there, we saw that these are highly correlated with each other, and this is a new sequence type. So, what happens is that, when we have different strains, and so, we define all those strains in typed type, because, suppose, if I am going to find out a strain from India and suppose, a person is going to find out a same strain from the America. So, we don’t know that whether those strains are exactly each other or they are going to be different from each other. So, we define them by a sequence type. So, if my strain and the strain are, which which is which has gone down in us are going to be completely close close to each other orthogonal to each other, they are going to be defined in a single time, they may have different strain names, but those two different strains have a sequence type which is which is same. So, but when we did this MLST and we we compared these isolates from the SP which are already there on the genomic level. So, we saw that there is a p does not this does not belong to any SP which is already there. So, we named it as a new it was founded as a new SD and it’s sp 691 net Yes, Now, coming to the marker part, that is that is why we did this study. So, we go we have gone through some of the literature’s and we saw that there are some of the important genes, which are more prominent in the I will say commensal to follow focus academically, and they are some of the some which are abundant in the pathogenic stream. So, we wanted to study that what is the status of all these strains in our strains or in our studies in our study, and just to recall in our study all the eyes loops that took us commands and they are divided into two different lineages that is feature one and feature two. So, as you can see the first gene that is FGH It is highly conserved in PG one lineage and all other

Dr. Shikha Sharma
all other genes, especially the for genetic genes, that is is 256 iesba and M Max a are completely absent from PG one lineage however, it is present in PG two lineage and we confirm these we did a lot of analysis, we wanted to see that whether these genes are actually present in the genome. So, we did a local study and all those things is a very technical part, so, I’m not going to discuss them in detail. So, but the point is that, well commensal genes is present in PG one lineage and the pathogen genes are present in PG two lineage next part then, we align the Indian isolate and the Western isolates to each other to see that, whether they are completely similar to each other and or not, and if not, then what are the differences. So, the blue one other Indian isolates and the yellow and other Western isolates, and you can see that there are some regions or some portions, like the white portion, which are there which, which are present in the Indian isolates, but are absent from the Western isolate. So, this is one area and we annotate these regions and notation means, we take out this particular region and then we try to find out that what are the genes which are present in this particular region. So, you can see two regions one is lanky peptide and another one is sexy peptide both of these regions are absent in western isolate, but they are present in Indian isolate Indian isolates firstly image and these are these and voted for lentic aside and sexy peptides and lenti peptide and sexy peptides are basically a new when we studied these sectors, so, these are new anti microbial peptides next part

Dr. Shikha Sharma
next Yeah, just just next slide. Yeah, so, these other clusters, first one the longest one is a lengthy peptide and the lower one is the sexy peptide. And when we did last analysis, we studied them into different types of metabolite prediction tools and etc. So, we came to know that these two lenti peptide and sexy peptide are not known till now are normal to each other. And of course, this requires a lot of experimental validation just to see that whether whether possible whether do they do technically exist, as well as whether they are beneficial against a specific particular specific disease or bacteria coming to the next part. So, until these two antimicrobial peptides are present in human isolates of India, Indian population, so, this particular study gathered a lot of media attention. And the point is that, we extended this part. And we isolated this particular SG 691 from different individuals randomly, just randomly after the study also, and they all came, came to have those lenti peptide and sexy peptides. So, this particular study was pretty interesting for us. And yes, it gives someone an idea that how, how different our fellow focus academics are from each other? Yes, coming to the next part. So, similar, similar kind of study was for oral microbiome, and oral microbiome is generally noted with this chapter focus and streptococcal, but we wanted to focus on lactobacillus. The reason behind is that lactobacillus is present throughout the body in the bird in the vagina in the different parts of body COVID wanted to study that whether this lactobacillus which is present in oral cavity is different from rest of the body part or not. So, for this, we collaborated with Dr. Pawan Singh, Judge, Institute of Dental sciences and hospitals. And sorry, I didn’t mention in the slide, but this study was completely collaborated with MTPC. In fact, they initiated study under Dr. Suresh properly, which, who is head of mtcc. So, what happens in this case is that by a lot of factors by a lot of enrichment and different types of media, somehow we were able to find out the lactobacillus from the oral cavity, and we did a whole genome sequencing, and I just wanted to discuss a small part that is lactobacillus Desiree, which is by Follow genomic markers that we studied in the previous study came out to be a new species. Next part yeah, so, this is the comparative analysis that I talked about that lactobacillus Desiree now it is para deciding it has to be classified as para guitare. So, it is presented oral cavity it is present in vagina it is present in gastro intestinal tract as well as so we wanted to study how the paradise area isolate of oral cavity is different from rest of the ice, so we didn’t find it overlap. And gentlemen, we wanted to study all the genes which is present in its genome. So, these are the genes which represent that how much gene is similar to the other icolor for example, 124 genes are common in this seven as well as JB v 03. And similarly, nine genes are common between oral cavity vaginal isolate, as well as the breast isolate. So, these this is how the this whole image is going to be called, I will just discuss next

Dr. Shikha Sharma
I will just discuss the two parts first of all the common gene which is there in oral cavity and the vaginal isolate, because there are a huge rest of them have shared genes like 9933 634 etc. But vaginal isolate and the oral cavity isolate shared maximum number of genes that is 24 and there are some very interesting results that we know the reason why first of all the question why is that why oral cavity and vaginal isolate has such huge number of common genes. The main reason is because the physiological involvement of these two niches somewhat similar. So the genes which were found in these common pools were PTS systems blood glycosyltransferases, which are generally involved in biofilm formation. But the most interesting result was to find a sub eg the sixth one and the fourth ABCD of the bite is interesting because some EEG is present in some pounds like is futile peridinium and slippery, might get ions, et cetera. So, this an EEG is resistant to all these compounds. And these compounds are present in any plaque or anti Catholic agents, which that we use generally in as a in which are present in mouthwashes and all those factors call you all those things. So, that is why a gene is present in these particular genome. So, another one is called ABCD operon. So, this particular operon helps in growth, cell wall formation, as well as biosynthesis of siding force and acid resistance. So, oral cavity as well as vaginal isolates, faces a lot of imbalances in terms of acid reflexes and a lot of competition from other bacterias etc. So, that is why they require a different kind of Oberon or a different system, which, which helped them in growth in cell formation in forming colonies. And that is why there is a significant of course ABCD operon over here. So, next, yes, coming to the part that why there are some genes which were unique to the oral cavity. So, around 142 Gene was unique to oral cavity and these encoded for PTS system to aquastyle Cronquist. But the most interesting results was that it has gene for cold shock, future oxidative stress etc. Now, we know that oral reading different kinds of food have different temperature etc. So, oral cavity is subjected to different kind of environmental pressure. So, that is the reason that over times of evolution or this, this seven the oral cavity estimate has undergone has acquired these and it also has high fluoride resistance to force us to fish that we use. So, they have fluoride in them. So, over the time seven or the lactobacillus which is present in oral cavity has acquired high provide next Yes, this is one of the most important part that seven encodes a complete class have to see better using cluster whenever there is a concept of provided omics. So, bacterial things are always going to be discussed, they’re always going to be studied. So, we can say that in this seven also, bacterial cells cluster was there. So, it showed activity against Pseudomonas aeruginosa MTCT, 1934, and six to 6% of its genes. It shows similarity to the SNP synthetic gene cluster. So, this is this was a cluster, it needs to be experimentally validated, but it was a complete cluster and ensure that it will this particular isolate has this bacteria. Next spot.

Dr. Shikha Sharma
Yes, this is one important study about the lactobacillus voudrais. However, this was a very in depth study, I’m just going to go going in a very brief part of this because of lack of time. So, we know why biotics are very significant has a very significant role in human health conditions. And because there is a sudden increase in drug resistance, not sudden, but there is an increase in drug resistance. So there is a need of probiotics right now, and there is a proven probiotic industry. However, there are some issues which are associated with probiotics right now, that there is no specific cure of a particular disease, then there is no specific knowledge of a mechanism of action, and it has some technical issues, I will say, like colonization, bacterial competition, competition, etc. And we also should know about the host microbiota and inter microbiota interaction, and it should focus on individual screens and fishes as well. So next. So yeah, okay. So the, I think the main problem of this probiotic, which are studies right now is that the last is the last ecological and evolutionary perspective, that means their adaptation, that whether they’re adapted to a particular host or human, because if we are going to use them from for human benefit, and even if they are adapted, so what are the adaptation, friends, their evolution, and all those kinds of studies. So when we’re finding adaptation trends, we study global omics analysis, horizontal gene transfer, exogenous omics, some of these these things are have discussed in the previous slide. So this is, this is one important aspect of probiotics, sciences. Next, it’s coming to trial. So it is a very well known probiotic. And it is an anti microbial compound, which produces sorry, it produces antimicrobial compounds like nutrient recycling, vitamin v2, and etc. But the most important and peculiar part of the DRI, is it, host adapted lineages that maintain only colonizes in their host. For example, I’m just going to tell you in the very brief and very straightforward part, that suppose if there are some strings, which are extracted from humans, so they are only going to colonize to humans, that means if they’re going to isolate it from pictures, they’re not going to turn us into humans. So, it is very important for us to know that if you are using a particular strain as a probiotic, it for human body. So whether it is electronics or auto corner to human body on next part. So, in order to do that, we wanted to trace the evolutionary history of diversification of Rudra into it into different hosts, for example, pigs, rodents, human etc, by comparing it to some closed species. So at that time, it was lactobacillus vaginalis. But it is not close enough to be hunted for another species that was in particular at that time by cysteine is an Adobe tree and after that, we did this whole genome sequencing. So it came out to be a new fishy NexBox cooler, just skip this part, because I’m just not going to because this part, this is very technical and it is just skip this part, move on to the next slide next. The next next. Yeah. So, once we did the whole genome sequencing of M 31. So, we studied some of the important clusters, that whether they are present in those genomes, in that isolate or not. So, these important clusters which is present in human isolates, they are present in for example, pgcbl cognitive cluster This cluster is completely present in human isolates, but this is this is absent from the rodent lineage. And then there are some other clusters, which are present in other lineages, but they’re completely absent from human lineage next. Next, next. So, this is the These are clusters and all these parts that I’m just going to skip over there. They’re very technical, and

Dr. Shikha Sharma
I’m just not going to discuss them, which are next. Next, Next, Next. Next, okay, your previous previous one, yeah. Yeah. So, by all these analysis, when we did this analysis, we find out we found out, I’m just going to discuss the results, that human lineage has more gene for biofilm formation, and it has to do CBI problem cluster which is also recorded in the previous research and which is there in the above table as well. And there is a lot of insertion sequences and mobiloans, which is present in this particular lineage, while the rodent lineage has a lot of gene for biofilm formation, and it has also it has also preserved its ancestral gene or genes. So this was a finding from this particular part where we track the D clusters, and how we did them and what are the results, I’m just not going to discuss those in detail. Next part. Okay, so after that, we did the pan genome analysis, we discussed that what are the genes which are common in all the URLs do try as well as it’s close wishes, and what are the genes which are unique to them. So, when you studied through try, so, these red genes, the genes, which are highlighted in red color, these are the genes which are unique to them that means, which are not present in other species, while the yellow ones are the shared one. So, when we studied these genes, which are shared as well as up, so, we saw that most of the genes were insertion the the one, the genes, which are highlighted in green color, so these are the genes which encoded for the insertion sequences. So around from unique Gene 10% Gene encoded for insertion sequences from three at 16 to 6% of genes and 44 insertions. Next part did the competitive and assist by aligning complete genomes as well. And the regions which were unique to human genome, the blue one, the light view of the human genome, the red one, or the poultry, human genomes, the yellow one, sorry, the red ones are the Yeah, poultry, poultry, human genome, the yellow one are the pig genome and the last blue one other rodents. So you can see r one, r two R three, R four and R five other regions, which are present in human genomes, but they’re absent from rest of the other genome. And when we did the annotation, or we tried to find out that these regions and voted for which genes. So we found out that all these regions and voted for parties and recombination and elements, insertion sequences, etc. So we can say that these particular areas are basically annotated for genome dynamics or mobile. Next part. I’m just going to skip it next. Next, okay. So, this is the conclusion of my last case study that was applied to the request, that human lineage did not have any jeeto biofilm formation, and it has lost most of its structures and but it has b2c by corporate structures, while the Rhoden lineage has been inherited and acquired by Phil formation. So, by this was my studying which showed that if we want to be wanted to extract out genes, which are human genes, so we can say the human isolated strains. So, these strains does not have these features. For example, it does not impose any supervisory formation, they does not have any clusters which encode for biofilm, they do have video CBI program clusters and I think that these important information is very important in order to find out that whether those strains which are there, which are which we have extracted from human so they’re autonomous to humans or not, and hence is whether they will be beneficial to us as a probiotic or not. So next

Dr. Shikha Sharma
Yeah, so I’m just going to be there so, this was my presentation. And this this basically showed a very basic give a very basic idea and different case studies that how bacterial research is done in NGS time by using NGS, especially probiotic, bastard eco Virgin Mobile. So if you have any questions, so you can just stop we thank you so much Dr. Shaker for his wonderful talk. And really paid for the time is what it is to share your thoughts and knowledge. Thanks so much I’ve been sent all the questions in the comments box. Okay. So, we have one question from Wireshark. Could you please talk a little bit about restaurant analysis regarding what data should we begin with for the analysis? Okay. Yeah, that is very important and very interesting question also. So, resist Tom, for all the students out there, I just want to say that resistance is basically the the gene set of all the resistant genes which are there and the genomic regions in a particular genome which encoded for those resistance genes. So, we can just start it out, if we have NGS data, we can just start it out with the genome or fruits also, there are different tools which are there like there is one very basic tool which is there in Patrick, there is where there is some very basic tools like God, which are NCBI basis, then there are some other tools which are Linux based as well, and then we can extract out all the information that what are the different types of resistance genes which are present. And suppose if we want to, if you want to see sometimes what happens is that over the evolution, those resistance gene changes themselves and there are some SNPs of mutations which are present in those genomes. So, if you want to pinpoint and specifically study that what are the mutations, which are present in those genes or if you want to see that whether those genomes, whether there is a new locus or new antibiotic resistant gene, so, you are just you should map all those genes with the reference antibiotic resistance genes and you should study their attributes that whether there is some SNPs are there or not? So there are different tools which are there and you can just mail me also I will, I can discuss those

BioBattalion
tools as well. Thank you so much for operating. Thank you, ma’am. Thank you everyone for attending this session. I’m sure it’s not been a must have been very insightful for everyone and also for the people who are going to see the recorded version of this in the future.

BioBattalion
And if you’re not on our mailing list, you can go sign it is given in the description box below the official link to our website, you can sign up so that you will get the you will get notified on our upcoming events. So see you all soon. Thank you, everyone. Thank you, ma’am. Thank you so much.