Lecture 09

Added: 2026-05-12

[00:00:21]What are the strength and limitation of the DGE?

[00:00:24]So the key strength of the DG DGE is uh are band excision enable direct identification.

[00:00:32]So you can have multiple band like 1 2 3 4 five bands. So band you you you just uh take out the bands on the gel uh maybe on agros gel or maybe on you know page polyacry electrophorosis. It depends on what kind of a you know size of the DNA you are looking into. So bend oxygen enable direct identification of the marker. Then accident resolution of dominant species.

[00:00:59]So you can resolve the multiple strains within the species. Say for example E.coli has multiple strains and subtype based on these signature sequences. You can you can you can separate them easily. Uh then visual interpretation of community patterns.

[00:01:15]uh just on the agros gel patterns or page on page gel patterns you can visualize the variation among these genome uh reproducible and reliable results. So your results are very very reproducible. Uh once you identify the markers and this marker can be reproducible uh with the same primers if you can identify through PCR and then sequencing or uh through some other mode of uh method uh cost effective for routine analysis.

[00:01:41]You can routinely do it uh you know just looking at the same standardized condition of DGE and you can identify these markers and you can find variation. For example, if you find a strain ecoli strain u strain ecoli eoli 1. Now how strain ecoli 1 is different from strain equali 2. This can be identified through this marker. So this marker.

[00:02:09]Now this marker are basically nothing but identified from the same DGE. All right. Now so this is the strength. So cost effective routine analysis can be done. Now notable limitations. What are the notable limitations? Now the complex and band patterns may be over overlapped. Sometime what happen these band patterns gel. So suppose lot of bands are being overlapped here. Now these overlap bands are belong to the same size same patterns. So sometime it is very difficult to look into the overlapped band pattern because of the similar size although they are different. So there might be for example a T g C for example this is one sequence having this band then you can have a T c G another band now both the size is same they they should come on the same pattern same size but the sequences from here see is different from a single sequence difference you find variation or we call the mutant.

[00:03:18]This is the drawback. This is the limitations. So complex band patterns may overlap because of the mutations in remain intact uh with remain intact the size of the sequences less sensitive to rare text detection. So rate extra detection are difficult for the reason because for many reasons. So for example sometime for the rare sequences are not available. Sometime if you want to compare with the databases the rare sequence data is not available. So these are the some reason where it might have less sensitive for prediction requires optimization for different sample. So you need to have a you know it's not like that you have one sample, two sample, three sample and you can optimize it. No, you need to have multiple uh you need to have a multiple uh uh steps to carry out the different samples to identify and this is how uh this is one of the limitations. Then again you have a time uh intensive gel preparation. So you have to have a lot of gel preparations taking place and so on. Now then come to the TGE. This is the second method. Now TGE is basically temperature gradient gel electrotophorosis. Now in case of temperature gradient gel electrophorosis you have uh exactly the same process as we talked about uh as we talked about in DGE.

[00:04:46]In the DGE, you have the chemical denaturing agent which is used in order to denature these sequences in the gradient mold from the top to bottom or from bottom to top from high to low or from low to high. Here in this case, similar work is being done by the temperature. So I have to prepare the gel with a temperature gradient. High temperature, low temperature, low temperature, low, low, low, low like this. So gradients in the temperature will again provide a certain precise point variation in the two different genome right to denature. So this is what the purpose the whole principle of this is based on the same. So once you differentiate these two uh then you can easily differentiate on the banding pattern and this banding pattern is basically will tell you okay this is one type this is second type this is third type and so on. So temperature gradient means uses controlled temperature uh difference like in gradients rather than the chemical dennaturation just like in other we have taken the chemical here we take the chem temperature melting behavior. Now DF fragments separate based on their specific melting temperature and what is the basis of melting temperature? The basis of melting temperature is the intrinsic character of the genome. This is what we are trying to find out GC content. High GC content, high melting temperature.

[00:06:13]Low GC content, low melting temperature.

[00:06:16]So we have to look into melting temperature. Then microbial ecology.

[00:06:19]What is microbial ecology? Frequently used interchangeably with TGE environmental microbial microbiology study. So you can have a different ecology which microbial ecology which microorganisms you are using in order to for that uh TGE.

[00:06:42]Now if you compare TGE versus DGE as I've told principle is same method is different mode is different here in one you have a chemical in another you have a temperature. So if you compare these two together you find TGE is a very simple gel simpler gel and DGG is uh sort of more stable gradient. So this is the benefit why it is more stable because you have a complete gradient of a temp not temperature but the chemical which denatured regularly the DNA and make a difference. Suppose one DNA come at this temperature the other DNA is also coming on the same temperature but this DNA is get denatured this DNA is not denatured so based on the denaturing and non- denaturing condition you can have a variation in the banding patterns mobility patterns and so you can identify this is one type this is two type then uh no chemical dennaturing handling this is one other advantage of the This where in this case of uh one of the advantage is that higher reproducibility then you have direct temperature control in this case. In this case you have a better better stabilized uh established protocols are available because all the chemicals there is standard concentrations to be used. So you can use the any protocol but in case of DNA temperature you need to have optimized lot of time is consuming optimization of the temperature. then reduced chemical waste in this case here wider literature support. So you have a lot of literatures available. So there's some benefits uh and advantages of the TGE and DGE uh in terms of like use of these techniques in your own lab in your own course and your own practical cell but the both methods provide the community fingerprinting and enable band sequencing for poly phoggenetic identification. So making differentiation uh mutations variations in these two just by differentiating with the banding patterns of this.

[00:08:49]Now uh TGD application in environmental microbiology is commonly used as a ecosystem monitoring. So tracking the microbial community dynamic in natural ecosystem including forest wetlands and marine environment. Right. uh whereas uh uh temporal studies uh you have monitoring seasonal and long-term changes in the microwave community because over a period of time they they keep changing. So when they keep changing you can identify the changes through these uh marker methods of DGE and TGE method. Global research widely adopted the environmental micro microbiology or ecology laboratories worldwide for comprehensive diversity comparison. uh look into the diversity comp comparison in the most microbiology and ecological labs where you can differentiate these two individuals through these methods. Uh they very commonly used throughout the globe.

[00:09:45]Now uh now we have another uh topic start here. So uh in my second lecture so this is my second lecture where we will be talking on uh understanding of a r and a r ia. In earlier talk uh we talked about the D G and T G. Here we talk about the A R D A and A R IA. Now A R D A is a new topic uh although it it it is also used for comparing the variation or the mutation among the microorganisms through different mode.

[00:10:21]So again we can call these two are the markers but these markers are based on certain different principle and different methods. So we'll discuss what are the different methods and different principle based on these markers.

[00:10:36]Uh the ARD method uh by name is called as amplified RDNA restriction analysis combined with the PCR amplification of the ribosomal DNA with the restriction enzyme. dation to generate the characteristic fragment pattern. Now uh if you go my just the earlier lecture where we talk about the DVD and TVGE that is the gradient and temperature gradient gel electrophoroses and and and denaturing gel gradient gel electrophoroses the basic principle that the variation in patterns of these band is same as that here.

[00:11:20]So in this case also method is different but outcome is same. What is the basis of visualizing the difference is same.

[00:11:29]For example again I can make the same gel here right and you have a different u sorry this is gel. So this is one pattern. So we call them as a uniform pattern. So 1 2 3 4 uniform pattern. Now in another case if you look into like this. So this is a non-uniform pattern. Now this is the uniform pattern. This is the non-uniform pattern. Now a non-uniform pattern means this band varies from this in their mobility.

[00:12:16]mobility why it different in mobility because it varies in terms of like denaturing conditions are earlier case or here in terms of like a size it may be you know 1 KB it may be 1.5 KB it could be plus minus so depending upon the size smaller is the size the smaller is the size SS smaller is the is faster is a run. FR faster is a run then larger is the size LS slow is run SR. So this is basically the basic principle behind this technique.

[00:13:03]Now what is happening here in ARD markers are I call it marker as well as a technique. the target DR RD DNA sequencing. Our target RDNA sequencing was amplified. You have to use a specific restriction enzyme. So once you get a sequence, use a specific recession enzyme and these for example you have uh say for example here you have this is uh one fragment. This is another fragment.

[00:13:35]This is another fragment. So these are the fragment from the same individual.

[00:13:38]we have RDNA sequencing. Now in case of one three so a specific restrictions for example here we are using EO R1 this Eco R1 sequencing R here in this case this Eco1 sequencing sequence is get mutated and it either shift or shift here or it is lost.

[00:14:16]In both the cases you can have it will cut like this and this and if it shift from here to here then it can have cut like this. So see the difference this is one this is two this is size of the fragment. See this this this fragment this and this. So the variation in the size of the fragment due to the change in the mutation identifying a specific restriction side give you different size and so different patterns of the band.

[00:14:50]So this is very precise very accurate uh and very very very advanced techniques.

[00:14:56]So we call amplified uh RDNA restriction analysis means you have to have amplification of the RDNA sequence or whatever size. Then you have to identify certain restriction site to to choose to in order to cut it and based on the variation in the gen genetic uh genome pattern or the gene pattern the size will be varying I think it's clearly low then uh create a fragment length polymorphism. This is that variation.

[00:15:23]This is called polymorphism. One band is large and another band is a small then you have a large small so on. So the variation in the band pattern reflects the phogenetic changes.

[00:15:36]You can visualize on a gel or page. Now in case of uh difference between page and aos. So suppose if I ask you a question when do you run a page and when do you run a uh agros uh agurus versus or versus page aurus versus page means a can can resolve large size and they can small size but there's a limit of large size and a small size the small size limit I can go large size limit has no limit it can go for you know 10 KB 20 KB even whole genome and all in this small size what is the resolving power of the agurros gel and resolving power of the page is important to understand the resolving power of agurros so we call it resolving ing power. Now resolving power means visualization limit.

[00:16:50]What you can suppose if if you have this hand like this. So it it looks like you have a two hands together like a single hand. But if we separate that separate that hands like this small one you can say okay these are the two. As long as they are not separated, it is very difficult to visualize them as their two hands are complex of a one hand. Similarly, when the two bands of equal size get gets get on the overlapping conditions in the overlapping condition, it it looks like a single band referred to the single type of genome but it is not. So suppose you have a single mutation and due to the single mutation there's a no variation either you deletion or insertion or some changes changes may also affect the dennaturation for example if you chain A to GC it will affect right or if you chain otherwise GC to 80 it will affect right due to melting dennaturation. Now what is the smallest limit for the page polyepide gel electrophorosis is the single SNP single nucleotide polymorphism right even a mutation variation in the single nucleotide will be catched by the page through this method and that's how the sequencing is done on the page through page now Arisa enhancement we call automated ribosomal intergenic spacial analysis focus on the intergenic spacer region of the enhanced resolution and automated processing. Similarly, ARD method ARISA is also one of the method where we can use this powerful tool in order to understand the identifying different markers.

[00:18:42]uh in case of uh in case of this again the workflow the processing of this ARD or AIA uh is the PCR amplification uh of ribosomal DNA or intergeneric spacer. Now what is the intergenic spacer? So this is the intergenic spacer like this. So RDNA sequences RDNA sequences are ribosomeal RNA sequences they have intergenic region introenic region IGR this intergenic region is basically the conserved regions right so just like RDNA we can also interpac regions or intergenic regions can also be used for the identification of the species every species has a very conserved interpacer regions interpace region are smaller They can run on the gross gel easily and can identify it. The ribosomal DNA are intragenic or intragenic spacer or space region amplified using the specific primer set. Now the uniform are we call the universal universal primer sets. So these primer sets are available and you can use these in universal primer set to amplify these interpac region or RDNA sequencing or RDNA region.

[00:20:06]Once you amplify these RDNA sequencing uh sequences region or interspace region then you can uh you can um perform the PCR using the primers and this PCR product when amplified we call amplicon.

[00:20:22]Now this amplon is again used to uh electrophorosis. On the electrophorosis you can when you run the embleicon they give you the different banding patterns and these banding patterns are basically nothing but they are the patterns where they you can show the variation in the banding pattern due to the difference in the size of these bands. Now why these size are different? These sides are different because of the mutation. So same individual same category ecoli ecoli ecoli but they're different strain having variation changes in the size of the genome mutations or chains so on so forth through these markers can be identified and you will end up with the variation. So this variation can be detected through these ard and ariser.

[00:21:08]Then after this you have to look into the patterns the fragment patterns compared and analyzed for the micro community assessment as I've told you in earlier slides like this. Uh here you show the bending patterns 1 2 3 different types of bending patterns.

[00:21:24]Similarly you can see here in uh in this method righta and ardna. So amplified RDNA restriction analysis and this is called automated ribosomal introgenic spacer. So the only difference is that here we are using in Arisa we are using the interpacion and ard we are using the uh same uh RDNA sequence uh but we digested with the restriction enzyme.

[00:21:51]Now you can ask a question single recession enzyme is used for digestion give you different pattern. Yes this is the beauty of this this this uh technique. Single resection enzyme. If you are not using singular section enzyme you will mess up the whole experiment. You cannot have you know um find a variation these two and you cannot have any rule out the possibility of the whether they belong to the same are they belong at the different now arda research applications what are the applications of this of course application is a strain differentiation.

[00:22:26]What is a strain? A strain is something uh beyond the species. So species is uh I call them as a species species like e coli. So coli is a species under the escarishia you have a number of species but the single escarishia coli species may have thousands and thousands of different stain. Now these thousands and thousands of different stains differentiate through this method through ARD through ARISA through DGE TGE methods. So differentiating or distinguishing closely related bacteria.

[00:23:12]Now what we call closely related means within the E.coli you can have a multiple bacteria of the same species or same genre. So very closely related. So on the fogetic tree if you look onto the branches you find on the same tree same branch you find the ecoli or seduminas are capsula and these ecoli clapsular sedumas they are very easily detected if they varies in terms of like the mutations and changes through these ard ard and arisa method. So strain differentiation among the related bacterial species right with high precision essential for the texonomic and epidemological state.

[00:23:53]Now what is epidemological studies?

[00:23:56]Epidemological studies means if you go back in 2019 2020 21 and so on there's a covid pandemic. Now in the covid pandemic you find the different variants different uh you know omicron omega and so on so forth. Now these three four five different variants of the sars cove 2 virus were found to be epidemic in a particular region. For example, omeicron is epidemic in India, Italy and all. Similarly, epidemiology is a study where you identified a specific strain of a species belong causing a certain disease or infection or breakthrough right or outbreak and this outbreak is basically is responsible for some disease some infection from a single strain identified by this method. So this is called epidemological significance or taxonomic significance. Then we have uh environmental assessment. In environmental assessment uh comprehensive microbial diversity it's well well known analysis in soil composed system and aquitic environment.

[00:25:10]All these environmental conditions can be identified in their variation through these methods. complimentary analysis work synergistically with DGE and TGE methods for complete community structure characterization. So you can also make cross check between a RDA and Arisa for for for basically uh comparing the two other method uh we call DGE and TGE for this purpose.

[00:25:44]Now what are the advantages and challenges of these two method?

[00:25:51]Uh the key strength is high throughput capabilities for large sample sets. So you can have you know large sample sets can be uh easily identified through this method.

[00:26:03]Relatively simple methodology and equipment accident for a strain level identification. cost effective for routine screening and automated processing available. Now uh though these are the strength but I think uh one thing I would like to share with you is the skilled requirement. This technique required lot of skills because you need to have the designing parameters and then then you have to have the you know sequencing and then you have to have resection digestions and to run the gel. So it need a good practice and good high skill development uh for a technical background.

[00:26:43]Uh what are the challenges? See the required careful selection of a restriction digestion. Which recession enzyme you are using? Which size of the cutter you are using? Four cutter, six cutters, eight cutters. This is again very important. So usually four cutters are used for that limited phogenetic resolution compared to the sequencing because if you compare the whole sequence or the whole genome sequencing or the whole metagenome sequencing that's more advantageous.

[00:27:12]This has a limited uh scope of identifying because it is very small sequences. It could have a variations in some other sequences as well which you're not touching about. Uh it may need multiple enzymes for complete analysis. So you have one enzyme, two enzyme, three enzyme. So multiple enzymes are required. So again every enzyme you need to have kind of a you know every time you have to set the reactions and do the reactions and if you lost one reaction then your results will be lost.

[00:27:42]Now what is the workflow?

[00:27:44]Basically uh workflow for that is the same as sequencing PCR and then uh running the gel after the digestion.

[00:27:54]Now compared to that if you talk about the T RFLP we call terminal restriction pro restriction fragment length polymorphism. This is the very old technique we call RFLP restriction fragment length polymorphism.

[00:28:09]Now what is restriction fragment length polymorphism? It is the polymorphism of the different for example this is uh three sequences one 2 3. Now in these three sequences you have like this.

[00:28:27]Now if you cut these at three this side, this side and this side, this side, this side, this side and this side. Now in these three sequences they are same sequences but variation in the recession site. Now their size is same. The sequence is same but the restriction dation size have different positions due to mutation due to variation. This can be detected through RFLP. You just cut 1 2 3 you got 1 2 3 four bands. You got three bands. You got three bands. And these three bands of this three bands of this might be variation in the size that can be detected identified on the gel or bending pattern. But this technique uh we call terminal fos terminal recession fragment length and polymers is fluoresence based techniques more precise more highly detectable and very you know precisely a very good technique highly sensitive technique. What is done in this technique? The fluorescent PCR is done. So you have a fluorescent probe to be attached. So first PCR amplification using fluorescently labeled primers used. So when you use a primer for example this is bind use a primer here. Now this primer is a fluorescently tacked. Now this fluently tech primer bind and it amplify. So when the amplon is formed and you digest it you find a fragment can be detected through fllorosense microscopy through fuosense dye through different fllorosence techniques. So this is the advantage the fllorosence tag is remain attached then you can cut with the restriction dion 1 2 3 so you'll get a different fragments. So the terminal fragment you identified of different size sometime say for example suppose if you have uh okay suppose if you have this fragment this fragment this fragment you have primer design here and you amplify it.

[00:30:36]Now here is a tag one two 2 3. Now when you cut with the recession digestion one fragment is got of this size one got this size one got this size. So now you got a three fragment. One fragment is coming this size then you have this size fragment.

[00:30:54]Then you can have this size fragment. So first fragment is basically two. So when you run this three fragment on the gel you find variation in the banding patterns.

[00:31:07]This is smallest and that can be easily in earlier case like restriction fragment length polymorphism are in other techniques we are not using fossis probe. So we have to look into the visualization through naked eye and this visualizing through naked eye sometime fail to identify. But here you need a fluorescent microscope. You need fllorescent technique to identify the floresence probe attached here.

[00:31:35]that can easily it's a very very sensitive technique. So pattern is same principle is same but the tech technique is very very sensitive. Enzyme detection enzyme digestions produce terminal fragment varying length. See is one length second length third length then you have to forest ontoes and you find the different. So DNA sequencing generate you can also get a sequence 1 2 3 another technique. So when you sequence this fragment you find a different peaks you find different peaks like this we don't have a different color of pan but you find this kind of a peaks and these peaks are can be easily a a t c so on so this is how you can identify these peaks now fluosis help to detect easily and this DNA sequence generate a peak representing the community members 1 2 3 4 5 6 and so on.

[00:32:38]So within the species number of members identified through these sensitive techniques.

[00:32:45]Now uh the RFLP method overview fuosence primer PCR amplification I have just explained restriction enzyme digestion next step then capillary electrophorosis separation is one thing then also you can go for the sequencing uh automated detection and analysis and electrogram electrophoroggrams that is the electrophorosis that you run so the gel of electrophorosis can be checked into the gel doc or chemoc or denstometer whatever instrument you have you can check it and then you can interpret based on the banding patterns and the variation in the band patterns.

[00:33:28]Now what is the outcome of this technique process uh we call data output. So the method produce detailed electropheroggram with the quantitative information about the microbial community compositions and relative abundance.

[00:33:44]Now I give you the like this. So suppose if you run three fragment 1 2 3 four fragment. Now these three four frag fragments are basically of different size. So they will come at different patterns. For example like this. So this is called the electropheroggram.

[00:34:05]This electrogram will give you the clearer picture what these three band are and belong. Okay. Now the TRFLP method overview means like you have to look into the what are the overview of this method. Okay. I've already explained details. So this is just like overview and this is the data outcome. So this is the uh data outcome in terms of like a blending patterns right.

[00:34:38]uh terminal restriction fragment length polymorphism data interpretation like you can have x-axis then y axis so in x ais you have a fluence size measured like this x-axis the y axis so in xaxis you have a fragment size uh measured in the base pair you know 10 20 30 40 50 indicating molecular weight differences size of the 10 kb 8 kb 2 kb 1 kb 800 base press and so on so forth on y axis you have a fence in density like this. So like this.

[00:35:12]So intensity and which is relative abundance of community members. Okay. And then peak analysis. So these peaks were analyzed.

[00:35:22]How intense the peaks are, right? What is the size of these peaks? Where does the peak come? This can be identified.

[00:35:29]So usually people do through sequencing and the sequencing method can easily identify that. uh but also through fluosense uh techniques also we can use this peaks what are the applications of these uh with the case studies we can explain uh so TFLP or terminal fragment uh terminal restriction fragment length polymorphism or we also call it fragment length polymorphism is basically a lot of applications one is a spatial monitoring so tracking the bacterial community variation that is changes, variations, mutations both in viruses and bacteria across the different uh ecosystem, different niches like soil locations, agricultures, then you have water bodies and clinical samples and so on. And that also has importance of the clinical epidemiology uh in medical sciences. In agriculture, you can compare the transenic plant with the non-trazenic plant, change in mutated plants and so on and variation in types of crops, quality of crops.

[00:36:32]Suppose if you generate a disease resistant crop with certain markers can be checked with this method. Then temporal changes for example monitoring seasonals and treatment induced shift.

[00:36:43]So how the treatment um and monitoring the seasonal and treatment induced means the during seasonal and treatment you can change the physiology of the individuals could be bacteria could be fungus and plant also which can shift the which can shift the microbial population over time due to variation due to changes that can be checked also from different timeline longitudinal study different time scale different regional and so on so forth.

[00:37:12]The clinical application is that look into the epidemiology uh environmental agriculture clinical microbiology application worldwide to look into the epidemic mode or epidemic nature of these microorganisms through this techniques. That's the best method to look into the epidemology.

[00:37:32]Now the RFLP advantage and limitation we talked about uh in earlier cases also similarly you have a fully automated high throughput processing quantitative uh relative abundance measurement then high reproducibility sensitivity uh stable it's highly stable for complex community analysis for example if you have a you know environmental different community for example water bodies seage water hospital seage water domestic seaweed water. Uh you can look into a different time periods log log studies and find the variations through RFLP remain very stable. Uh statistical analysis is compatibility. So you can also statistically signify u you know significance can be checked statistical analysis can be done on that. Now what are the limitation which may sometime make the scientists not to do this technique or you know get rid of this technique and try to think something else the different texa different taxa may share identical fragment size sometime it happen there's a lot of overlapping of these taxa size genome size uh that is here that is where we can uh have to differentiate with a different technique now peak overlap Just like a band overlap in the case of gel then multiple enzymes often require for resolutions. So not like earlier you can have a multiple enzymes may also so for example you need to have one enzyme or two enzymes or three enzymes of different cutters can be identified.

[00:39:07]Then you can have requires expensive sequencing equipment also cost around 1 crores or more than two crores equipment depending upon what kind of a sequencer equipment you have. uh in spite of uh despite of the limitation of these uh this terminal fragment length polymorphism remain a very powerful tool very sensitive and very very uh good tools though it is very expensive but from research point of view it's very sensitive and very precisely uh which give very precise results data so micro community can be easily identified through these methods now if you compare these methods like DGE TGE ard DRA and ARISA and DR RFLP you find little variations and every method has its own merit and demerit and every method had its own advantage and applications. So for example in DGE TGE you have visual band patterns right uh in case of RD and array size we have the recession fragment length uh patterns and various the fragment of the enzymes used for the recession digestion. Then we have automated processing takes place in RFLP through sequencer band sequencing possible. So you can take a band and sequence that band for further analysis for mutations for variations. Uh superior uh strains differentiation.

[00:40:33]Then quantitative measurement right uh excellent for dominant texa right uh which is not in case of uh RFLP. Then you have a cost effective analysis right in this case of ARD and ARSA we have high throughput capabilities available.

[00:40:52]then you have a simple methodology right then in case of TR RFLP we have high throughput analysis and statistical statistical compatibility so these are some applications and strength and you know uh things uh attached to these methods uh to understand the microbial diversity uh quickly we move to the which is the right method so if you look the when you're doing research in the lab are as a as a master students or as a UG students and look into the projects or to identify the marker to differentiate between the microbial diversity. Uh so which method to be opted right then you have to look into to observe first the what is your hypothesis so look into the sample complexity which sample you are working on. So consider the community diversity level and expected the species the niche uh richness in your environmental for example if you're taking the wastewater treatment plant you're taking the environmental plant if you're taking the wastewater uh bodies or if you're taking the rivers and ocean bodies there you have to look into the diver chances of the diversity is more so you have to look into what is the complexity of the genome right then research objectives what are the research objectives so determine the need for the Texonomic identification versus the community fingerprinting analysis. So whether you are going for the taxonomic identification or for fingerprinting or the molecular analysis right so what kind of experiment you are preparing what is your hypothesis right so that you have to look into before you pick up the method that you are working on then laboratory resources so whether you have sequencer or not you can't go for uh terminal fragment length polymeri you cannot go for sequencing so look into the what are the do you have a PCR if you have a PCR you can do it. If you don't have a PCR, you can't do it.

[00:42:41]Right? So, look into the look into the laboratory conditions and infrastructures and physibility of these experiments, right? Accordingly, you have to choose the method. Not you should not go to directly to the merit and demerit of the method. But to look into the infrastructures and the facility that you have budget constraint suppose if you go to TRLP or ARISA the budget required is very high. It's a very expensive method. You have fluoresence probe, you have a primary design, then you have a sequencing and so on. So all these things are important taking into consideration before you choose the method to be used whether it is a DGE TGE ard or Arisa or terminal length polymorphising whatever method you are using for these mark identification you have to look into the constraints budget equipment facility also is skill technical support and what kind of assembly you are working on what is the complexity of your samples right whether it is a clinical sample Whether it is a environmental sample, whether it is some other divers diversified microbial samples, whether it is a gut micro microbiome, it is a vaginal microbiome or it is a oral microbiome.

[00:43:48]So, every microbiome has a different complexity. Accordingly, you have to choose the method. I think this is all uh with this uh I'm closing this uh talk today and we'll meet again. Thank you very much.