So I took mere fine chin because someone asked me to and I hope you like the like to break But welcome back. So we will be talking about secondary structures and a little bit more but The thing about primers is is that they work in pairs, right? Yeah, I'm recording. So I press record So primers work in pairs. So you have a forward and a reverse primer, right? And both are used in the PCR reaction So you need to ensure that and besides them not being able to either hybridize to each other You also have to make sure that they are suitable for the reaction And that means that the melting temperature and their annealing temperature cannot vary too much You cannot have a forward primer that is 30 base pairs long has an annealing temperature of like 63 degrees and a reverse primer which is only 20 base pairs long and has an annealing temperature of like 50 degrees because that won't work. So the critical feature here is is that if you are designing primers Then their maximum difference in annealing temperature is Kind of three degrees Celsius if there's a bigger difference than three degrees Celsius This is not going to work So the closer their annealing temperatures are to each other the better your primers will work And generally you want to be within three degrees Celsius Because if you're like outside of three or if you're like five or six degrees Celsius difference Then your PCR reaction might still work, but your yield will be very low So had the amount of DNA that you get will just be lower than what you would normally get All right, so very basic summary of primer design is Make sure that your primer is unique that it only binds to the target DNA that you want to amplify and not to Other DNA which might be floating around and especially human DNA if you're not working on humans So if you're if you're working on on plans make sure that your primer does not bind to humans The length of a primer needs to be between 17 and 28 base pairs But this varies and you can go a little bit longer and you can go a little bit shorter Of course the shorter you go the less the uniqueness will be or the harder It will be to find a primer which is unique The base pair composition needs to be around 50 to 60 percent You need to avoid long stretches of A's and T's and G's and C's so you cannot really amplify like repeats in the genome You need to optimize base pairing to minimize false priming So like I told you guys you have to have a low stability at the three prime end And this is just because the polymerase won't work when the D are when the primer is tightly bound to the to the template DNA The melting temperature of a primer needs to be between 55 and 80 degrees Celsius Generally, you don't want to have it above 70 degrees Celsius But like as a rule of thumb if the melting temperature of your primer is between 55 to 80 degrees You should be able to have a successful PCR reaction Because primers work in pairs You have to have their annealing temperatures to be very similar But never design or never order a primer pair When the difference between the annealing temperature is more than three degrees Celsius And you have to minimize internal structures So you have to avoid hair pins and dimers, right? So they those should not occur a primer should not be able to bind to itself It should not be able to fall back on itself because that will just make your PCR reaction not work alright, so Advanced primers so advanced primers are primers where you are doing multiple things in one go For example, if you use multiplex PCR Then you are using primers to not just amplify a single region of the genome, but you're amplifying for example multiple parts of the genome Or you're for example, trying to amplify not just one virus, but a whole family of viruses You have universal primers you have semi universal primers and you have gasmers So we will go through these four primer structures order to these four primer designs And this is relatively advanced and normally you would not be able to to do that, especially gasmers nowadays like I think that no one Really designs gasmers anymore because sequencing is so cheap that you don't have to guess anymore However, um in the past, um, I designed a whole bunch of gasmers, which is really fun Alright, so multiplex PCR is when you have multiple primer pairs in the same tube, right? So you want to amplify two parts of the genome? So you want to amplify gene x and you want to gene amplify gene y in the same go? um and Yeah, I'm sorry commando. I think it broke. I think you can't use the wizard command at the moment Let me see if I can if I can reset my overlay Let me let me do that Oh, no. Yeah. Yeah. No, I made a coding box every time that I that I go away Yeah, yeah, don't don't throw it all in the chat then like wait for me to reset the thing.  All right. So I turn it on again. So now It should work So does it work? Yes, it works. So now you can use your your wizard and your hard eyes and these kinds of things again I like it. It's just so silly, but it's it's it's something which is fun All right, so I have multiple primers in the same tube So we do this sometimes and we sometimes use the three primer systems where we have A primer which is in the middle and if there's kind of a deletion then this primer cannot bind But multiple or multiplex PCR is very common Especially in sequencing projects where for example, you're not only interested in a single gene But you're interested in like two or three genes at the same time And you want to amplify these two or three genes and then want to sequence them afterwards So the application for using multiplex PCR is genome identification So for example, the the genetic test panel that is being used by the police Also targets multiple parts of the of the of the genome So here what they what they do is they have a primer mix of 20 known primer pairs And the distance between these 20 primer pairs is slightly different for every individual So some individual have a length of 50 between one and two Other people have 60 and this varies for each of the primer temperatures So the main design difficulty when you do multiplex PCR is to have to make sure that The melting temperature should be similar for all of the primer pairs that you are using And of course the dimer for me The dimer formation Will be much much More common right if you have two primers then the chances of two primers binding together is relatively low But if you're using 20 different primer pairs Then you have to check each primer against 39 other primers to make sure that they cannot form a dimer So it's it's just uh, it's just more Involved in making a multiplex PCR But in theory multiplex PCR is nothing else than just having multiple primers In a single go so instead of amplifying one part of the genome You're amplifying two or three or four parts of the genome and this is very useful for genome identification A universal primers is normally primers are designed to amplify one product But when you are dealing with universal primers, um, you can, uh, amplify multiple products And we call such primers universal primers and for example, uh, one of the main Usages here is to amplify all different huma and papilloma virus genes Huma and papilloma virus like the flu, um, has very different viral variants So there's not just one virus, but there's happy if a one two three four five six up until like 36. I think Um, yeah, so you have all you have all kinds of viruses, which all look very similar. Um, But they are slightly different Right, so the strategy here is that you have to align your sequences that you want to amplify And then you find the most conserved ends at the five and the three prime end Then you design a forward primer at the five prime conserved region Um, and you do the same thing for the for the three prime end You match forward and reverse primers to find the best pair And then you have to still ensure the uniqueness in all template sequences and you have to ensure Uniqueness in positive possible Contamination sources. So, um, how does this work? Fortunately, I have a board, right? So we have for example, the huma and papilloma viruses And so we have for example virus one, which looks like this and then we have virus two Um, which is very similar, but it has a small deletion in the middle Right. So when we align it to the first sequence, then there is no sequence in the second one And then in the third one we see the same thing But now it has a bigger deletion at the end And for example, we want to amplify a couple of these and then we have to find the region at the beginning Which is shared between all the sequences We have to find a region at the end which is shared between all the sequences And we can then design a primer a forward primer here In this region and we design a reverse primer here And of course this primer can bind to all four this primer can bind to all four So now we can amplify any of the four hapeve viruses that we are interested in Is that clear? It's it's just the same But now we're using a single primer pair And we first have to align all the target sequences with each other To find a region or two regions one region in the front one region in the back Where we can more or less target our primers. I hope that it's visible Actually, otherwise have to switch to full screen and then the overlay will kind of get worked again But so let's not do that But that's making universal primers You have semi universal primers, which is the same but now you want to have for example You want to only amplify The first six human papilloma viruses, but not the other one. So you don't want to target number seven So again, you do the same thing. So you have to align all the all the hapeve genes So you have to align every all of these viral genomes to each other And then you have to identify a subset that are more similar to each other than the other subsets So in this case, we want to look at type one to six And then we want to find the region which is conserved and then we have to design forward primers here And so I imagine that we would have a third sequence or we have now one two three and four So imagine that we have number five Which is slightly different So it might have like a little deletion here And it might have like a large deletion at this point, right? So what we want to do is we now want to find a region for the forward primer where the first four are similar to each other But the fifth one cannot be similar to the first four And so here we are kind of doing the same thing But now we are looking to find a primer Which is only amplifying the first four and this quickly becomes very difficult, especially if you want to Do multiple genes in the same go So if you use semi universal primers and you combine this with for example a multiplex PCR Then it becomes a real puzzle to figure out where you should exactly target your primers and How they should look like to kind of avoid amplifying sequences that you are not interested in But these are called semi universal So the strategy is more or less similar to the universal primers But now you don't only want to identify the region at which everything is similar But now the other virus variant should be different at this point So then you are talking about a semi universal primer My favorite primer design is the gesmer and the gesmer design is when you do not have a dna sequence available Right, so if if you are working on a species, which no one has been working on before so there is no There is no No template dna available. You don't have any sequence data available But you you do know that this animal for example has a certain protein and you want to kind of Amplify this protein in this unknown species. So here we are using the homology trick again. Yeah, because we know that for example Hemoglobin is not that different from humans to mice Imagine that we do not have the genome sequence available for mice and then we would use the human sequence for the gene of interest so for example like hemoglobin So we use the human hemoglobin sequence the protein sequence And then we design primers based on the human hemoglobin sequence Of the protein and then we translate that back, right? So hey in case we are interested in Hemoglobin not only in mice but in other species which do not have a genomic sequence available And so what we then do is dna sequences are unavailable A single group of related proteins can be back translated into nucleotide sequences And then this will be used as a template to design our primers Translation from proteins to dna is possible, but it has its problems because there will be There will be slight differences, right? So Like I told you guys every amino acid is coded by a triplet But the last base pair right the last base so this one no this one for you guys I don't know. How does it look on stream? Is this the last one or is this the first one? I don't know But when you have a three base pair codon then the third base pair in the codon is called the wobble base And this is more or less free to choose right because of the way that the That the ribosome works So every like third base pair you could make an error You could say well, I'm targeting a c but the animal that you're looking at is actually coding a t there So what you have to do then is to either know the codon bias of the animal, which you probably don't But here you can use that to back translate So here we are designing then based on the protein sequence of another animal Which we know the protein sequence of and then we use that We use the codon table to go back from protein sequence to dna sequence And then we design a primer based on this hypothetical dna sequence Of course, there might be bugs there or had there might be mismatches. So we have to make our primers longer Yes, so we back translate the protein sequence using the corresponding codon table Had we identify five prime regions and three prime regions where we're most likely to not have made a mistake And then we design and match forward and reverse primers as before And but now we we make our primer around 30 or 35 base pairs long had just to avoid that if there are any Nucleotides that do not match that these mismatches can be Can still work right because the primer can if you have a primer, which is 30 or 40 base pairs long Had then as long as like 30 out of 40 base pairs bind It is still possible for the dna to bind So it's still possible to get a a product Um, yes, so, um You use longer primers not only that you use a slightly higher annealing temperature Because of the slightly higher annealing temperature you increase the primer annealing stringency So it it becomes a little bit more stringent But gasmers are really fun to make Because there's an additional step of going back from because you go from protein to dna code And then based on this hypothetical dna code you are starting to design your primers and This is an iterative process. So you do you have to do it a couple of times You're never getting it right in the first go so But it's very fun to make gasmers All right, so have primers can be designed to serve a multitude of purposes You can do multiplex pcr you can design semi universal primers You can design gasmers and there are actually many many different other Strategies to design primers for different different solutions Hey, but there's many different fields where primer design skills are required For example, if you are going to do real-time pcr For example measuring corona viruses. Hey florian, welcome to the stream How's it going Do you have any mood that you want to share? Bad, uh, still bad Well, we're not going to talk about that but Just uh, just throw something so if it goes bad, then what do we have for that? We have the kind of intensive emoticon for you then so you can you can throw something like this in chat and then Then you look i'm using the overlay now All right, so and there's many different fields where primer designs are are necessary Especially when you work in a lab Head then you have to design your own primers Normally for phd students Well, you have to type it in first uh florian So just just type in a word with like capital letters like me like pensive And then you will be added to the mood box on top of me All right, but many fields for primer design skills are required So real-time pcr population polymorphisms where you target microsatellites or aflp or snips Too old for twitch. Yeah, i'm too old for twitch as well, but i'm still doing it But the basic rule in every primer design that you do achieve the appropriate hybridization Specificity so make sure that your primer is unique It can only bind to the dna of the target species and not of any contamination sources and make sure that there is enough Stability so make sure that your primer can bind and can bind properly And that the three prime end is not tightly bound to the dna Because that's one of the reasons why most primers don't work Is because the primers just bind to the template too strongly for the polymerase to be able to kind of extend the dna All right, so searching in databases um because hey, of course, we we have to deal with the databases So had databases are genome browsers Hey, we are going to look at ensemble How do we find our genomic location that we want to target our primers on and how are we going to export our sequences? And I was initially wanting to do a live demo, but I just put some of the screenshots in the slides because otherwise have to switch between The firefox and the normal window So of course when you want to design primers, you need to be able to figure out what part of the genome you want to target So you can do this using a genome browser Um genome browsers like ensemble or ucsc. They visualize genetic information, right? So there's you have a genetic sequence and these this genetic sequence codes for different um different Different genes right or different micro RNAs So what a genome browser does is that it takes the genetic sequence and then adds Information on top of that sequence saying that well at this region. There's a certain gene Here's an axon here's an intron and then there's an axon again And this is the promoter region And so it allows you to use different scales and you can zoom in and out in a genome browser But everything here is based on a coordinate system. So the coordinate system that we use in in in genetics Is not fixed in a way And this is a little bit annoying because some genome browsers ensemble The first base pair is base pair number one while in ucsc the first base pair in the genome So on chromosome one base pair one is coded as being zero So and the first thing that you have to remember is that there can always be like a shift in where the the genome starts But the idea behind the genome browser is just to integrate different information sources So to have an information source Like a protein database, which is then incorporated and shown on top of a DNA code, right? So you have your DNA code and you have the the Introns and axons then you have the protein level on top of that and when you go to ensemble, this is the way that they present the information to you So very basically how does a thing work? Well, you have your lab experiments which are fed into a computer Then of course you get some textual data or xml or json have representing your experiment and people put this data into a database Of course, you don't have one database, but you have many many different databases like ensemble pdb PubMed Hey, you have medlin and all of these databases. So all of these different databases They have their own web services and you with your laptop can use all of these databases or can reach these databases via the different web services Yeah, so you go to a website and you use it or you use an api So hey, you connect r directly to the database and to queries directly to the database So when you choose a database for your research you have to know The availability and how up to date it is So have which organisms are in a database and the ensemble database is for many many different organisms But there are databases which are unique to human or which are unique to mouse or which are unique for livestock species But you you you have to when you choose a database for your research You have to make sure that your database is available and that it's up to date And this is one of these things that goes often wrong because in the end we want to have Reproducible information and reproducible results And often many databases do not provide access to like the old data sets that they have And so they for example used to have the information based on genome build number five But then they switched to genome build number six and then to genome build number seven But when you publish your paper and when you did your analysis you did that based on genome build number five So the database needs to have old versions available Right, so you have to have either a backup of the database yourself to redo your your research Or you have to have a database Or a data provider which has old data sets available. So fortunately ensemble is very good in this So had the ensemble database every time that they update their database they they keep their old database available And they have like this this Structure where you can just say well, I want to go back to the database As if it were 2016 right because then you can redo your your analysis In the old days and that this is becoming less and less important is that the location of the database was very important Um, if you are located in europe then a database which is located in Japan or in china is of course far away And especially if you're if you're dealing with a lot of data transfer, right? If you want to download a whole genome sequence, um, and then you're talking about like two gigabytes of data And transferring two gigabytes of data is easier when the database is physically located very close to you Nowadays with Things like the amazon web services have which are available all over the world the same as google data centers And had this becomes less of an issue because many databases are more or less replicated all over the world And had these these databases are more or less always close to you But like five or ten years ago. This wasn't that easy. So if you would use something like keg Nowadays keg is really really good because hey, you always have a local version Which is hosted on a computer more or less in your own country or within your own like zone of the world But it used to be that keg would be only hosted in japan and then hey if you would go to the database Then that would take time and downloading data would be really really slow So databases when you choose a database you also have to look at which software they have available Which analysis tools they have available, but in the end choosing a database is more or less Hey, you have your own personal favorite or your own personal flavor of database that you like Like i am someone who uses ensemble a lot But i know a lot of the people that i work with they prefer ucsc So that's they contain more or less the same data But a different coordinate system And slightly different organism and so your personal flavor and the flexibility of the surface matter when you when you choose a database And like i said, you have many different. So hey, you have the ucsc database which you see here You have map viewer Oh, no, this is map viewer here. You see ucsc and here you see ensemble and all of these databases look slightly different They have very similar information in there But choosing one is very up to you and you can choose one and someone else chooses another one But then you run into the issue that sometimes you have slightly different coordinate systems So ensemble starting with base pair position one while ucsc starts with base pair position zero So that will that will have a gene start at three thousand in ensemble and at three Are two thousand nine hundred and ninety nine in in ucsc. So it creates some issues So ensemble is the main database that that i am using So i will be using that today for your overview when you go to ensemble it kind of looks like this It doesn't really look like this anymore. They they change the database, of course every so often So when i made a screenshot like two years ago for the first presentation that i did it looked like this But the thing is is that if you for example click on a species So you select the species you can go to the assembly information and here It will tell you all of the information that you kind of need to know But the most important information is this line So hey here you see the assembly that we're currently looking at so we're looking at b tau 4.0 Which was published october 2007 so when you are writing a publication and you are saying that we use the ensemble database for boss Tautus then you have to add this information to your publication Because people need to know exactly which genome version you are working at because genomes get updated Not very frequently but frequently enough that you have to mention which version you used because every new genome build Will see changes to where genes are located has sometimes the genome becomes a little bit longer Because people were able to sequence a part which was not sequenced before So you you have to mention the assembly that you're currently or that you are working on when you write a publication And you also generally mention the database version that you used So these two are more or less the most important ones that you have to use in in a publication So when you search an ensemble very easy We already did this you can search by gene symbol by database id or position in the genome And so they they give some examples here like you can search for the gene name or you can Search for just a single term like prion and searching in databases becomes easier and easier Because they are more or less And they're getting smarter every day So searching by gene symbol used to be a pain in the ass because genes Symbols are not standardized. They used to not be standardized For humans you have the h c n c. So the human gene nomenclature committee that assigns Names to genes Um, has so a gene symbol is an approved name which is approved by the committee for a verified human gene um And of course this has a massive a massive advantage has so it is a unique reference for all for a genes In in scientific articles. Hey, you can easily search for a gene in a database And you can actually identify several gene families, right? If i'm searching for sip and then a number had then these all point to cytochrome Genes if I search for hawks, um, which are genes which control like development of different types of body tissue And these are all homeobox genes So there are genes in the genome which which kind of shut down another part of the gene And it it allows you to clarify orthologous genes in other species This did not used to be the case It used to be the case that a single gene could have up to like 15 different names. So someone would call it bbs7 Other people would call it differently. Um, so let me let me just go quickly to ensemble and show you the Or not show you but tell you the diversity in a certain gene So how bbs7 is one of these genes that we've been working on a lot Um, yes, so can I show you firefox? Yeah, there we are Um, yes, so if we look in ensemble for bbs7, let me scale this up a little bit for you guys Um, not too much actually So here we see, um bbs7 right, so it's called bbs7 now, but it used to be called 8 4 3 0 12 4 0 6 and 16 R i k So this and bbs7 are the same gene It's now called bbs7 based on the human gene nomenclature committee, right? Because they decided that all of these beadlet part alert associated syndromes associated Jeans should be called bbs But it used to be called this and and this holds for for many genes and especially genes which have been studied a lot They sometimes have five or six different names That pop up in literature and then of course it becomes very hard to kind of understand Which gene people people are talking about if everyone's using their own name But that fortunately got better when the human Well the human community so the community of human geneticists came together and decided to form this human gene nomenclature committee Yes, so it has many many advantages So here when we want to search for a gene we can for example search for abcg2 Which is a gene which is involved in milk production in cattle Hey, so it's called boss towers at the bay binding cassette. It is located on chromosome 6 in cow And this is based on the chromosome um umd3 coordinates so In the boss towers 4 genome build this gene might be located somewhere else might be on a completely different chromosome The name of the gene is a p a abcg2 And in this case, they don't have any any synonyms. So fortunately just gene only has one name And so when we search for the gene here on the side, we see all of the different options that we can do So one of the things that we can click on is for example click on this ortholog gene right that will show us the orthologs or the the gene in different species When you click on that and then you see here that that there are many different animals in which this gene occurs It highlights the gene that we that we had selected and you can see that when we look at this abcg2 gene in cattle And then the closest known related variant is the the same gene in sheep And then the next closest relative is um in dolphins So and genetically you would not think that cows and dolphins are very Related, but they actually are cows and dolphins and secesians and like Whales and dolphins and so all of the Mammals who are living in the sea currently They are very closely related to cows So and that that's something that you can learn when you when you have a database filled with all of these sequences for different genes and for different genomes All right, so when we when we look at this abcg gene and we go back to the main gene page Then you see here that this gene has two transcripts. So there's two different splice variants, right? That means that um when you click on the show transcript table you you get this little overview And here you see that there are two different versions of the gene Both code for a protein which is 658 amino acids long But the length of these genes are different on the genome. So this means that this is a gene which has a Which codes for two kind of different proteins. They're the proteins are more or less of similar length But they are not the same proteins. So here we have a situation where in one case Certain axon might be skipped or there might be um an intron which is included So transcripts so every gene can code for an n number of transcripts So an n number of proteins that are being made And so here we see the two different transcripts and so we can see that both of them start more or less at the same position habit the first Gene actually skips a whole part of the genome and then the first axon is located here While the second version of this gene already has its first axon very much Closer to the gene right so we can see that some of the some of the axons are shared Right. So this part is always included into the abcg two gene However, this part is only available in variant number two and it is not available in variant number one And so although they code it's the same gene Coding for two different proteins and these proteins are coded very differently But of course there are axons which are shared and there are axons which are unique to one of the two gene variants So these transcripts and you can just get them from ensemble And so there's a lot more information from example So you can go to the external references where you can go to either literature or uniprot to show the the protein sequences Or the protein domains You can go to wiki gene which has some information about the gene in a in a kind of wiki-pedia format And you can go to genomic alignments here You see the location of the genome so you can you can look at the gene on sequence level And then here when you click on this phenotype button, then you have the phenotypes which are associated with this gene Yeah, so this is um the part of ensemble which is built up by cutiel analysis, right? Because cutiel analysis allows you to associate a region of the genome with a certain phenotype And so here there are all these associations will be there And then you go to the variation table to show all of the known single nucleotide polymorphisms inside of this gene So the uniprot database we already saw it before but the nice thing about uniprot that it has a very kind of clear description of what the protein does and so Had this high capacity urate exporter functions in arena and external urite secretion plays a role in polyform homestations enables to mediate the export of Ppix for both from the mitochondria. So it gives you an overview of what the gene is doing had the known snips Generally when we are talking about snips in the in the in primer design Of course When we know that there are snips In this gene then we do not want to target the primer at the location where there is a snip And so the snips that we are most interested in are the snips which are the the miss sense variants Which are changing the protein, but we also need to account for not for for standard variants so so Snips single base pair changes into the genome because this will affect the hybridization of our primer And we can imagine that if we work on two different species of cows So I am working on holstein and someone else is working on a road bontus fleck fee or or another cow species head and of course then the issue comes in is when these two cow Species have or cow breeds have very different snips So very different or relatively different sequences then a primer pair might work in holstein But it might not work in in one of the other species. Yes So when you are designing primers always make sure that your primers are not targeting a region Where there is a single nucleotide polymorphism or where there is a known deletion. So And you can find that in the snip table, which is located here in the variation table All right So if we look at a certain gene or if we look at a certain snip for example this snip here Then we can see it is a miss sense mutation meaning that it changes the amino acid structure And then we can use db snip to get more information about what is exactly changing Yes, so when we search for this snip Yes, so when we want to get the region for a certain gene For a certain snip so imagine that we want to pcr out This part of the genome which has the snip in there, right so that we can send it in for sequencing Imagine that holstein has the reference allele and another Cow breed has has got a different allele and then we can target primers to amplify this part of the genome And then by sequencing we could figure out if The animal in in has the holstein allele or if it has the rhodopontus fleck v allele Yes, so We can search for a certain snip Then we choose the region in detail button And then we have to verify that the snip is is in the picture Or that we can see the snip there and then we can export the data To a fusta sequence and now we can use this fusta sequence to create primers Had to extract this piece of dna for sequencing or other Things that we might want to do with it so If we are designing primers There is one big disadvantage and that is that there are repeats in the genome, right? I told you guys that when you when you design a primer your primer needs to be unique Of course Repeats are up to around 50 percent of the mammalian genome So if I just look at a a region in the dna and I just randomly select like 50 base pairs Then the chances that this 50 base pairs occur somewhere else in the genome is around 50 percent Right because every every if you just randomly select a part Then there's like a 50 chance that this part will be in the genome twice And so pcr primers cannot contain repeats themselves So and we do not want to target primers in these areas which are repeated So we need to get rid of them and you can use something like repeat mask or for that Nowadays in ensemble. You can also just repeat mask it when you export your sequence So let's show you guys since All right, so hey imagine that we Let me make this a little bit bigger so that fits a little bit better into the screen A little bit more a little bit bigger here right, so imagine that we want to Go for this this one snip which was in the presentation. So the snip was called Let me move this to the side so I can see so the snip was called rs 4 3 7 0 7 0 2 3 3 7 All right, so we just search for this snip and then here we see that it's a cattle variant So it's it's a it's a snip which occurs in in cows All right, so we just click on it And ensemble is relatively slow at the moment, but here we have the rs snip, right? So it says that the reference genome has an a and some animals have been detected which have a t What does it do? It's located at this point in the genome And this this And this this is the name of the variant and it overlaps seven transcripts. So it does affect the coding of the of this gene Of this abcg 2g So what we can then do is of course we can now say Head that we can now Export so we go to region in detail So where is it region in detail? It doesn't have the region in detail anymore. It's so nice that they changed the database I think it's now Yeah, it's probably called genomic context. All right, let's click on it We have to wait a little bit for the component to load And then here we see that this snip is located here. So when we hoover over it Oh, you don't get the pop-up because it's not capturing the the pop-up, but here at this position The the snip is located so it's Can we zoom in a little bit? No, we can't zoom in allowed to zoom in So here we see the snip located in the middle and then one we want to go and get the region for this snip And then we can go to the primary assembly to the location And we just click on it here, right? So then we go back to the standard ensemble website Where again it loads in this component that kind of shows where it is located And so we can see here that here. There is this snip that we were interested in And just yeah, so now you see it. So here you see our snip, right? 4 3 7 0 2 3 3 7 It's located exactly here And you see also here that there's an overview for all the phenotypes that have been associated through qtl mapping And so we can see for example that this is This this snip is associated with Difference in milk protein percentage. So having a certain variant of this snip increases or decreases your milk protein And it also Increases or decreases the milk fat, right? But when we click on it, we can see that okay So this thing is in the middle now And so we want to export the sequence here so that we can design two primers to pcr out For example this piece of the genome, right? So we can then go to export data Right and then we have to Make sure that we we want to pcr out A part of the genome and the snip is located at this exact location So what you would normally do is say well if you do sequencing then you can sequence very cheaply like 200 300 base pairs So what we want to do is we want to go like 100 base pairs in front of this one. So we say, um, oh we go from Let me reset that five six. So it actually oh no, it's not in the middle So it's already in the middle. So it already selected 100 base pair region Where the snip is located at position number 50 So but I want to make the region slightly bigger So i'm going to say well go 50 base pairs earlier have give me not a sequence Which is 100 base pairs long, but which is like 200 base pairs long So I want to start 50 base pairs earlier And then I want to say 7 4 to be 50 base pairs later, right? Then we say next And then we say well, we want to have it in text and then it opens up a window where we have The primary assembly now we have to make sure that we find our snip in this sequence Um, yes, so if we would go back and we would zoom into this snip then we would know that this snip is located at exactly position Um 640 So 640 so that means that it is 100 base pairs Before the end and 100 base pairs after so that should be perfectly fine, right? But we we do want to check that the location here is exactly in the middle of the location here All right, so then the next step would be is to now do repeat masking on this sequence So we just copy this sequence and then we go to uh repeat masker So let me open that up. Can I just click on it here? Yep, so it here we go to the repeat masking website Here we take our sequence of interest that we want to design primers for Um, yeah, so we go here we put in the sequence And then we say well, we want to use um had the dna source in this case is um Mammal other than below Had the sensitivity is going to be default and well, we just leave everything in standard So we just submit the sequence and then it will start repeat masking our sequence. So it will look in this 200 base pair sequence if there are any repeats And we just have to wait until it finishes And so it looks for simple repeats and then have full length and disperse repeats So then it just goes through all the possibilities that what what might not be suitable for primary design Yes, so it takes a little bit little bit of time, but It should be to refresh it Just to make sure and it says no repetitive sequence was detected. So that's really good So in this case this region of the genome has no repetitive sequences And then here we can go and it says no repetitive sequences So we can just use the sequence that we have as if Because there are no repetitive sequences But you always have to do this step You have to make sure that there are no repeats because you you can't design primers based on the repeats All right, so then the next step would be to start designing a primer And because now we have our sequence we know that our snip is exactly in the middle And so that means that the snip is somewhere around so this is 60 So then 40 base pairs in so it's around here where our snip is located So we can design a primer all the way up to where our snip is all the way So we have a forward primer that we can design here and a reverse primer that we can design here All right, let's go back to the power point So make sure that you always do the repeat masker step Um, again, how does it work? So you take oh, I took a thousand base pairs. So let's go back because Then I can show you that there are a lot of repeats. So we go to primary assembly, right? We take the location of this snip So we just go to export data We say we want to have the location of the snip location of the snip and we want to have a thousand base pairs upstream And a thousand base pairs downstream.  So now we get a much much longer sequence than before So we take the much longer sequence and again, we are just going to first go to repeat masker and make sure That there are no repeats. So we just put it in Other please specify Well, we're just going to say other than below. So it's a mammal All right, so we just submit our sequence We'll take a little bit of time not too long. So again, no repetitive sequences detected Why is that because in the original one, why does that not work? That's strange because there used to be a repetitive sequence in here Did I take another snip compared to the one that I was looking at? That might be that the genome build got better. Um, that is interesting Let me try this once more. So I'm not going to use repeat masker. I'm just going to hard repeat mask this region Um, so that that should be and then we go to look at text Yeah, so actually ensemble does find a repeat, right? So the repeats here are these regions where it says nnnnn So ensemble nowadays has its own built-in repeat masker Which is a little bit better than the repeat masker that I point to in the in the slides But here you can see that if we would design a primer without masking the repeats And we would pick a primer which would have been located here And then of course our primer would not have worked because it would be binding not only this sequence But also other sequences in the cow genome. So let's just use this one then for For future reference, right? So again our snip should be in the middle And of course we need to make sure that we check that as well All right, so let's go back and Disable firefox. So this is what it used to look like when I when I ran it with the old version And then it would take the DNA sequence and then here you would see that there would be a big repeat Which was also in another place in the genome All right, so when we design primers and primer design you can do by hand So it is possible to design it by hand, but you can do it much better by a computer And so a computer is much better at designing primers and there's like things like primer three Have which is a web application. You have oligo, which is also a standalone application But there are like literally dozens of tools to do primer design. So have where You can select any tool that you want And you can calculate the melting temperature using for example the promega Calculator to calculate your melting temperature for your primer to make sure that your forward and reverse primer are Of suitable temperature, but nowadays programs like primer three will also do a first prediction of the melting temperature So we don't have to deal with that the recording time. Yeah, we're at 52 minutes. So i'm just going to continue Um, yeah, so an example of primer three Um, yeah, we will do that after the break. Um, I guess you're just interested in getting the uh Getting the animated gifs to cheer up a bit Um, let me change at least my um My my my mood box. Um, so I will be doing this one Yeah, all right, so I will stop the recording and we will be back in like five to ten minutes. Um, so I will


Welcome back. So we'll continue where we stopped. So chapter eight, we were looking at the giving. So two things that Paul asked the believers, or he asked them to notice about the Macedonian Church in their giving, was that they gave in spite of their poverty and they gave in a way that Christ himself had given of himself to the church. We see in Philippians 2.5 to 8. Let this mind of Christ be in you, which was also in Christ Jesus, who being in the form of God did not consider robbery to be equal with God, but made himself of no reputation, taking the form of a burden servant and coming in the likeness of men. And being found in appearance as a man, he humbled himself and became obedient to the point of death, even the death of the cross. And so we see where Christ himself emptied and gave himself fully for us in that same way where the Macedonian Church was giving out for place of not that they were having abundance, but even in their poverty they were able to give to the church in Jerusalem. So we go on from there to those last few verses that we were looking at. So the last verse, verse 15 says, he who gathered much had nothing left over and he who gathered little had no lack. Now that is a reference to Exodus 16, 16 to 18, where the Israelites are instructed to go out and collect their mana for each day. And this is the instruction that's given to them. If someone can read Exodus 16 verses 16 to 18, please. Exodus chapter 16 verses 16 to 18. This is the thing which the Lord has commanded that every man gathered according to each one's name. One oma for each person according to the number of persons that every man take for those who are in his tent. Then the children of Israel did so and gathered some more, some less. So when they measured it by omas, he who gathered much had nothing left over and he who gathered little had no lack. Every man had gathered according to each one's need. So the principle here is important, right? So each of them had as much as they needed. And if we see in verse 16 where God commands them, he says, gather as much as you need. Take an oma for each person you have in your tent. So God had given them a specific quantity that they were supposed to take for each person. So they were not going to go collect as much as they could or they were not going to collect any amount of food for each person. They had a specific amount. God knew how much each person needed. And so if there were five people in the family, they had five omas. Three people in the family, they had three omas. So to say that you take as much as your family needs. And so the same principle is what Paul is talking about here. Church or each believer should have as much as they need so that there is equality within church. Don't give more than you can give. So don't reduce your whatever you possess to the extent that you have a lack and you're giving so much to others. Rather keep as much as you need and give to others out of the excess or whatever extra is there to. Share with others. And that is the goal to have equality. Another thing if someone can read Ephesians 4, 15 to 16. Ephesians 4, 16. 15 and 16. Okay. But speaking the truth in love may grow up in all things into him who is the head Christ. From whom the whole body joined and knit together by what every joint supplies according to the effective working by which every part does its share. Causing growth of the body for the edifying of itself in love. Amen. Thank you. So we see an Ephesians 4 where Paul talks about the church being like a physical body and each part brings something to supply to the rest of the body. Each part does its share. Each part contributes to the growing of the body and for the growth of love within the body. So in that same way we look at this teaching on giving is that each local body also supports one another so that we are all growing together as the body of Christ. And we're all growing in love. We are all growing towards Christ like this and we're all able to share and build each other up. So that's the beautiful thing about these churches as well is that they were so connected to one another although they weren't completely different parts of geographically. They were still connected because these apostles were traveling across to different churches and they could vouch for the churches. They could speak on their behalf. They could share their needs. So we go on from here versus 16 to 24 if someone could read that for us please. Second Corinthians chapter 8 verses 16 to 24. But thanks be to God who puts the same earnest care for you in the hearts of Titus for he not only accepted the exhortation but being more diligent. He went to you of his own accord and we have sent with him the brother whose praise is in the gospel throughout all the churches and not only that but who was also chosen by the churches to travel with us with this gift which is administered by us to the glory of Lord. Avoiding this that anyone should blame us in this lavish give which is administered by us. Providing horrible things not only in the sight of the Lord but also in the sight of women. And we have sent with them our brother whom we have often proved diligent in many things but now much more diligent because of the great confidence which we have in you. Anyone inquires about Titus he's my partner and fellow worker concerning you or if our brethren are in kind of work their messages of the churches the glory of Christ therefore show to them and before the churches the proof of your love and of your boasting on your behalf. So here Paul just concludes this part about giving and talks about the people who will be coming to receive the funds from them. So first he talks about Titus in verses 16 and 17 so Titus as somebody who cares for the church not someone who's going there because he's being forced to being sent but he willingly wants to go he wants to go see them and in seeing them and being with them. To be able to carry these funds back and then verses 18 and 19 he talks about somebody who was sent by the churches to travel so in doing this he is basically saying these people can be trusted. I'm introducing them and I'm telling you who they are the authority that they're coming with so that when you're giving you know that you're giving to trustworthy people. Right and that they will take it to the church there won't be any misuse of the money there won't be any loss of the money that you have given. So verse 18 we have sent within the brother whose praise is in the gospel. So this in the NIV it says praised by all the churches for his service to the gospel. So we don't have his name but we have this recommendation that Paul gives as he has served for the sake of the gospel and recognized through all the churches. And so he has been sent he was chosen by the church in Jerusalem itself to travel with them and take this gift that was being given. In the end of verse 19 administered by us to the glory of the Lord himself and show your ready mind. So this gift is being given for the glory of God. Right while we're doing all of these things we're giving sacrificially we're doing generously it's always for the glory of the Lord and to show that we are ready to give that we have a willingness to share we have a willingness to bless others in need. And then verse 20 avoiding this that anyone should blame us in this gift that is administered by us. So verses 20 and 21 is just to say they have been put in charge of administering this gift right of collecting it from the churches or gathering that money together of talking to the churches raising up people who will get it. They get these funds will do all that. But then they want to do all of that in a way that is blameless that nobody can question the way they've handled their finances. And so the way they do it in verse 21 he says providing honorable things not only in the sight of the Lord but also in the sight of men. So being very careful in the way they are doing it so that nobody can question how they've handled these funds. This is very important as we also deal with finances in ministry that we do it with such wisdom that there's always in the way we're handling it. There's always transparency there's no confusion about where did the money go how did we deal with it. So when people are giving money to us how are we accountable to how are we reporting to them where the money went how it was used who used it all of those things having that kind of openness about the way the money is being used how the money is being taken care of and then having accountability to be able to report about how how we have used the funds that people have given. So this is something that we shouldn't take lightly especially in ministry because we want people to know that we are in ministry for Christ's sake. They're not there for the money they're not there to to get rich or to promote ourselves in any way right and if people can trust us and can see that our motives are pure then they can trust the ministry that we're doing. But if we don't have these kinds of practices in place where we are taking care of what God has entrusted to us then it's very easy that our funds get misappropriated even if we are not doing it. If the transparency that accountability is not there in our ministry somebody else who comes on board can easily misuse money. So have good processes in place have clear communication with those we are ministering to that there will be no hindrance to the work that we are doing. Finances or questions of doubt or misuse of funds will not come come in the way of what we're doing in serving God and serving people. And then verses 20 to 24. We have sent with them a brother whom we have we have often proved diligent so along with Titus and this other brother who was sent by the churches there's one more brother being sent. And here he's saying he's proved to be diligent. And so we're sending him to you along with them. And then 23 and 24 he says OK if somebody has questions about these three people and wonders can we give them money or who are they what authority do they have can we trust them. He is saying what you can answer about these three people. So Titus is Paul's partner and fellow worker concerning you. So he's a partner and fellow worker with Paul in serving Church of Corinth. And then the brethren who are going with them are messengers from church have been sent by the church itself. And then and then he says so show them your love. Approve your love and are posting on that behalf. So honor them respect them take care of them because they are people you can trust their people we have trusted and sent to you. And just one important description here in verse 23 the church is the glory of Christ. So just a reminder that that is what the church is called to be the revelation of the glory of Christ which is the powerful beautiful description of what we are called to be as a church. We go into chapter nine from here. Someone can treat versus one to five. Second Corinthians chapter nine versus one to five. Now concerning the ministering to the saints it is superfluous for me to write to you. For I know your willingness about which I boast of you to the Macedonians that a care was ready a year ago and your zeal has stirred up the majority. Yet I have sent the brethren lest our boasting of you should be in vain in this respect that as I said you may be ready.  Therefore I thought it necessary to exhort the brethren to go to you ahead of time and prepare your generous gift beforehand. Which you had previously promised that it may be ready as a matter of generosity and not as a grudging obligation. So here Paul is concluding all of these arrangements about collecting the funds. And just as he has used the Macedonians as an example for the Corinthians to follow. He's also used the Corinthian church to encourage the Macedonians. So looking this is something we can learn is to look at other believers who exemplify faith or strong faith in Christ. We know the faith that is worth emulating and to be encouraged and stirred up to follow their example. That's what Paul is doing here. So encouraging one church with the other church's example. So he says in verse 2, I've boasted about you to the Macedonians saying that you were already ready a year ago. And that your passion, your excitement, your zeal for giving has actually stirred up many people to give. And so we see Okia here referring to that region, right? So telling the region in Macedonia, the churches in Macedonia about the region of Okia that they were ready to give. And then verse 3. So even though you were already ready a year ago, I've sent these brethren, that is Tychus and the two brothers. Because we don't want a boasting, so we want you to be ready. So you had spoken about it. We don't know if they had already started the process of collecting, but so that the process would be completed. All the funds would be brought together and they would be ready to give when it was time for them to actually go and collect the money. So he doesn't want them to be unprepared. If he takes the Macedonians there that whatever he had been talking about them would prove to be something that was untrue. And then the last part he says, we want your giving to be out of a willing heart and not a grudging obligation. So Tychus and those brothers have come there to prepare you so that you can make a decision and give according to your willingness. Rather than us coming when it's time to collect and then you feel forced to give out that type. And you don't give it with a willing heart. You don't give it with a thought beforehand of how much you want to give and giving with that kind of intentionality. So that is something also for us to learn where we are giving with generosity. But we're giving willingly and cheerfully not out of any feeling of obligation with holding any grudges against people. Feeling like we are forced to give. That should never be the way we are asking for funds or the way we are giving funds as well. Move on from here to verses 6 to 15. Maybe we'll split that up. We'll just do 6 to 9 for now. Someone can read that. But this I say, he who so sparingly will also raise sparingly. And he who so wonderfully will also rebound to flee. So then each one gives as he purposes in his heart, not grudgingly or of necessity for God loves a cheerful giver. And God is able to make all grace abound toward you. That you always having an old sophistication in all things may have an abundance for every good work as he is written. He has dispersed a board. He has given to the poor his righteousness in years forever. Thank you. So here we see Paul just talking a little bit about how we should give. And so he begins with saying that if we saw a little bit, we will read a little bit using the example of a farmer. But if you saw more, you can look forward to a greater harvest. So likening that to how we bless others. We are sowing into their lives and what comes out of that will be a great harvest. And then and we can share in that harvest. So we can share, we can rejoice in that harvest. So that is regarding how much to give. And he talks about how to give in verse seven. And he says, not like we read in the previous verses, not with the sense of necessity, not like you're being forced to give, but to give cheerfully with the heart that is willing, with the heart that desires to give. And then he verse eight says how not to give. Verse seven, sorry, says, you should not give gradually and you should not give out of any sense of pressure, whether it's from the leadership or from the other church members, or even feeling a sense of obligation towards a person in need. It should never be from that place of pressure. It should be from your own heart that is willing. So Paul's this, what he does to send them ahead of time is a very, very important thing because it's not pressuring them to give immediately, right? So a lot of sales in our day is kind of this make an immediate decision. This discount will end in 15 minutes or will end today. And so they put that kind of pressure on you to make a decision and to feel like, okay, I'm going to lose out on this if I don't do it today, if I don't do it right now. So Paul takes away that kind of time pressure. They have time to think about it. They have time to process it to make a decision as they within their own hearts or with their families and then give a very good example of how we can we can encourage people to give as well. And when we are, when we want to raise funds, want to give money for whatever it is to have that kind of attitude to not adopt the methods and means the world uses to pressurize people, rather to adopt the ways that we see in scripture from people of God. And then we see Luke, Luke 638 is where Jesus talks about giving. He says, given it will be given to you, good measure, pressed down, shaken together and running over will be put into your bosom. But with the same measure that you use, it will be measured back to you. So the same principle of the more you give, the more you will be blessed, the more will be given to you. So Paul is kind of speaking in line with that kind of thinking. So verse eight, he says, he will make all grace abound to you that in all sufficiency, you may have an abundance for every good work. So he's not saying that you're going to give and then you will get back a lot of money in return. He's saying you will find sufficiency in everything, you will be blessed in every way. And in being blessed in every way, you will have an abundant supply to do all for every good work that God calls you to. So when we're giving funds, not to expect that we're just going to get back a lot of money, rather to just believe that God is going to bless us in every way. He's going to take care of all of our needs and he will supply whatever is needed for us to continue to be a blessing, to continue to do his work. And then verse eight, he has dispersed the priorities given to the poor, his righteousness endears forever. So that is reference to Psalm 112. If we can just read, or someone can read Psalm 112 verse five and then we connect that to what this says. Psalm 112 verse five, a good man deals graciously and lends. He will guide his affairs with discretion. Thank you. So we see in Psalm 112, those first few verses are describing a righteous person. And verse five talks about the generosity of that righteous person who will lend their money freely, will conduct their affairs with justice. So it's one of the things about righteous person. So if you look at that Psalm, there are different things that are said about the righteous person and one of the things is generosity. And so the blessings that come upon that person also are an overall blessing. It's not only in one area of their life. And so when we read Psalm 112, nine, it's talking about that righteous person who is righteous in every way, who is also generous. That he has given to the poor and his righteousness will be an eternal righteousness, not a righteousness just for his lifetime. It's going to be a blessing for generations to come. And his horn will be exalted with honor. So using that passage to say that you will be blessed as scripture has promised, you can trust in the promises of scripture that you will be blessed in every way. And you will be blessing generations to come as well in this giving. From here, let's go on to verses 10 to 15. Someone can read that please. The wicked will see it and be grieved, will gnash his teeth and melt away. The desire of the wicked shall perish. No, from chapter 9 verses 10 to 15. Second Corinthians chapter 9 verses 10 to 15. Sorry. Now may he who supplies seed to the sower and bread for food supply and multiply the seed you have sown and increase the fruits of your righteousness while you are enriched in everything for all liberality, which causes thanksgiving through us to God. For the administration of this service not only supplies the needs of the saints, but also is abounding through many thanksgivings to God. While through the proof of this ministry, they glorify God for the obedience of your confession to the gospel of Christ and for your liberal sharing with them and all men.  Thanks be to God for this indescribable gift. Thank you. So we see that Paul is going back to remind them that God is the one who supplies our needs. So he says in verse 10, he supplies seed to the sower. So even though you are the one sowing, the fact is that God himself has given you that seed to sow. So the way we look at our finances is that God has entrusted these finances to me, and I need to be sowing it in a way that God that pleases God. Whatever I'm doing with it, whether I am investing it for my own purposes for my family, whether it's it on different things or whether I'm giving it to people. How am I sowing what God has entrusted to me, what God has blessed me with. And he says he supplies seed to the sower and bread for food. So the seed is that initial thing that is put in and bread is the product of what is what comes out of the seed. So he not only gives us that seed to sow, but he also gives us that finished product, that food, that will nourish our bodies, that will meet up physical needs. So remember as we are dealing with finances that this is the God we serve. He is the one who supplies our needs in every way, right from start to finish. He is the one who supplies our needs. And so he will also supply and increase your store of seed and will enlarge the harvest of your righteousness. So he will increase what is available to you to sow and he will also increase the harvest that comes from what you're sowing. So when we are looking at this as we are giving, we look at both aspects of it.  And what is beautiful about this is in verse 10 is the harvest of your righteousness, right? It's not just a harvest of crop, it's not a harvest of money. It's a harvest of our own righteousness, our own standing with God will improve and be blessed as we are giving. And that will happen not only for ourselves but for other people. There will be a harvest of righteousness. Verse 11, you will be enriched in every way so that you can be generous on every occasion. Again, this reminder that the blessing is an overall blessing. It's not only going to be a financial blessing. It's a blessing of every need of every aspect of our lives so that we can be generous in every aspect of our lives. Whether it is in mercy, in kindness, in helping others, in serving others in every way that we will experience God's generosity towards us and then we will be able to be generous towards others. And through us, your generosity will result in thanksgiving to God. So the end result is that God himself is being praised. When we are giving to people, it results in praise to God. Verse 12, the service that you perform is not only supplying the needs of the Lord's people but is also overflowing in many expressions of thanks to God. So as we are giving, we are giving with an attitude of gratitude to God. Like what we said that whatever we have, we recognize God has given us. And so when we are able to share that with others, we say we are giving it with that thankfulness to God. Thank you Lord for what you have blessed me with and thank you for this opportunity to share it with others to be able to bless somebody else with what you have given me. So thank you for blessing me so that I can bless others. So it's an expression of gratitude to God and it results in gratitude to God as well because the other person will thank God because of what we have done. Then verse 13, because of the service by which you have proved yourselves, others will praise God for the obedience that accompanies your confession of the gospel of Christ and for your generosity in sharing with them and with everyone else. Again here, an important thing to remember that faith should result in works of faith in things that actually are evidence of the things we believe. So if we believe that God is our provider, then how are we reflecting that in the way we deal with the things that He's given us? How is our generosity? How is our willingness to share with others reflecting the fact that we believe God is our provider? So our faith should be evidenced in our works. And so it says here others will praise God because they see your obedience, they see your generosity that you not only believe, you not only accept the gospel but that is impacting the way you live and you are actually living in a way that reflects that gospel of Christ.  So not only will they thank God but they will also bless you. They will pray for you, they will remember you when they are praying, when they are thanking God because of the grace that God has given you to bless them. So when we are blessed by somebody and we go to God with gratitude, our heart is also then to remember that person who has blessed us and to remember them before God. And so he says that is an additional blessing that you will receive where people will remember you in prayer. And then verse 15, thanks be to God for this indescribable victory. So here Paul is just kind of bringing all of this giving, teaching and giving together. He is reminding them that it is actually a blessing to give. At the end of this giving you are the one who is being blessed with this indescribable gift, right? You have things that were blessed, were given to you by God. They are not things that you yourself have somehow received by your own strength or wisdom. It's things God has given you. You have the opportunity to bless others. And as you bless others, you are being blessed. God is being glorified and you share in the gospel of Christ. And the example of Christ which was this act of generosity of Christ himself giving his life for us, for the world. And so we get to be a part of that and to live that out of the way we deal with our finances, the way we deal with the things that God has blessed us with and how we use those things. So any thoughts, anyone wants to share anything from what we've covered so far? Any thoughts on finances, whether how we use it personally or in ministry, something that we've looked at today? Something that you can take away from that? Yeah. I think there's a lot of takeaways that I got from this class. Like how you said giving from the place of surrendering to God is one of the new things I have heard. And even the point where you said by giving to the same, there is a share in the sufferings of each other. Mostly I think of a way of only giving to church which might have been a little wrong. We are also sharing with the saints and they are taking care of their needs. So those are some of the few points that I have left. Thank you. Does anyone else want to share anything before we move on to chapter 10? Let's move on then. Just a quick thing. I think these chapters just really are a great teaching on how in ministry we both deal with finances, we talk about finances and how as believers as well we use our finances of how we employ our finances. So it is from a ministry perspective as well as from a believer's perspective and how as ministers we can talk to people about finances. It's a very good and holistic teaching I think on finances for us. So it's by the example of the ministers of the leaders that we can lead others to deal with their finances. So when people are saying how are we as a ministry, how are we as leaders using our finances, then they are encouraged to follow our example or to approach their finances in a similar way. So how are we setting an example as ministers and leaders? So from here we will go on to chapter 10. With chapter 9 was the conclusion of Paul's teaching on giving and encouraging the church to prepare their gift for the church in Jerusalem. And then in chapter 10 he goes on to talk about some of the issues again that were coming up with the people who were opposing him and to encourage some right understanding and right teaching for the current things. We don't have a lot of time so maybe we'll just do the first six verses. Someone can read that please. Now I pour myself and pleading with you by the makiness and the gentleness of Christ. Who in presence am lowly among you, but being absent am bold towards you. But I beg you that when I'm present I may not be bold with that confidence by which I intend to be bold against some. The paper got disconnected from the call. So we'll just read and discuss simultaneously. So Paul says, by the humility and gentleness of Christ I appeal to you. So Paul is sharing in those qualities of Christ, meekness and gentleness of Christ. So he wants to come to them with that kind of approach rather than one who is bold in the eyes of the world. So when we hear those words, meekness and gentleness, they usually from the outside are looked at as weakness. As someone who can be taken for granted or someone who is very weak. But here he is using that as words to describe Christ and he is coming to them with that attitude of meekness and gentleness. And then he talks about what other people have been saying about it.  So this seems to be what other people were saying about him, maybe because he had gone and met them and addressed those issues with them. And then afterwards he'd gone away and sent them to them. So some people were talking against him in that way. But he says in verse 2, I beg you that when I come, I may not have to be as bold as I think, as I expect to be towards some of you who think that we live by the standards of this world. So that is an important thing, standards of this world. So when the people were looking at him, they were judging him. This was something that we see repeatedly in his letters to the Corinthians, not the judge by standards of this world. So if he was being gentle towards them, if he was being humble, it was the way of Christ. He was following the way of Christ. But if he was to follow the ways of the world, then he would be like the other leaders, the opponents who were coming against him, who wanted to post, who wanted to put other people down, who were putting Paul down as the leader of the church. They had come in and they were putting him down. So he could be like that. He could be someone who was bold and confident and putting other people down. And like Jesus talks about, the rulers of this world lauded over the people. They take their authority and dominate over their subjects. So he could have been like that, but that was not the way of Christ. And so he chose to not follow in that way. And then verse 3 says, though we live in the world, we do not wage war as the world does. So he is not going to go against these opponents with that same attitude of posting or talking against them in the way that they are doing against him. Verse 4, the weapons we fight with are not the weapons of the world. On the contrary, they have divine power to demolish strongholds. So we use weapons that have a power that is divine. So we don't have to resort to using our words. We don't have to resort to using our authority, our strength. We don't have to resort to using influence, using money. We don't have to use the ways of this world. We depend on God to break down the strongholds, to break down things that are standing against us. To see victory in whatever area we're fighting against, to see victory in that area. And then we see verse 5, we demolish arguments in every pretension that sets itself up against the knowledge of God. We take every thought captive to make it obedient to Christ. And we will be ready to punish every act of disobedience once your obedience is complete. So he's talking about using spiritual weapons rather than physical worldly weapons or using weapons of the flesh. So we'll talk more about this next week, but that is his approach. That we are going to approach this battle as a spiritual battle and we will fight it with spiritual tools rather than with physical fleshly tools. And then that last part is to bring to obedience, bring people to obedience and punish those who have been disobedient. So we'll talk more about this next week. That will be our last class next week. So we'll try and finish up all our content and I'll also be posting the streets. So please, thank you.