hey good morning I hope uh everyone are done with our assignments and you are done with watching the videos So far we have seen how to use a DNA sequence to find its identity through blast analysis How to construct a fog using the DNA sequence and reference sequences taken from the NCBI database How to study the pair wise distance data and how to prepare the Excel sheet using color scale bars Okay I hope everyone is clear till now and before we start our own project which is today I want you guys to practice this bioedit Okay what is this bioedit last class we have seen how to extract the DNA sequence from the chromatograph Okay as you have seen we are actually doing two-way sequencing What is this two-way sequencing we are going to know sequence both the ways Why to know DNA occurs in double standard double standard DNA and we are going to sequence both forward and reverse strand Why okay even people who are working in say for example 500 base per gene they will again go for two-way sequencing As I told you in last class if it is 1,500 base sequencing both is very much essential to you know obtain the completeness of the entire length Okay as the modern day DNA sequencer they may produce up to 900 or,000 base So if you want to cover 1,500 better sequence both strands from both ways Okay when I say both ways usually the DNA strands are right from five prime to three prime Okay So by now you should be very clear that in molecular biology when you say DNA extraction you are extracting the entire genome which contains thousands of genes Okay How we particularly amplify our own desired gene or our target is by using primer during the PCR Okay This is a primer sequence in the sequence in the picture where the forward primer bind to a specific region and the reverse primer complement to the particular gene and they will just amplify the particular fragment Okay So using primer that's like baiting you are getting what you want which gene to amplify is decided only in PCR Okay Even people working with 500 base per gene they will always sequence both stands because sequencing two ways also ensures accuracy Okay If this primer is committing a mistake mistake when I say mistake it means instead of thine it will be R9 here instead of R9 if it is adding guanine you can verify this position by comparing with your reverse complement What is this reverse complement let's now understand just the reverse sequence When you have a reverse sequence it's it act as a you know one more layer to check whether the forward sequence is correct or not And it is true vice versa Okay So for error correction accuracy completeness you need to go for two-way sequencing Meaning when you are done with your PCR the PCR amplons are split into two samples and they are again subjected to single strand amplification Okay So single stand either forward stand or reverse stand is amplified separately Again both the samples are injected into the sequencer ABA sequencer So you should know when you submit a sample to the company they actually isolate DNA they go for PCR then if necessary they will do PCR purification then they will go for single standard PCR where your sample is split into two single strands In one PCR tube only forward primer is added in another PCR tube only reverse is added That's how the single strand amplification is facilitated and that amplon is subjected to uh two sequencing reaction So every sample in the sequencer when they are loading it will be two samples two single standard PCs So for every sample you will be getting two machine output files Okay Now this bio edit is all it's entirely about how to use these two strands how to compile them and obtain our target DNA complete length of target DNA or how to check our accuracy of the DNA sequence and everything Okay So I will um to understand I hope this picture is very clear One stand is a forward stand other is reverse stand So you'll be having two machine output files Okay For analysis So before we proceed I just want to recall again what is this reverse complement Okay See here say for example consider this 10 base DNA strand Okay A a triple c Okay This time I hope you know this complementation Adinine always pair with thymine Adinine thymine Adinine thymine Cytosin guanine Cytosin guanine Cytosin guanine Guanine cytosin Guanine cytosin Thymine adinine Okay So adinine always pass with thymine and cytosine always pass with guanine in triple hydrogen bond Adine is always double hydrogen bond You know that Okay So what is this reverse complement reverse complement is nothing but if I am reading the this is a forward sequence a a c C cc GT okay and the reverse sequence we are seeing this reverse complement is something you have to read the sequence in reverse and also in complemented manner okay so what is this okay see here I'm going to read the sequence in reverse reading the sequence of this sequence in reverse is t G G triple C W A W A okay instead of reading like this instead of T I'm putting A here instead of G C I'm complementing the reverse that is called reverse complement so T A for G C here G C triple C triple G W A T okay it's all about ATGC all four nucleotide this is reverse complement why to do this reverse complement you will know Okay Only by reverse complementing the reverse strand you are actually uh merging or you are actually completing your forward strand I will tell you how it is done and I will show you how it is done Okay So I hope this is uh very clear now Okay DNA are double standard Okay Uh so positive stand forward strand stand is something like this In another example just to make my point Okay A G T C A G T A A C T A G This is the forward strand and this is reverse complement to the forward strand Read it from the reverse G C A complement is T T complement is A C complement is G So this is reverse complement of the forward ST Okay I hope this is very clear now U so how to merge the two machine output file it is a it is a well-known fact now that you have to dedicatedly sit and edit each and every baseware in the uh machine output file After doing that you can extract the sequence of forward and reverse and you can do the merging How to do this merging that's when the bio edit comes into play Okay So these are the eight important steps you need to follow to merge the DNA sequence Instead of reading each and every step I know it is will going to freak you out Instead of reading I'm going to show you how to merge the sequence Okay So what I'm will do is uh I will take an example file here Uh I have a client's uh sequencing file here Okay So I sit I edited each forward sequence and reverse sequence I copied those edited sequences in the Notepad file Okay So here I have a forward sequence Okay Here I have a reverse sequence I always believe word document is an excellent tool for DNA compilation and counting number of base pair So I am going to use my word file to show you how it is done Okay So I'm opening my word file First you copy both forward and reverse sequence in the notepad file to word file Okay So I have copied this is my forward machine output file This is my reverse machine out file I have edited each and every chromog I got this Okay So the first step is as you have copy and paste the for reverse sequence into the word file Done Open bioedit and reverse complement the reverse sequence what it is I will tell you So before opening bioedit you should know where how to download the bioedit So I'm showing you in a Google just type bioedit Okay The biodedit download biodedits software.infirmware.com This is an official website Download go here Okay This first option download the latest development This is the latest file Okay 15 MB Yes Click it Now download is in progress Okay I can see it here Once downloaded or download is done they will appear as a uh zip file Okay there are multiple files downloaded maybe because I clicked multiple times but just unzip the file Extract all So extraction is done The file is opening Okay it will be a zip file uh double clicking the setup you can easily uh install the okay once the installation is successful you will uh see a uh Okay You can uh check the availability of so this is because I already have wire edit installed Okay So just for demonstration I showed you how to install the bio edit Once the installation is done you will see something like this Yeah window something opens like this Okay So this is bioedit and what is been the second step open the bioedit I have opened it Reverse complement the reverse sequence Meaning copy the reverse sequence Reverse sequence alone and reverse complement it You know if it is a 10 base pair I can sit here and I can compl reverse complement the sequence But it is around 600 base or 700 base here So how to reverse complement using bio audit just go to file new from the clipboard Since I have copied the sequence already I have copied by copy Then it is in the clipboard of the uh computer Now from the clipboard new from the clipboard If I click this now the sequence is been imported into the bio edit Okay I will click the sequence title I'm going to reverse complement the sequence I will increase the font for you to see how the reverse complement is done See sequence nucleic acid the third option I clicked third option sequence come down nucleic acids in nucleic acids reverse complement I click it I'm going to click it just observe the text here they will be complemented immediately right 2 3 1 can you see that reverse complemented now the entire sequence is reverse complemented now I want to comp copy this sequence again paste it in the word so go to Second option edit copy sequences to the clipboard Then I will open the file and pasting it here Yes this is the reverse complement of the reverse sequence See it starts from CC but this is T GG If you see here this D actually T CG G actually represents to A G G C Can you see that the entire sequence has been reverse complemented here Now I don't need the reverse sequence I'm deleting it I have a forward sequence I have a reverse complement of the reverse sequence I would say reverse complements are extension of the forward sequence How just copy the last fragment 10 base Okay Around 10 base I'm copying it here in the word in the word document Ctrl F I'm pasting it here See So this means the sequence ends by C A G C A G after this is an extension So I don't need these portions now Okay I'm going to merge it That's it Okay that's how the forward and reverse sequence are merged See this is possible only if you have meticulously prepared the chromograph If you have left one two base pair no differences we'll be in trouble Okay this is we can also merge this in mega and we can also merge this merge and prepare a consensus sequences in bioedit but I prefer to do it in word because many more things can be done Now I'm checking the world length it is,470 base which is more than 90% length or 85% length of the the 6S RNG Okay So that's how the merging is done That's what the instruction is also about Once uh delete the original file Okay From the forward sequence copy 10 base pair Use control F to find this 10 base for the reverse complement Once match copy the remaining part of the reverse complement After the 10th base over it to the end of the so that's how the merging is done Okay So today it's a new tool bio edit because we want to compile both for and reverse chromatoggrams by reverse complementing it Okay these are the steps for reverse complementing I hope uh you understand this Uh today I will be sending you guys the uh forward and reverse extracted sequences So you reverse complement that and you go for blast analysis let me know how this uh blast results uh turns out Okay So today again we will uh fix on our individual topic I will share one more video regarding that Thank you