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hello this is David coil and welcome to the next video in our series on aluminum multiplexing in this video we're going to go over the background and principles of barcode design just a quick review the videos in this series consists of the overview this video which is the background and the details of barcode design and the next video will be a tutorial on using the barcode generator and then finally we'll have a video that shows some applications and examples of bar coding we should note that all of these videos assume a basic familiarity with Illumina sequencing but just as a quick review here is the Illumina Y adapter and here's the barcode this is the sequence that you're going to add to the adapters to uniquely identify a particular pool of sample so there are a few considerations when designing barcodes the first is the balance between the four nucleotides at each position in the barcode this is because the way Illumina determines clusters is based on the first few bases and if the first few bases are the same then Illumina can't determine what belongs to one sequence versus another for example if the readout from four different barcodes is as shown here then the first two nucleotides of every sequence in the run will be 80 and Illumina can't determine what a cluster is we should note and we're going to go over this later that this is the readout of the barcode the actual name of the barcode will be the reverse complement of that but we'll get to that so one way to avoid this is if you're only mixing two libraries you should use two barcodes for each library to improve the nucleotide balance so that each of the four bases is represented about a quarter of the time in the initial cluster determination however an appropriate customer protection can still be a problem with some versions of Illumina software particularly the newer high SiC machines but we anticipate that this will change with time so other considerations when designing a barcode the length of the barcode is important we use five bases an odd number is better than even for avoiding palindromes barcodes could be as short as four or a little longer if necessary the difference or the genetic distance between barcodes this type of li called the hamming distance we find the two bases is usually sufficient but in some cases three basis is fine but if you're using short barcodes and you try to make them too different you won't have enough combinations a couple of technical notes one is that you need to order the adapter with a five prime prospect on adapter a normally a doctor a and this is our nomenclature is adapter a for the paired and Illumina tapped or the first one normally that comes with a five prime phosphate because we're going to add a barcode to it we still need to remember to add a five prime phosphate upstream of that barcode and no special purification is needed the standard desalting is fine so a quick note on nomenclature we name the adapter by the way it shows up in the top sequence what we call adapter a however the sequence in the read would be the reverse complement of that so let's look down here at the Illumina paired and adapter sequences in blue this is the standard adapter a and adapter be no at the five prime phosphate on adaptor a we had a barcode to this we add five bases and then an A and this a is to allow the sequencing primer to bind there's normally a teed overhanging present there then here an adapter B you can see that you have the reverse complement of that and then we've added the key overhang again and this is the equivalent of this T overhang here so by convention we call the barcode this see see but in reality all the reeds in the sequence will be gg and we'll get to that in a minute so when you want to design barcodes you have three choices the first is they can be designed by hand and this works fine for simple experiments just choose an appropriately balanced series of bases perhaps even simpler than that we've created a list of 24 well tested barcodes available on our website at the URL shown here and the most common option is to use a barcode generator and we've made a simple python barcode generator available on our website shown at this tiny URL here and in the next video we're gonna go through how to use this barcode generator and this takes into account several parameters for length number genetic distance etc something important to note when dealing with barcodes is the critical importance of measurement if you think about a sample that has 96 different barcodes and it that means that any given bar coded sample only represents about one percent of the total sequence that runs so if you want to be able to compare between samples you need highly accurate measurement and so there's a couple of pitfalls that we've encountered that we wanted to share with you one is that and now I drop spectrometer measures free nucleotide as well as DNA so if you're going to use an down and drop be sure to purify your samples before using the nanodrop another is that rt-pcr which is commonly used can amplify some sequences more than others so it's also risky we recommend the use of double-stranded DNA specific flora for example we use cyber green this is a simple replicable plate friendly method for measure during the concentration of DNA in your library so let's go over the bar coding workflow real quick you design the barcodes using one of those three methods order the new adapters being sure to get a five prime phosphate in the right place so your standard library prep and quantification preferably using cyber green or some equivalent pull your samples and submit them for sequencing and then we get to data analysis we'd like to say a quick word about that here we're dealing with bar coded sequence you need to first process the sequence file to remove the bar codes the first thing to do is verify that the bar code is intact and again we already went over this make sure that you're looking for the reverse complement of the name of the bar code so in the case of TTT ECC you're looking for GGG a a on all sequences even those that came from adapter B because the way the Illumina primer is line up then you're going to remove the bar code from the read paste in the name and don't forget to remove the extra T as well and these six matching characters from the quality mutation line I'll show you what that looks like here here's an actual Illumina read you can see I've highlighted in blue the barcode and then the key overhang so for processing we simply cut this add it to the sequence ID remove the T and then cut out those six characters orientation and now you have a process sequence and now you can sort in two different files or whatever it is that you want to do now that you've removed the barcode from the sequence so that's it for this video in the next video we're going to go over how to use the barcode generator thanks for watching and see you then you