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Multiplex initials
hello this is David quail and welcome to the first video in our series on aluminum multiplexing in this first video we're just going to do a simple overview of multiplexing and then in subsequent videos we'll get into the details so what is multiplexing multiplexing is simply a way to save money by sequencing multi samples in a single unit so in our case an Illumina flow cell lane the same technique can be applied to four five four pyrosequencing or whatever we're going to talk specifically about Illumina and at $1,000 per Lane per run this is an attractive idea so there's two ways to think about multiplexing the first is just to mix highly divergent sequences and sequence them together and then separate them by analysis you could take a bacterial sequence and a human sequence sequence them together and then just align them to the respective sequences however you have to be really careful of homology that can mess up the alignments so more common is what's called barcoding Illumina uses a slightly different technology which they call indexing but in reality that two terms used interchangeably so we'll stick the bar coding so what is bar coding bar coding is simply the addition of a short nucleotide sequence shown here in purple that is added to the adapters that are used for PCR amplification this is the Y adapter that's used by aluminum in every sequence that's amplified with this adapter will have this unique barcode on it so basically you create different libraries each with a unique barcode you pull the libraries and sequence them together and then you separate out this different libraries by the barcodes when you do analysis so just to show sort of what this would look like here we have 1 2 3 different libraries each with a different barcode we pull them together and about 1/3 of the resultant sequences have each of the different barcodes so what does this look like an analysis so shown here at the top is a FASTA output from an aluminum where we used barcoding the first five basins are the barcode and the sixth base is the t overhang so all we do in processing is we cut the barcode we put it up here we remove the t overhang and we remove these first six quality scores so now we have a processed output now we can make everything with this barcode put in a one file everything with a different barcode put it into a different file and we can do our separate analyses so just to summarize multiplexing is a way to save money by sequencing multiple samples in the same aluminum flow cell lane we do this by using a short nucleotide barcode with in the primers and we can have many barcodes in a single experiment in fact you can take an entire 96-well plate everything with a different bar code sequence all the samples together and let separate them during analysis so that's multiplexing in subsequent videos we're going to talk about all the details thanks for watching and see you then you
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