Hangover barcodes are added to the primers during library preparation for sample-specific multiplex sequencing but still during PCR why some researchers add different unique barcoded primers to the different individual samples. Please help me make clear the use of this individual barcoded primer in PCR. What could be the major difference in sequence results between using barcoded and non- barcoded primers?
Are you are asking why you barcode samples in multiplex sequencing or some kind a second layer of barcoding?
Sample level barcoding is to differentiate samples and allow for demultiplexing and assigning reads to a specific sample. This can be single, dual, unique dual to prevent barcode hopping and correct assignment of reads to the correct sample.
There are also Unique Molecular Identifiers (UMIs) that are used to identify PCR duplicates and potentially remove them from your dataset depending on your study
Hello, I had the same question while performing my first NGS using illumina. Currently, I am amplifying a CRISPR KO plasmids library and I had to perform NGS to verify that my library contains 100% of initial non-amplified library plasmids. When you prepare your sample for NGS.... you must perform 2 different PCR : the first PCR used usual primers (20-22 NT, without barcode) to amplify your targets in the library. The second PCR used forward and reverse primers containing an additionnal barcode sequence : usually we use a combination of different barcoded-Forward primers (same priming sequence but different barcode) to increase the coverage of your library (to be sure that all your PCR products will be target by PCR2) and a single barcoded reverse primer (for each sample you will use a different single barcoded reverse primers).
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