Dear board,
I am trying to develop a method for adding 6mer barcodes to RNA-seq protocols with the illumina platform. From what I understand, indexing under illumina's methodology has an entire adapter and reaction to read the 12 different sequences that illumina chose for their specific indexing. Is there an easy way to insert my own hexamer barcode which would be read under the main sequencing reaction i.e. in-between the bridge PCR adaptors and the library strand. We would be doing paired end sequencing and I would be using a few different protocols initially to test efficacy (while our lab chooses which method for our RNA seq to use) and need an easy method to simply insert our desired barcode.
Does this make sense? Any help?
I have initially thought that we could do a site directed mutagenesis insertion but I don't know how cheap or practical that is for our RNAseq.
I am trying to develop a method for adding 6mer barcodes to RNA-seq protocols with the illumina platform. From what I understand, indexing under illumina's methodology has an entire adapter and reaction to read the 12 different sequences that illumina chose for their specific indexing. Is there an easy way to insert my own hexamer barcode which would be read under the main sequencing reaction i.e. in-between the bridge PCR adaptors and the library strand. We would be doing paired end sequencing and I would be using a few different protocols initially to test efficacy (while our lab chooses which method for our RNA seq to use) and need an easy method to simply insert our desired barcode.
Does this make sense? Any help?
I have initially thought that we could do a site directed mutagenesis insertion but I don't know how cheap or practical that is for our RNAseq.
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