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  • Illumina standard barcode sequences not completely base-balanced, why?

    I am aiming to sequence 72 amplicon libraries in one lane, and I plan to use in-line barcodes, 12 on 5'-end and 6 on 3'. As I understand, first 4 nucleotides of each read are crucial for cluster identification and therefore should be base-balanced. I checked standard Illumina Truseq barcode sequences, they are supposed to be well-balanced, but it seems that they aren't (or I'm missing something).
    See:

    RPI01 ATCACG
    RPI02 CGATGT
    RPI03 TTAGGC
    RPI04 TGACCA
    RPI05 ACAGTG
    RPI06 GCCAAT
    RPI07 CAGATC
    RPI08 ACTTGA
    RPI09 GATCAG
    RPI10 TAGCTT
    RPI11 GGCTAC
    RPI12 CTTGTA


    on position 3, A is overrepresented, at the expence of G. Is there any reason for this? What's better, to leave it this way, or to order better-balanced barcode sequence instead of RPI02, CGGTGT?

    For 3'-end barcodes I need 6 sequences, and as I understand they should be base-balanced as well. There are no way to get equal amounts of each nucleotide in 6 barcodes, so 2 should come in duplicates and 2 should be unique.
    The first 6 barcodes do not meet this requirement due to overrepresented A, so I came up with this set:
    RPI01 ATCACG
    RPIUU CGGATT
    RPI03 TTAGGC
    RPI04 TGACCA
    RPI08 ACTTGA
    RPI09 GATCAG

    I had to make custom barcode, RPUU, since there were no matching sequences within 48 Illumina's barcodes.

    The questions that I have: Did I get it right about balancing requirements for amplicon starting nucleotides, or there are some tricks that I missed?
    Are my barcode sets well-suited for multiplexing?

  • #2
    "Well" balanced does not mean 100% perfectly balanced. You're over thinking this. Pick a set which gives a decent mixture but don't try to make it perfect.

    Comment


    • #3
      Well that's relieving.
      I don't actually have to pick, because I'm ordering my custom primers with included barcodes. So then I'll go ahead and order this set.

      Comment

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