Illumina Technology

Library Structure
  • Illumina Adapter Sequences: a comprehensive list of all Illumina adapters and indices sequences from various products (including nextera, truseq, etc.)
  • Standard Index oligos:
    • P7: 5’ CAAGCAGAAGACGGCATACGAGAT[index][adapter_sequence]3’
    • P5: 5′ AATGATACGGCGACCACCGA[dual_index][adapter_sequence] 3′
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Laser and Dyes

Nextseq and Novaseq – 2 dye

  • Rather than a separate dye for each base, 2-channel SBS uses a mix of dyes. Images are taken of each DNA cluster using red and green wavelength filter bands.
  • Clusters seen in red or green images are interpreted as C and T bases, respectively. Clusters observed in both red and green images are flagged as A bases (appearing as yellow clusters), while unlabeled clusters are identified as G bases).


  • Miseq – 4 dyes/2 lasers
    • G/T share one laser and C/A share another. When choosing indexes, make sure each base has diversity between the two lasers.



  • iSeq
    • One dye and two chemistry step