Index Pooling Guidelines (NEB #E6631)
For the HiSeq®/MiSeq®, Illumina uses a red laser/LED to sequence bases A and C and a green laser/LED to sequence bases G and T. For each cycle, both the red and the green channel need to be read to ensure proper image registration (i.e. A or C must be in each cycle, and G or T must be in each cycle). If this color balance is not maintained, sequencing the index read could fail. Tables 3.1, 3.2 and 3.3 list some valid combinations (up to 4-plex) that can be sequenced together. For combinations > 4 choose any 4-plex combination and add any other index as needed.
Tables 3.4, 3.5 and 3.6 lists each index sequence color coded to correspond to the red/green channel.