Index Pooling Guidelines - 96 Single Index Kit (E6609)

For a link to download a sample sheet with the index sequences for use with the Illumina Experiment Manager (IEM) please go to our FAQ's tab on – NEBNext Multiplex Oligos for Illumina (96 Index Primers) (NEB #E6609).

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. Table 2.1 lists 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.

Table 2.1
Table 2.1
Table 2.2 lists each index sequence color coded to correspond to the red/green channel. For combinations of valid indices, ensure that you will have signal in both the red and green channels in each cycle. See examples below:

Table 2.2 Index Sequences