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 www.neb.com/E6609 – 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.

For the NovaSeq^® NextSeq^®/MiniSeq^® which utilize 2 color chemistry, valid index combinations must include some indices that do not start with GG in the first two cycles. Use Table 2.1 for some suggested combinations.

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