Index Pooling Guidelines (NEB #E6631)

A sample sheet with the index sequences for use with the Illumina Experiment Manager (IEM) can be downloaded from the "Other Tools and Resources" tab, under "Usage Guidelines and Tips" on the NEBNext Direct Custom Ready Panels product page.

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.

Table 3.1 Valid index primer combinations for NEB #E6631S.


Table 3.2 Valid index primer combinations for NEB #E6631L.


Table 3.3 Valid index primer combinations for NEB #E6631X.


For combinations of valid indexes, ensure that you will have signal in both the red and green channels in each cycle. See examples below:



Tables 3.4, 3.5 and 3.6 lists each index sequence color coded to correspond to the red/green channel.

Table 3.4 Index sequences for NEB #E6631S.


Table 3.5 Index sequences for NEB #E6631L.


Table 3.6 Index sequences for NEB #E6631X.