A sample sheet with the index sequences for use with the Illumina® Experiment Manager (IEM) can be downloaded from the FAQs tab on the NEBNext Direct Cancer HotSpot Panel 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 #E7000S.
Table 3.2 Valid index primer combinations for NEB #E7000L.
Table 3.3 Valid index primer combinations for NEB #E7000X.
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 #E7000S.
Table 3.5 Index sequences for NEB #E7000L.
Table 3.6 Index sequences for NEB #E7000X.
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