Sequencing Chemistry
Illumina sequencing systems use one-, two-, or four-channel chemistry to perform base calling. During sequencing, a separate read called the Index Read sequences the index. Dual-indexed sequencing includes Index Read 1 and Index Read 2.
|
Chemistry |
Sequencing System |
|---|---|
|
One-channel |
iSeq 100 System |
|
Two-channel |
NovaSeq X Series NovaSeq 6000 NextSeq Systems MiniSeq Systems MiSeq i100 Series |
|
Four-channel |
MiSeq System HiSeq Systems |
For more information on sequencing chemistry and base calling, refer to the system guide for your instrument. For information on indexing workflows on Illumina systems, refer to Indexed Sequencing on Illumina Systems.
One-Channel Chemistry
One-channel chemistry requires one dye and two images to encode data for the four bases. Intensities extracted from one image and compared to a second image result in four distinct populations, each corresponding to a nucleotide. Base calling determines the population that each cluster belongs to.
When sequencing on a one-channel system, the first two cycles of the Index Read cannot start with two G bases. Otherwise, intensity is not generated.
| • | Make sure that at least one index sequence in a library pool does not start with two G bases. |
| • | Select balanced index sequences so that signal is present in at least one image (preferably both) for every cycle. |
Two-Channel Chemistry
Two-channel chemistry requires two dyes and two images to encode data for the four bases. The MiSeq i100 Series, NovaSeq X Series, and NextSeq 1000/2000 use one image from the green channel and one image from the blue channel. Other two-channel systems use one image from the green channel and one image from the red channel. Intensities extracted from an image and compared to another image result in four distinct populations, each corresponding to a base. Base calling determines the population that each cluster belongs to.
Guidelines for index cycle base selection on a two-channel system vary by sequencing system.
For the following sequencing systems, either of the first two cycles of the Index Read must start with at least one base other than G:
| • | NovaSeq X Series |
| • | NovaSeq 6000 |
| • | NextSeq 1000/2000 |
| • | NextSeq 550 |
| • | MiniSeq |
| • | MiSeq i100 Series |
If an Index Read starts with two G bases, signal intensity is not generated. Signal must be present in the first two cycles. Combine index sequences so that signal is present in at least one channel (preferably both) for every cycle:
| • | Red or Blue channel—A or C |
| • | Green channel—A or T |
When sequencing on a NovaSeq X Series, or a NextSeq 1000/2000 using XLEAP-SBS chemistry, combine index sequences so that signal is present in at least the green channel (preferably both) for every cycle:
| • | Blue channel—A or C |
| • | Green channel—C or T |
For more example index combinations for two-channel systems, refer to the Library pooling guidelines for the NextSeq and MiniSeq systems bulletin on the Illumina website.
Four-Channel Chemistry
Four-channel chemistry uses four dyes and four images per cycle to observe which dye is incorporated into a cluster. A green laser sequences G and T bases, while a red laser sequences A and C bases. To ensure proper image registration, each cycle must include at least one of two nucleotides per color channel.
When sequencing on a four‑channel system, make sure that pooled libraries contain unique and color‑compatible index combinations for each Index Read.
